Tierärztliche Hochschule Hannover Density gradient centrifugation of stallion semen INAUGURAL – DISSERTATION zur Erlangung des Grades einer Doktorin der Veterinärmedizin - Doctor medicinae veterinariae - ( Dr. med. vet. ) vorgelegt von Gesa Stuhtmann Uelzen Hannover 2011
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PAS (FITC-PNA pos) 11.5±4.3a 4.8±1.6b 11.6±3.9a 10.3±3.6a
SCSA/DFI 10.5±6.0a 4.0±2.0b 11.2±8.4a 11.7±9.5a
(n=6 stallions per group; 3 ejaculates/stallion).
DS: diluted-non-centrifuged semen
EPP: EquiPureTM Pro centrifugation
CC: cushioned centrifugation
WSSM: centrifugation without sperm-selective medium
Sperm recovery: in %
PMS: progressively motile sperm (%)
VCL: curvilinear velocity (µm/sec)
VAP: average path velocity (µm/sec)
ALH: amplitude of lateral head displacement (µm/sec)
PMI: percentage of plasma membrane intact sperm (PI neg)
PAS: Percentage of sperm with positive acrosomal status (FITC-PNA pos)
DFI: denaturation fragmentation index of sperm DNA (%) a, b, c Values with different superscript differ significantly within rows (p≤0.05).
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First Experiment: Cooled-stored semen samples
In Figure 3 to Figure 5 are shown the percentages of progressively motile, plasma membrane
intact sperm (PI neg) and sperm with positive acrosomal status (FITC-PNA pos) during stor-
age at 5°C for up to 72h of sperm obtained after the different preparation procedures.
Upon 72 hours of cooled-storage, spermatozoa prepared by EPP centrifugation showed a
higher percentage of progressively motile, plasma membrane intact sperm and a reduced per-
centage of sperm with positive acrosomal status compared to CC, WSSM and DS (p<0.0001).
After 48 h cooled storage, a higher percentage of progressively motile sperm and lower per-
centage of sperm with positive acrosomal status could be observed in semen samples prepared
by CC, WSSM compared to diluted non-centrifuged semen (p≤0.05).
Upon storage for 72 h, sperm VAP was found to be higher (p≤0.05) for the EPP prepared se-
men than the other treatments. For semen samples centrifuged with EPP slightly higher VCL
values were also found after 24 h compared to diluted semen, although differences between
the four treatments methods were after 48h and 72 h (p≥0.05). Furthermore, in comparison
with the velocity parameters determined direct after centrifugation (0h), semen treated with
any of the three centrifugation methods showed higher VCL as well as VAP values after 24 h.
After 24 h, ALH in the CC and WSSM samples was different (more head movement) from
diluted and EPP prepared semen (p≤0.05). Furthermore, significant differences could be ob-
served after 48 h of storage time between the semen samples treated by EPP and CC, as well
as between DS and EPP treated semen after 72 h (less movement for EPP treated semen).
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Figure 3: Effect of preparation method on the percentage of progressively motile sperm (PMS in %) for semen samples that were stored at 5°C, for up to 72 h. Means ± standard deviation are shown for n=6 stallions per group, 3 ejaculates/stallion, DS: Diluted-non-centrifuged semen, EPP: EquiPureTM Pro cen-trifugation, CC: cushioned centrifugation, WSSM: centrifugation without sperm-selective medium,
a, b, c Values with different superscript differ significantly within days (p≤0.05).
Figure 4: Effect of preparation method on the percentage of plasma membrane intact sperm (PMI neg) (%) for semen samples that were stored at 5°C, for up to 72 h. Means ± standard deviation are shown for n=6 stallions per group, 3 ejaculates/stallion, DS: Diluted-non-centrifuged semen, EPP: EquiPureTM Pro centrifugation, CC: cushioned centrifugation, WSSM: centrifugation without sperm-selective medium centrifugation,
a, b, c Values with different superscript differ significantly within days (p≤0.05).
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Figure 5: Effect of preparation method on the sperm with positive acrosomal status (FITC-PNA pos in %) for semen samples that were stored at 5°C, for up to 72 h. Means ± standard deviation are shown for n=6
stallions per group, 3 ejaculates/stallion, DS: Diluted-non-centrifuged semen, EPP: EquiPureTM Pro cen-trifugation, CC: cushioned centrifugation, WSSM: centrifugation without sperm-selective medium,
a, b, c Values with different superscript differ significantly within days (p≤0.05).
First Experiment: After resuspension with freezing extender
The semen quality of samples before freezing after dilution with freezing extender was as-
sessed. Progressively motility, ALH, plasma membrane and chromatin integrity were not dif-
ferent for any of the three centrifugation methods. Only semen centrifuged using EPP showed
a higher percentage of sperm with a positive acrosomal status after dilution in freezing ex-
tender (p≤0.05). In all semen samples VCL was reduced after dilution in freezing extender
(p≤0.05).
First Experiment: Frozen–thaw semen samples
Sperm quality parameters that were determined after freezing and thawing are shown in Table
2. For sperm that was selected using centrifugation with EPP reduced DFI values were found,
similar as before freezing. The percentage of plasma membrane intact and motile cells, how-
ever, were lower after freezing and thawing using EPP. WSSM also resulted in a lower per-
centage of plasma membrane intact sperm as compared to CC (p≤0.05).
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Table 2: Effect of EquiPureTM Pro density gradient, cushion centrifugation and centrifugation without
sperm selective medium on frozen-thawed sperm quality parameters (means ± standard deviation).
(n=6 stallions per group; 3 ejaculates/stallion).
DS: Diluted-non-centrifuged semen
EPP: EquiPureTM Pro centrifugation
CC: cushioned centrifugation
WSSM: centrifugation without sperm-selective medium
PMS: progressively motile sperm (%)
VCL: curvilinear velocity (µm/sec)
VAP: average path velocity (µm/sec)
ALH: amplitude of lateral head displacement (µm/sec)
PMI: percentage of plasma membrane intact sperm (PI neg)
PAS: percentage of sperm with positive acrosomal status (FITC-PNA pos)
DFI: denaturation fragmentation index of sperm DNA (%) a, b, c Values with different superscript differ significantly within rows (p≤0.05).
Centrifugation techniques
EPP
330 x g
15 min
CC
1000 x g, 20min
WSSM
330 x g
15 min
PMS 9.7±5.0a 29.3±11.4b 25.2±9.0b
VCL 105.5±19.3a 131.8±24.8b 131.1±22.6b
VAP 77.9±13.1a 82.8±13.2a 83.9±14.1a
ALH 2.4±0.5a 3.8±0.3b 3.9±0.2b
PMI (PI neg) 29.0±8.3a 41.5±7.9b 32.1±5.7a
PAS (FITC-PNA pos) 32.5±12.5a 21.2±4.6b 23.1±6.1b
SCSA/DFI 3.1±1.6a 13.6±9.0b 14.1±8.1b
- 32 -
Second experiment: Fresh semen samples
As described for experiment 1 after centrifugation with EquiPureTM Pro, progressive motility
and plasma membrane intact sperm were increased, whereas the percentage of sperm with
positive acrosomal status and the DFI values were reduced compared to the diluted non-
centrifuged semen. However, neither these differences nor the changes in sperm velocity pa-
rameters were significant (p≥0.05).
Second experiment: Frozen–thaw semen samples
After thawing, a stepwise increase in progressive motility and plasma membrane integrity was
seen with different amounts of freezing extender (p≤0.05) (Table 3).
The % DFI values of EquiPureTM Pro treated spermatozoa after thawing were similar to the
results of the fresh semen samples independent of the dilution ratio of freezing extender.
Table 3: Effects of varying quantities of freezing extender on frozen-thawed sperm quality parameters of
EquiPureTM Pro treated semen (means ±standard deviation).
Spermienselektives Aufbereitungsverfahren von Hengstsperma durch Dichtegradient-
zentrifugation
Das Ziel der beiden vorliegenden Studien war die Untersuchung der Spermaqualität von
Frisch-, Versand- und Tiefgefriersamen nach der Aufbereitung durch Zweischichtendichte-
zentrifugation.
Als Dichtezentrifugationslösungen wurden EquiPureTMPro und Iodixanol hinsichtlich ihres
Einflusses auf die mittels CASA (SpermVisionTM) analysierte Spermienmotilität, auf die
Spermienmorphologie (Versuch 2) sowie durchflusszytometrisch ermittelte Plasmamembra-
nintegrität, den akrosomalen Status und die Chromatinintegrität (SCSA-assay) untersucht.
In der ersten Studie wurden die Spermienqualitätsparameter nach Dichtezentrifugation mit
EquiPureTMPro (EPP, 330 x g, 15 min), Zentrifugation ohne Dichtezentrifugationslösung
(WSSM, 330 x g, 15 min), „Kissenzentrifugationstechnik“ (CC, 1000 x g, 20 min) und von
verdünntem Sperma (DS, INRA 82, 25 x 106 Spermien/ml) miteinander verglichen sowie der
Einfluss variierender Volumenanteile des Tiefgefrierverdünners (INRA 82 mit 5 % Eigelb
und 2,5 % Glycerin) auf die Samenqualität der EPP zentrifugierten Spermien untersucht.
Im ersten Versuchsteil war die Spermienrückgewinnungsrate nach der EPP Zentrifugation
(26,6 %) niedriger als nach CC (95,5%) und WSSM (74,0%) (p≤0,05). Frischsamenproben,
die durch EPP Zentrifugation aufbereitet wurden, zeigten im Vergleich zu DS, CC und
WSSM aufbereiteten Samenproben eine Verbesserung der Spermaqualität in Hinblick auf
Vorwärtsbeweglichkeit, Plasmamembranintegrität, akrosomalen Status sowie Chromatinin-
tegrität (p≤0,05). Ähnliche Ergebnisse wurden bei der Untersuchung gekühlt-gelagerten Sa-
mens über eine Lagerungszeit von 72 h (5°C) festgestellt. Bei der Analyse des aufgetauten
Tiefgefrierspermas, welches vor der Samentiefgefrierung mit EPP zentrifugiert wurde, konnte
eine höhere Chromatinintegrität im Vergleich zu mit CC und WSSM aufbereiteten Samens
ermittelt werden (p≤0,05). Im zweiten Teil der ersten Studie wurde ein positiver Einfluss der
Zugabe höherer Verdünneranteile auf die Vorwärtsbeweglichkeit und die Plasmamembranin-
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tegrität (p≤0,05), sowie auf den akrosomalen Status und die Chromatinintegrität der Spermien
(p≥0,05) festgestellt.
In der zweiten Studie wurde der Einfluss der Dichtezentrifugation mit Iodixanol (IODIX,
1000 x g, 20 min) auf die Spermaqualität mit derjenigen nach Aufbereitung durch die routi-
nemäßig eingesetzte Zentrifugationsverfahren (CENTR, 600 x g, 10 min) und der Verdün-
nung von Spermien (EXTEN, EquiProTM, 50 x 106 Spermien/ml) verglichen. Die eingesetzten
Hengste konnten aufgrund ihres vorhergehenden Einsatzes in einem kommerziellen Samen-
tiefgefrierprogramms in Gruppen mit guten (durchschnittlicher Anteil von progressive vor-
wärtbeweglichen Spermien nach dem Auftauen > 35%) oder schlechten Samentiefgefrierei-
genschaft (durchschnittlicher Anteil von progressive vorwärtbeweglichen Spermien nach dem
Auftauen von < 35%) zugeordnet werden.
Nach der IODIX Zentrifugation wurde eine Spermienrückgewinnungsrate von 33,1 % und
nach der CENTR Zentrifugtion von 74,4 % ermittelt (p≤0,05).
Nach IODIX Zentrifugation wurde im Vergleich zu den herkömmlichen Samenaufberei-
tungsmethoden EXTEN und CENTR eine Verbesserung der Spermaqualität im Hinblick auf
die Plasmamembranintegrität, den akrosomalen Status, die Chromatinintegrität sowie den
Anteil an Spermien mit abgelöstem Akrosom ermittelt (p≤0.05). Ebenso konnte ein positiver
Einfluss der IODIX Zentrifugation auf die Samenqualität nach 72-stündiger, gekühlter Lage-
rung bei +5°C hinsichtlich der Vorwärtsbeweglichkeit, der Plasmamembranintegrität und des
akrosomalen Status festgestellt werden (p≤0.05). Desweiteren wurde nach dem Auftauen von
Tiefgefriersamenproben eine Verbesserung der Plasmamembranintegrität, des akrosomalen
Status, der Chromatinintegrität sowie des Anteils an Spermien mit abgelöstem Akrosom
durch IODIX Zentrifugation ermittelt werden (p≤0.05), wobei insbesondere Samen von
Hengsten mit schlechten Tiefgefriereigenschaften ein positiver Einfluss der IODIX Zentrifu-
gation im Vergleich zu herkömmlichen Zentrifugationsverfahren CENTR beobachtet wurde.
Zusammenfassend führte die Dichtezentrifugation mit IODIX und EPP, die mit einer redu-
zierten Spermienrückgewinnungsrate einhergeht, zu einer Verbesserung der Spermaqualität
im Vergleich zu den Aufbereitungsverfahren DS, CC, WSSM, CENTR und EXTEN. Die
durch IODIX und EPP selektierten Spermien wiesen sowohl am Tag der Samengewinnung,
als auch nach 72-stündiger Lagerung bei +5°C und nach Spermatiefgefrierung eine Verbesse-
- 62 -
rung in standardspermatologisch und durchflusszytometrisch ermittelten Spermienqualitätspa-
rametern auf.
- 63 -
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8 Appendix
8.1 EquiPure EquiPureTM Pro centrifugation, a method for sperm clean-
up and its effects on sperm quality of cold- stored and frozen- thawed
stallion sperm
First Experiment: Fresh semen samples
Figure 6: Effect of preparation method on the sperm curvilinear velocity (VCL, µm/sec) for semen sam-
ples that were stored at 5°C, for up to 72 h. Means ± standard deviation are shown for n=6 stallions per
group, 3 ejaculates/stallion, DS: Diluted-non-centrifuged semen, EPP: EquiPureTM Pro centrifugation,
CC: cushioned centrifugation, WSSM: centrifugation without sperm-selective medium,
a, b, c Values with different superscript differ significantly within days (p≤0.05).
- 82 -
Figure 7: Effect of preparation method on the sperm average path velocity (VAP, µm/sec)
for semen samples that were stored at 5°C, for up to 72 h. Means ± standard deviation are shown for n=6
stallions per group, 3 ejaculates/stallion, DS: Diluted-non-centrifuged semen, EPP: EquiPureTM Pro cen-
trifugation, CC: cushioned centrifugation, WSSM: centrifugation without sperm-selective medium,
a, b, c Values with different superscript differ significantly within days (p≤0.05).
- 83 -
Figure 8: Effect of preparation method on the sperm amplitude of lateral head displacement (ALH, µm/sec) for semen samples that were stored at 5°C, for up to 72 h. Means ± standard deviation are shown for n=6 stallions per group, 3 ejaculates/stallion, DS: Diluted-non-centrifuged semen, EPP: EquiPureTM Pro centrifugation, CC: cushioned centrifugation, WSSM: centrifugation without sperm-selective me-dium, a, b, c Values with different superscript differ significantly within days (p≤0.05).
- 84 -
First Experiment: After resuspension with freezing extender
Table 7: Effect of dilution with freezing extender of sperm treated with EquiPureTM Pro density gradient,
cushion centrifugation and centrifugation without sperm-selective medium on sperm quality parameters
(means ± standard deviation).
EPP
330 x g
15 min
after
centrif.
EPP
330 x g
15 min
after
resus.
CC
1000 x g 20min
after
centrif.
CC
1000 x g 20min
after
resus.
WSSM
330 x g
15 min
after
centrif.
WSSM
330 x g
15 min
after
resus.
PMS 80.1a
±5.7
76.8a
±7.2
59.8a
±12.2
54.9a
±14.6
59.3a
±12.0
56.7a
±17.2
VCL 213.9a
±29.3
168.6b
±34.7
223.1a
±30.6
186.2b
±31.6
223.8a
±30.9
182.4b
±33.2
VAP 138.7a
±19.4
117.2b
±18.8
127.0a
±21.8
103.8b
±19.2
125.9a
±21.4
101.7b
±19.2
ALH 3.6a
±0.5
3.4a
±0.6
4.6a
±0.5
4.8a
±0.7
4.6a
±0.5
4.9a
±0.8
PMI
(PI neg)
83.0a
±4.4
81.2a
±5.8
70.4a
±11.4
69.0a
±12.0
71.5a
±12.0
70.9a
±10.7
PAS (FITC
-PNA pos)
4.8a
±1.6
8.5b
±3.5
11.6a
±3.9
13.6a
±4.5
10.3a
±3.6
11.8a
±3.4
SCSA/DFI 4.0a
±2.0
4.2a
±2.0
11.2a
±8.4
12.2a
±8.3
11.7a
±9.5
13.5a
±8.6
(n=6 stallions per group; 3 ejaculates/stallion).
EPP: EquiPureTM Pro centrifugation
CC: cushioned centrifugation
WSSM: centrifugation without sperm-selective medium
After centrif.: values after centrifugation
After resus.: values after resuspension with freezing extender
PMS: progressively motile sperm (%)
VCL: curvilinear velocity (µm/sec)
VAP: average path velocity (µm/sec)
ALH: amplitude of lateral head displacement (µm/sec)
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PMI: percentage of plasma membrane intact sperm (PI neg)
PAS: Percentage of sperm with positive acrosomal status (FITC-PNA pos)
DFI: denaturation fragmentation index of sperm DNA (%) a, b, c Values with different superscript differ significantly within treatment groups (p≤0.05).
Second experiment: Fresh semen samples
Table 8: Effect of EquiPureTM Pro density gradient on sperm quality parameters (means ± standard de-
viation).
Initial value After centrifugation
Sperm recovery - 30.1±4.0
MVO 82.1±8.2a 89.0±4.0a
PMV 69.2±8.9a 76.9±6.0a
VCL 187.7±22.7a 157.3±6.0b
VAP 120.3±13.0a 109.2±3.4a
ALH 3.6±0.4a 3.4±0.6a
PMI (PI neg) 74.6±8.2a 84.4±1.3a
PAS (FITC-PNA pos) 6.1±2.2a 2.4±0.6a
SCSA/DFI 6.6±1.3a 2.4±1.2b
(n=2 stallions per group; 2 ejaculates/stallion).
EPP: EquiPureTM Pro centrifugation
Sperm recovery: in %
PMS: progressively motile sperm (%)
VCL: curvilinear velocity (µm/sec)
VAP: average path velocity (µm/sec)
ALH: amplitude of lateral head displacement (µm/sec)
PMI: percentage of plasma membrane intact sperm (PI neg)
PAS: Percentage of sperm with positive acrosomal status. (FITC-PNA pos)
DFI: denaturation fragmentation index of sperm DNA (%) a, b, c Values with different superscript differ significantly within rows (p≤0.05).
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Danksagung
An dieser Stelle möchte ich allen Personen danken, die zum Gelingen dieser Arbeit beigetra-
gen haben.
Mein Dank gilt meinem Doktorvater Herrn Prof. Dr. Harald Sieme für die Überlassung des
interessanten Themas und die Unterstützung bei der Abfassung der Dissertation.
Ein besonders großes Dankeschön geht an Frau Dr. Gunilla Martinsson für die geduldige Be-
treuung und grenzlose Unterstützung in allen Lebenslagen.
Bei Herrn Landstallmeister Dr. Axel Brockmann und allen Mitarbeitern des Niedersächsi-
schen Landgestüts Celle bedanke ich mich für die Ermöglichung der Versuchsdurchführung
und die gute Zusammenarbeit.
Ebenso möchte ich mich bei Barbara für die Hilfe bei der statistischen Auswertung und Chris-
tian für die Unterstützung bei der Formatierung bedanken.
Genauso bedanke ich mich bei Jane, Jutta, Steven und Harriette für die ständige Diskussions-
bereitschaft und Hilfe bei der Fertigstellung.
Bedanken möchte ich mich weiterhin bei Pamela, Sophie, Wasyl, David, Steffi und Gesche,
Camilla, Daphne und Christiane, die mich während der Doktorantenzeit und auf dem Landge-
stüt stets unterstützt haben.
Mein größter Dank gilt meiner Familie und Heinrich für ihre Unterstützung und den Rückhalt,
den sie mir während der ganzen Zeit gegeben haben.