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University of Veterinary Medicine Hannover
Freeze- drying of equine sperm
and sperm chromatin structure during dried storage
Inaugural-Dissertation
to obtain the academic degree
Doctor medicinae veterinariae
(Dr. med. vet.)
submitted by
Katharina Narten
Minden
Hannover 2017
Academic supervision: Prof. Dr. Harald Sieme
Clinic for Horses
Unit for Reproductive Medicine
Dr. Ir. Harriëtte Oldenhof
Clinic for Horses
Unit for Reproductive Medicine
1. Referee: Prof. Dr. Harald Sieme
2. Referee: Prof. Dr. Dagmar Waberski
Day of the oral examination: 2017/05/11
A contribution from the Virtual Center for Reproductive Medicine, Lower Saxony
III
Meiner Familie
IV
V
Parts of this thesis were presented at the following congresses and published at the
corresponding scientific journals.
OLDENHOF, H., NARTEN, K., BIGALK, J., WOLKERS, W.F., SIEME, H. (2017):
Preservation of sperm chromatin during dried storage. Chromatinintegrität gefriergetrockneter
Spermien während der Lagerung.
50. Jahrestagung Physiologie und Pathologie der Fortpflanzung, gleichzeitig 42. Veterinär-
Humanmedizinische Gemeinschaftstagung, München, 15.-17.02.2017
Reprod Dom Anim; 52 (Suppl. 1):38.
OLDENHOF H, ZHANG M, NARTEN K, BIGALK J, SYDYKOV B, WOLKERS WF,
SIEME H (2016):
Freezing-induced uptake of trehalose by stallion sperm.
Proc. 7th International Symposium on Stallion Reproduction (ISSR), Illinois, USA.
J Equine Vet Sci 43, 73-74
VI
CONTENTS
CONTENTS
1. INTRODUCTION: AIMS AND OUTLINE ................................................................................. 1
2. LITERATURE SURVEY ......................................................................................................... 3
2.1 Preservation of sperm/semen of domestic animals for artificial insemination ... 3
2.2 Osmotic behavior of stallion sperm ......................................................................... 4
2.3. Cryopreservation process ........................................................................................ 5
2.4 Cryoprotective agents for freezing/cryopreservation of stallion sperm .............. 6
2.5 Freeze-drying process ............................................................................................... 7
2.6 Protectant properties for dry preservation ............................................................ 8
2.7 Sperm DNA structure ............................................................................................. 11
2.8 Methods for detecting chromatin integrity and DNA damage ........................... 12
2.8.1 Sperm chromatin structure assay (SCSA) .................................................... 12
2.8.2 Sperm chromatin dispersion (SCD) or halo-test .......................................... 13
2.8.3 Single cell gel electrophoresis (SCGE) or comet-assay ................................ 13
2.8.4 Further methods for detecting DNA damage ............................................... 14
3. MATERIAL AND METHODS ............................................................................................... 16
3.1 Semen collection and processing ........................................................................... 16
3.2 Sperm cryopreservation ......................................................................................... 17
3.3 Hydrated storage of sperm ..................................................................................... 20
3.4 Sperm freeze- drying .............................................................................................. 21
3.5 Computer assisted sperm analysis of motility (CASA) ....................................... 24
3.6 Flow cytometric analysis of membrane integrity (FCM) .................................... 24
3.7 Sperm chromatin structure assay (SCSA) ............................................................ 25
VII
CONTENTS
3.8 Sperm chromatin dispersion test (SCD) ............................................................... 25
3.9 Single cell gel electrophoresis (SCGE) .................................................................. 28
3.10 Statistical analysis ................................................................................................. 30
4. RESULTS............................................................................................................................ 31
4.1 Sperm cryopreservation using various sugars and albumin .............................. 31
4.2 Sperm chromatin structure and stability during hydrated storage at 37°C ..... 33
4.3 Sperm chromatin structure and stability after freeze- drying and dried storage
at 37°C .................................................................................................................... 39
5. DISCUSSION AND CONCLUSIONS ...................................................................................... 45
6. SUMMARY ......................................................................................................................... 51
7. ZUSAMMENFASSUNG ........................................................................................................ 53
8. REFERENCES ..................................................................................................................... 55
9. APPENDIX .......................................................................................................................... 76
10. DANKSAGUNG ............................................................................................................... 80
VIII
ABBREVIATIONS
ABBREVIATIONS
a.u.
BSA
arbitrairy units
Bovine Serum Albumin
CASA computer assisted sperm analysis
COMP αt cells outside the main population of αt
DA diamide
DBD-FISH DNA-breakage detection fluorescence in situ
hybridization
DFI DNA fragmentation index
DIC
DMEM
differential interference contrast
Dulbecco's Modified Eagle's Medium
DNA deoxyribonucleic acid
DTT dithiothreitol
e.g. exempli gratia
et al. et alia
Fig. figure
FTIR
FCS
Fourier transform infrared spectroscopy
Fetal calf serum
GLU
ICSI
Glucose
Intracytoplasmic sperm injection
mRNA messenger ribonucleic acid
PBS phosphate buffered saline
r.u. relative units
ROS reactive oxygen species
SCD sperm chromatin dispersion test
SCSA sperm chromatin structure assay
IX
ABBREVIATIONS
SCGE single cell gel electrophoresis
SUC sucrose
Tg glass transition temperature
TRE trehalose
INTRODUCTION: AIMS AND OUTLINE
1
1. INTRODUCTION: AIMS AND OUTLINE
Preservation and long term storage of (bioactive) molecules, macromolecular
assemblies, cells and tissues for possible later use is of great interest for applications in
pharmacy, agriculture (e.g. food sciences, breeding industry), (regenerative) medicine (e.g.
biobanking), as well as scientific research. In the equine breeding industry, this includes
preservation of gametes and embryos from specific (valuable) individuals for distribution and
storage, both for the existing genetic pool as well materials from deceased animals. For short-
term storage and transportation over moderate distances, stallion sperm can be stored
hypothermically at ~4°C after dilution in a so-called extender, which is buffered and contains
antibiotics, nutrients and milk/protectants (AURICH 2008). For long-term storage,
cryopreservation is typically used. Freezing extenders include additional protective agents
like egg yolk and glycerol (HAMMERSTEDT et al. 1990). With cryopreservation, specimens
are stored in liquid nitrogen, which therefore needs energetically expensive freezers/liquid
nitrogen tanks. Dry preservation and storage under ambient conditions (i.e. at room
temperature) offers an attractive alternative to cryopreservation, since it would allow for easy
and low-cost handling. In nature, anhydrobiotic organisms and organs exist which can
withstand desiccation and resume metabolic activity upon rehydration (CROWE et al. 1992).
Such organisms typically accumulate disaccharides like trehalose and sucrose, and (high
molecular weight) proteins which facilitate formation of a highly viscous glassy matrix when
water is removed, as well as antioxidants which protect against oxidative damage. In the
anhydrobiotic state, molecules and organelles are immobilized and preserved (CROWE et al.
1992; CROWE et al. 1998). Formulations that include disaccharides are widely applied for
dry preservation in pharmaceutics and food sciences. Dry preservation of mammalian cells,
however, is more challenging (MARTINS et al. 2007). Drying of sperm completely abolishes
motility, and no membrane intact sperm are recovered. However, their chromatin and genetic
integrity can be preserved successfully (CHOI et al. 2011). Sperm chromatin stability is
increased if formulations for freeze-drying are supplemented with calcium chelators and
antioxidants (SITAULA et al. 2009) or disaccharides (CROWE et al. 2001; MCGINNIS et al.
2005; MARTINS et al. 2007; SITAULA et al. 2009). Freeze-dried spermatozoa have been
successfully used to fertilize oocytes via intracytoplasmic sperm injection (ICSI), in multiple
INTRODUCTION: AIMS AND OUTLINE
2
species including horses (KANEKO et al. 2003; CHOI et al. 2011). However, not a lot is
known about stability of freeze-dried sperm for long-term storage. The so-called DNA
fragmentation index which is derived using the sperm chromatin structure assay (SCSA)
closely correlate with fertility rates (EVENSON et al. 1980; SPANO et al. 2000). SCSA is the
‘gold standard’ for evaluating DNA damage and involves acid/lysis treatment, and flow
cytometric analysis after staining with acridine orange to distinguish between double stranded
native and single stranded damaged DNA (EVENSON et al. 1980; EVENSON et al. 2002).
We hypothesized that freeze-drying of sperm using formulations containing non-
reducing disaccharides in combination with albumin might improve sperm chromatin stability
during dried storage. Therefore, protective effects of various sugars with(out) albumin were
tested during cryopreservation and hydrated storage, as well as after freeze-drying and dried
storage. In addition to evaluation of sperm viability, special emphasis was placed on
assessment of chromatin structure and DNA damage. For the latter, various assays were used
and compared. In addition to SCSA, the sperm chromatin dispersion test (SCD) or Halo-test
was used as well as single cell gel electrophoresis (SCGE) also known as the comet-assay.
SCD and SCGE were used to visualize differences in sperm chromatin structure
microscopically for single cells.
LITERATURE SURVEY
3
2. LITERATURE SURVEY
2.1 Preservation of sperm/semen of domestic animals for artificial
insemination
Artificial insemination allows for insemination of mares with (valuable) genetic
material from a stallion, irrespective of their locations. Semen can be stored for a couple of
days at 4°C, after dilution in a so-called extender, or cryopreserved and stored for multiple
years in liquid nitrogen (ARMSTRONG et al. 1999; AGARWAL et al. 2008). Extenders are
buffered, and contain antibiotics, nutrients and protectants. Dilution of semen in an extender
is done to preserve sperm function during storage. Furthermore, from one ejaculate, multiple
insemination doses can be produced.
Liquid preservation and storage at 4°C allows for transportation of sperm over
moderate distances and use within 2−3 d (KOTHARI et al. 2010). Sperm viability decreases
progressively when stored at room temperature (FORD 2001), because of metabolic activity
and exposure to oxidative stress. The rate at which viability decreases is slowed during
hypothermic storage, with reduction of the storage temperature to 4°C. Also, addition of
protectants including antioxidants to the extender may have a positive effect on sperm
viability and longevity.
In the equine breeding industry, in recent years, the use of cryopreserved semen has
increased drastically (BARKER and GANDIER 1957; SAMPER and MORRIS 1998;
VIDAMENT 2005). Cryopreservation is advantageous since samples can be preserved and
stored indefinitely after collection and processing, even after castration and/or if the animal is
deceased. Typically, glycerol and egg yolk are used as cryoprotective agents, and samples are
stored in liquid nitrogen at −196°C. At this temperature, the samples are in a glassy state. For
cryopreservation of semen, slow cooling rates (~40−60°C min−1) and low concentrations of
permeating agents are used (e.g. 2−5% glycerol). In addition, reports exist in which semen is
preserved via ice-free cryopreservation or vitrification, which involves fast cooling rates (up
to 100°C min−1) and use of high concentrations of protective agents (e.g. up to 7.5% ethylene
glycol) (FAHY et al. 2004; FAHY and WOWK 2015; SANFILIPPO et al. 2015).
Vitrification, however, is typically used for cryopreservation of tissues and embryos.
LITERATURE SURVEY
4
Long term storage of sperm in the dried state at ambient atmosphere (i.e. room
temperature) would eliminate the need for use of liquid nitrogen tanks, and make transport
easier (KUSAKABE et al. 2001). Sperm drying results in non-viable sperm. Death sperm
with intact DNA, however, can be used for intracytoplasmic sperm injection or ICSI (CHOI et
al. 2011; HOCHI et al. 2011; KESKINTEPE and EROGLU 2015). Several formulations have
been tested for freeze-drying of sperm. Commonly used are TRIS-buffered solutions
supplemented with a calcium chelator such as EGTA or EDTA (KUSAKABE et al. 2001;
KANEKO and NAKAGATA 2006).
2.2 Osmotic behavior of stallion sperm
Upon ejaculation and deposition in the female reproductive tract, as well as with
dilution in extenders (containing high concentrations of protective agents), sperm are exposed
to osmotic stress. Osmosis is passive diffusion of water along the concentration gradient,
through the phospholipid bilayer or water channels, to achieve equilibrium between the intra-
and extracellular solute concentration. This results in shrinking or swelling of a cell in case of
transport of water out or into the cell, respectively (MAZUR 1984; HOFFMANN et al. 2009).
When cells shrink or swell, changes beyond their osmotic tolerance limits can be lethal. The
osmotic range in which cells behave as so-called linear osmometers is described by the Boyle
van ’t Hoff equation. Stallion sperm behave as linear osmometers in the 150 to 900 mOsm
kg−1 osmotic range, and have an osmotically inactive volume of 70−80% (POMMER et al.
2002; GLAZAR et al. 2009; OLDENHOF et al. 2011). The functional integrity of the sperm
plasma membrane can be evaluated as sperm swelling in hypotonic medium (RAMU and
JEYENDRAN 2013). The hypo-osmotic swelling (HOS) test has been used to predict fertility
rates (NEILD et al. 2000) and cryosurvival (VIDAMENT et al. 1998). Stallion sperm motility
drops below 50% when cells are exposed to osmolalities below 200 or above 400 mOsm kg-1
(BALL and VO 2001; ERTMER et al. 2016).
Membrane permeability for cryoprotective agents as well as water affects the extent of
osmotic and cellular damage. Permeating protectants like glycerol, ethylene glycol and
dimethyl formamide can move freely across cellular membranes, whereas sugars typically
cannot. Recently it was found that there is freezing-induced uptake of membrane-
LITERATURE SURVEY
5
impermeable disaccharides (ZHANG et al. 2016). Membrane hydraulic permeability (i.e.
water transport) is affected by the presence of (permeating) cryoprotective agents, as well as
the membrane lipid composition and cholesterol content (GLAZAR et al. 2009; AKHOONDI
et al. 2012).
2.3. Cryopreservation process
When semen is cryopreserved, cells are exposed to cold shock, ice crystal formation
and cellular dehydration, which all can cause irreversible damage (MAZUR 1984;
HAMMERSTEDT et al. 1990; AMANN and PICKETT 1987). Furthermore, passage through
membrane phase transitions has been associated with leakage of solutes to the extracellular
environment, which is detrimental to cells (CROWE et al. 1989; DROBNIS et al. 1993).
Upon extracellular ice formation, sperm are exposed to hypertonic conditions because the
solute concentration in the extracellular unfrozen fraction increases. This causes movement of
water out of the cell and dehydration, in order to retain equilibrium between the intra- and
extracellular solute concentrations. During thawing, the reverse process takes place, and
sperm are exposed to hypotonic conditions. For cells undergoing freezing, a two-factor
hypothesis of damage has been developed (MAZUR et al. 1972; MAZUR 1984). At high
cooling rates, viability losses are associated with intracellular ice formation. For cells cooled
slowly, damage is described as ‘solution effects injury,’ which is related to cellular
dehydration. Typically, there is an optimal cooling rate for maximum survival.
Semen collection for cryopreservation typically takes place during the non-breeding
season. After a period of sexual rest prior to use for cryopreservation, regular semen
collections should be performed to reach a steady quality of ejaculates. Furthermore, to ensure
quality, semen collections should be performed at 48 h intervals. After collection and dilution
with at least an equal volume of ‘primary’ extender of 37°C (containing nutrients, milk and
antibiotics), diluted semen is centrifuged to remove most of the seminal plasma and to obtain
concentrated sperm samples. After centrifugation, sperm is diluted to the desired final
concentration (e.g. 100×106 sperm mL−1) using extender containing cryoprotective agents like
glycerol and egg yolk (SIEME and OLDENHOF 2015; SIEME 2011). Then, samples are
cooled to 4‒5°C at a rate about 0.1−0.3°C min−1, packaged in 0.5-mL plastic straws, followed
LITERATURE SURVEY
6
by freezing at a rate of 10−60°C min−1 to temperatures below −80°C. Finally, straws are
plunged and stored in liquid nitrogen.
2.4 Cryoprotective agents for freezing/cryopreservation of stallion sperm
To minimize cellular damage during freezing and thawing, cryoprotective agents are
used. Cryoprotectants play a role in minimizing exposure to osmotic stress, preserving
biomolecular and cellular structure, affecting ice formation and limiting damaging effects of
reactive oxygen species (AMANN and PICKETT 1987; HAMMERSTEDT et al. 1990;
PARKS and GRAHAM 1992; WOELDERS et al. 1997; WATSON 2000; MARTINEZ-
PASTOR et al. 2009). The membrane hydraulic permeability at low temperatures is one of the
limiting factors of the sperm survival during freezing (MAZUR 1984; WATSON et al. 1992).
Cryoprotective agents increase the permeability of membranes for water and allow cells to
dehydrate at lower temperatures therewith facilitating them to respond osmotically for a
longer time (AKHOONDI et al. 2012; OLDENHOF et al. 2013). Permeating cryoprotective
agents (e.g. glycerol, ethylene glycol, dimethyl formamide) can move through cellular
membranes. For liposome model systems, it has been described that glycerol may form
hydrogen bonds with membrane phospholipid headgroups, facilitating stabilization
(ANCHORDOGUY et al. 1987). Cellular membranes enter a packed gel phase upon
extracellular ice formation. This indicates that cryoprotectants do not replace hydrogen bonds
nor facilitate entrapment of water around the phospholipid head groups in frozen state
(OLDENHOF et al. 2010; AKHOONDI et al. 2012). In addition to permeating cryoprotective
agents, non-permeating sugars (e.g. sucrose, trehalose) and polysaccharides (e.g. HES) or
polymers (e.g. BSA, PVP) affect ice crystal formation and/or the glass transition temperature
(Tg) of formulations. If formation of a stable glassy state occurs at higher subzero
temperatures, this would allow for storage at higher temperatures and handling at suboptimal
conditions (CROWE et al. 1997; STOLL et al. 2012; OLDENHOF et al. 2013). Antioxidants
like albumin or catalase may help against oxidative stress during handling and reduce
(mitochondrial) membrane damage occurring with exposure to temperature changes during
freezing and thawing (UYSAL and BUCAK 2007).
LITERATURE SURVEY
7
2.5 Freeze-drying process
Freeze-drying involves a freezing and drying step, after which samples can be stored
in the dried state. It is typically used to store heat-labile materials such as hormones, vaccines
and enzymes (ADAMS 1995, YADAVA et al. 2008). In addition, there is an interest in
stabilizing mammalian cells including sperm in the dried state. For biochemical activity (i.e.
metabolic processes, degradation reactions) water is essential. Therefore, reduction of the
sample water content aims to reduce such activities to stabilize samples in a ‘senescent’ state.
Metabolic activity is resumed upon addition of water. Air-drying using high temperatures is a
simple and inexpensive method, which is typically used for food products. With this
approach, however, the chemical and physical properties will be affected, which makes it
unsuitable for dehydration of products which should retain biochemical/metabolic activity
after rehydration. For the latter type of materials, freeze-drying can be applied which uses
sublimation for removal of water from the sample (ADAMS 1995).
Figure 2.1 depicts the water phase diagram with indicated the solid, liquid and gas
phase. During freezing, samples in a solution containing water convert from the liquid to ice
phase. With freeze-drying, during freezing, the sample temperature should be lowered below
the eutectic, glass transition and melting temperature (i.e. ‘triple point’). This immobilizes
components within the freeze-drying formulation in a stable ice crystal structure and prevents
foaming upon later application of vacuum. Furthermore, it reduces thermal denaturation.
Then, below the triple point temperature, the pressure is lowered (i.e. vacuum is applied)
which results in the direct transition from the solid to vapor phase (i.e. ice is replaced by gas).
Following this, for further removal of water, either the pressure can be further lowered or the
temperature can be increased (HOCHI et al. 2011; KESKINTEPE and EROGLU 2015). After
return to ambient temperature (and maintenance under vacuum), samples are sealed to prevent
moisture uptake during storage.
LITERATURE SURVEY
8
2.6 Protectant properties for dry preservation
Freeze-drying is done in formulations which preserve specimens both against freezing
and drying. In Table 2.1 a listing is presented on formulations that have been used for freeze-
drying of sperm from different species. Initially CZB or DMEM medium was used
(WAKAYAMA and YANAGIMACHI 1998). Later, TRIS-buffered solutions were used.
Addition of calcium chelators (EGTA, EDTA) to such media was found to improve sperm
stability and improve fertilization rates with use of freeze-dried sperm for ICSI (KUSAKABE
et al. 2001; KANEKO and NAKAGATA 2006). Further supplements include disaccharides
and antioxidants. Disaccharides like trehalose and sucrose facilitate formation of a stable
Figure 2.1. Overview of the freeze- drying process, which involves converting specimens from the
liquid to solid phase by freezing below the eutectic temperature, followed by lowering the pressure
below the triple point and then subject to a vacuum (i.e. lower the pressure) or supply heat to
convert from the ice to gas phase.
LITERATURE SURVEY
9
glassy matrix (CROWE et al. 2003; OLDENHOF et al. 2013). Antioxidants like catalase may
counteract oxidative stress (SITAULA et al. 2009). Also albumin (e.g. BSA or FCS) may be
added as a reactive oxygen species scavenger. Protection against oxidative DNA damage is
especially important in case of preserving sperm fertilization potential (GONZALEZ-MARIN
et al. 2012; AITKEN et al. 2016). It should be noted that extracellular protectants may not
preserve intracellular structures. Therefore, for freeze-drying of mammalian cells, several
methods have been employed for loading of cells with protective agents before exposure to
freeze-drying. Recently it was found that membrane-impermeable disaccharides are taken up
by cells upon exposure to freezing-and-thawing (ZHANG et al 2016).
Species No References Pressure
[mbar]
Drying
Time [h]
Agents
Mouse
and Rat
1 Kaneko et al.
(2003a,b)
0,030 -
0,033
4 EGTA-TRIS- HCl buffer plus diamide or DTT
2 Kaneko and
Nakagata
(2005)
0,037 4 EGTA-TRIS- HCl buffer or EDTA- TRIS- HCl buffer
3 Kaneko and
Nakagata
(2006)
0,030 and
0,045a
4 EDTA
4 Kaneko and
Serikawa
(2012)
0,038 and
0,058a
4 EDTA- TRIS
5 Kawase et al.
(2005)
0,040 and
0,001a
8 and 6a
6 Kawase et al.
(2007, 2009)
0,37 and
0,001a
13 and 6a EGTA- TRIS- HCl buffer
7 Kusakabe et al.
(2001, 2008)
0,032 -
0,040
4 EGTA- TRIS- HCl buffer
8 Wakayama and
Yanagimachi
(1998)
0,001 12
9 Ward et al.
(2003)
0,030 -
0,033
4 without protectants
Table 2.1. Listing of freeze drying conditions and formulations (i.e. protectants), which have been
used for freeze-drying of sperm from different species. In addition to the pressures used, times for
both primary (a) and secondary (b) drying times are indicated.
LITERATURE SURVEY
10
10 Hochi et al.
(2008)
0,37 and
0,001
14 and 3a
Rabbit 11 Liu et al. (2004) 0,023 -
0,040
4 EGTA- TRIS- HCl buffer
Dog 12 Watanabe et al.
(2009)
0,37 and
0,001a
- EGTA- TRIS- HCl buffer
Cat 13 Ringleb et al.
(2011)
0,16 4
Pig 14 García Campos
et al. (2014)
0,015 -
0,005a
24 and 6a EDTA buffer plus trehalose, lactose (EDTA- TL), EDTA
buffer plus sucrose, lactose (EDTA- SL), EDTA buffer
plus lactose (EDTA-LL)
15 Kwon et al.
(2004)
0,039 - Ca- Ionophore
16 Men et al.
(2013)
0,013 and
0,13a
19 and 3a EGTA plus trehalose
Horse 17 Choi et al.
(2011)
0,13 30 EDTA- TRIS- HCl buffer, Chatot-Ziomek- Bavister
medium plus BSA (Sp- CZB), DTT, leupeptin,
antipain,soybean trypsin inhibitor (NIM)
Cattle
18 Abdalla et al.
(2009)
0,37 and
0,001a
14 and 3a
19 Hara et al.
(2011)
0,37 and
0,001a
14 and 3a EGTA- TRIS- HCl buffer, NaCl
20 Hara et al.
(2014)
1,98, 0,57
or 0,12
6 EGTA- TRIS- HCl buffer, NaCl (EGTA buffer), EGTA-
TRIS- HCl plus trehalose (m EGTA buffer)
21 Keskintepe et
al. (2002)
0,19 12 - 18 Hepes- TALP- medium, modified Eagle- medium with
10 % FBS
22 Martins et al.
(2007a, b)
0,35 12 - 16 TCM 199 with Hanks salts plus 10% FCS with/ without
trehalose and EGTA
LITERATURE SURVEY
11
2.7 Sperm DNA structure
The success of fertilizing an oocyte (i.e. fusion of nuclei of the male and female
gamete) is dependent on sperm quality. This includes sperm motility and morphology as well
as chromatin integrity. Chromatin consists of DNA and proteins. DNA consists of nucleotides
which are composed of the sugar desoxyribose, residues of phosphate groups and four
different bases (adenine, cytosine, guanine and thymine). The nucleotides are connected via
phosphate and hydroxyl groups, and hydrogen bonds between the bases, resulting in a three-
dimensional double helix with the bases located inside and the sugar-phosphate backbone
outside (ALLIS et al. 2008).
In somatic cells, DNA is wound around histones to form the nucleosomes. This
packaging results in a 6-fold decrease in DNA-length (PIENTA et al. 1991). Further
packaging of DNA around histones results in condensation and a negative supercoil, which
can be easily separated for replication or transcription ( LIU and WANG 1987). Furthermore,
octomers control conformation during DNA transcription (CHEN et al. 1991). Formation of a
so-called solenoid fiber, further increases chromatin packing (FINCH and KLUG 1976).
For sperm, telomeres are longer as compared to those of somatic cells (DE LANGE et
al. 1990). During spermatogenesis, histones of somatic cells are replaced by highly basic,
arginine-rich protamines. This allows formation of compact doughnut-shaped loops of DNA
around protamines, resulting in sperm nuclei with a 40-fold smaller volume as that of somatic
nuclei (WARD and COFFEY 1991; WARD 1993). This ‘crystalline state’ protects DNA
during transport through the female reproductive tract (BJORNDAHL and KVIST 2014).
Different types of protamines are found in stallion (BALHORN 1982, 2007;
GOSALVEZ et al. 2011), of which protamine 1 (P1) and 2 (P2) have been correlated with
sperm chromatin stability (CASTILLO et al. 2011) and fertility (PARADOWSKA-DOGAN
et al. 2014). P1 is rich in positively charged arginine which can interact with the negatively
charged phosphodiester and cysteine residues which lack SH-groups. Intra- and
intermolecular disulfide bonds play a role in chromatin packing and stability (KUMAROO et
al. 1975; WARD 1993). Compared to P1, P2 has low numbers of arginine residues. Protamine
1 to 2 ratios in sperm have been correlated with the level of sperm chromatin condensation or
packing, which in turn affects susceptibility for (induced) DNA damage and sperm quality.
LITERATURE SURVEY
12
Accumulation of Zn2+ during spermiogenesis facilitates further stabilization. Histidine has
imidazole and SH-groups which can bind Zn2+. Furthermore, Zn2+ prevents formation of too
many disulfide bonds; facilitating DNA unfolding after fertilization the oocyte
(BJORNDAHL and KVIST 2010).
2.8 Methods for detecting chromatin integrity and DNA damage
2.8.1 Sperm chromatin structure assay (SCSA)
The sperm chromatin structure assay (SCSA) originally described by EVENSON et al.
(1980), is the ‘gold standard’ for evaluation of chromatin integrity. It was found that sperm
nuclear DNA from fertile men and bulls was more resistant to heat- and acid-induced
denaturation as compared to sperm DNA from their infertile counterparts. With SCSA, sperm
samples are diluted, after which they are acid-treated (pH 1.2, for 30 s) to open the DNA
strands at damaged sides (i.e. at strand breaks). Then, the DNA intercalating fluorescent dye
acridine orange is used, to distinguish between single-stranded denatured DNA and double
stranded native DNA regions in sperm chromatin (DARZYNKIEWICZ et al. 1975;
BUNGUM et al. 2004). Stained sperm samples are analyzed using flow cytometry, and the
extent of DNA damage is calculated as the ratio of red florescence versus total (red plus
green) fluorescence. SCSA data can be presented graphically as scatter plots obtained with
flow cytometric analysis. In such plots, the x- and y-axis represent the red and green
fluorescence intensities of each particle, respectively. Sperm with normal chromatin form the
main population, while sperm right from this population exhibit increased red fluorescence of
damaged DNAND This is expressed as the DNA fragmentation index (DFI), and is also
referred to as the percentage of cells outside the main population (COMP αt). Parameters
derived with SCSA analysis are considered the most valuable parameters for assessment of
male fertility (LOVE and KENNEY 1998; EVENSON et al. 2002). According to the
classification described by LOVE (2005), highly fertile stallions have DFI-values around
12%, whereas sub- and infertile stallions have DFI-values around 17% and 25%, respectively.
The quality of sperm DNA/chromatin structure from fresh and diluted semen as well as
cryopreserved semen can be evaluated with this assay (EVENSON and JOST 1994).
LITERATURE SURVEY
13
2.8.2 Sperm chromatin dispersion (SCD) or halo-test
With the sperm chromatin dispersion (SCD) test, chromatin structure is visualized
microscopically for single cells. Therefore, spermatozoa are embedded in agarose on slides
and treated with acid and lysis solution, after which specimens are stained with DNA
intercalating dye (FERNANDEZ et al. 2003). Treatment with acid solution facilitates opening
of sperm DNA and removal of nuclear proteins (FERNANDEZ et al. 2005), while treatment
with lysis solution containing Triton-X100 and DTT results in chromatin decondensation. The
test is based on the principle that sperm with intact chromatin undergoes less DNA
fragmentation during acid/lysis treatment and exhibit large ‘halos’ of dispersed DNA loops
which are visualized by DNA intercalating dye. In contrast, sperm with fragmented DNA
exhibit small or no ‘halos’. This assay is commercially available as the ‘Halomax’ kit (from
Halotech DNA SL, Madrid, Spain). An improvement of the initial SCD protocol which used
fluorescent DNA intercalating dyes (e.g. DAPI, SYBR-14) was the finding that staining with
‘Wright’s solution’ and light microscopic analyses of halo-sizes worked well, while for
quantitative analysis of halos-sizes image analysis software can be used (FERNANDEZ et al.
2003).
2.8.3 Single cell gel electrophoresis (SCGE) or comet-assay
The ‘comet assay’ as described by OSTLING and JOHANSON (1984) uses gel
electrophoresis to visualize DNA strand breaks and fragmentation. Initially, electrophoresis
was performed using neutral conditions, whereas later electrophoresis under alkaline
conditions was also described (LINFOR and MEYERS 2002; RIBAS-MAYNOU et al. 2014).
For this assay, cells are embedded in agarose and treated with lysis and alkaline solution,
followed by alkaline electrophoresis. During electrophoresis, DNA fragments are separated
according to their size and charge. Small damaged DNA fragments move away from the
nucleus/head to the anode more rapidly resulting in a tail with DNA fragments and comet-
shaped structure (GYORI et al. 2014). To visualize and observe comets, specimens are
stained with DNA intercalating fluorescent dye; for analysis using fluorescence microscopy.
LITERATURE SURVEY
14
Sperm with intact chromatin have no/smaller ‘comets’, whereas sperm with damaged
chromatin exhibit larger tails and higher relative DNA contents in the tail as compared to the
head. Such parameters can be measured for single cells with need of only low numbers of
cells per sample. Commercial image analysis software is available for such analyses, as well
as freeware (GYORI et al. 2014).
2.8.4 Further methods for detecting DNA damage
Figure 2.2 shows a schematic presentation various assays for evaluating chromatin
structure and DNA damage, and processes involved to reveal similarities and differences.
The DNA breakage detection-fluorescent in situ hybridization (DBD-FISH) test
includes embedding of the sample in agarose, followed by incubation in alkaline buffer as
described above for the comet-assay. With this method, however, samples are hybridized with
fluorescently labeled DNA fragments which bind their complementary single stranded
counterparts if present and intact (i.e. not damaged) (CORTES-GUTIERREZ et al. 2014).
With the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay,
samples are incubated with terminal deoxynucleotidyl transferase which can incorporate
(fluorescently) labeled deoxyuridine triphosphate nucleotides in case of presence of DNA
nicks. Presence of DNA damage (i.e. nicks) therewith can be quantified using microscopy
and/or flow cytometry (GORCZYCA et al. 1993; ERENPREISS et al. 2004).
In addition to approaches which visualize (induced) DNA damage, there are assays
which aim to detect differences in chromatin packing. With Chromomycin-A3 (CMA3), sperm
chromatin condensation anomalies are reported to be detected. CMA3 is believed to compete
specifically with protamines for binding to DNA, which is seen as decreased fluorescence in
case the chromatin is very tightly packed and condensed by protamines. The CMA3
fluorescence intensity thus is a measure for chromatin packing (MANICARDI et al. 1995).
Also non-fluorescent DNA intercalating dyes like toluidine blue can be used. Furthermore,
DTT-treatment prior to staining with DNA intercalating dyes may be performed. As described
above, (further) reduction of disulfide bonds and chromatin ‘loosening’ makes DNA available
for dye binding (KRZANOWSKA 1982; BARRERA et al. 1993).
LITERATURE SURVEY
15
Spectroscopic techniques, like Raman and Fourier transform infrared transform
(FTIR) microspectroscopy can be done directly without the need of a sample preparation, and
have been applied for evaluating sperm chromatin structure and damage (SANCHEZ et al
2012; OLDENHOF et al 2016). These techniques are based on interaction between light and
molecular groups present within the sample. In case of spectra collected from (individual)
sperm, this gives information of presence and conformation of endogenous biomolecules.
Oxidative DNA damage and the degree of chromatin decondensation, for example, are
characterized by specific changes in spectral bands arising from the phosphate backbone of
DNA (SANCHEZ et al. 2012). It has been suggested that sperm can be selected for ICSI
based on their spectral fingerprint (LIU et al. 2013).
Figure 2.2. Overview of processes involved in different assays for evaluating sperm chromatin
structure and detecting DNA damage. See text for details.
MATERIAL AND METHODS
16
3. MATERIAL AND METHODS
3.1 Semen collection and processing
Semen was collected from stallions of the Hanoverian warmblood bred that were held
at the National Stud of Lower Saxony, Celle, Germany. Stallions were kept in box stalls
bedded with straw, were fed with grain and hay three times a day and had water ad libitum,
according to institutional and national regulations. All semen samples were aliquots from
routine semen collections performed for the artificial insemination program of the stud.
Semen collections for the studies described in this thesis took place from September through
December 2015, during the non-breeding season. To stabilize extra gonadal sperm reserves,
semen collections were performed for two weeks before use for experiments. Semen
collection was done using an artificial vagina and a breeding phantom (both model
‘Hannover’ Minitüb, Tiefenbach, Germany), and ejaculates were filtered to remove the gel
fraction. Directly after collection, semen was evaluated and the sperm concentration was
determined using a NucleoCounter Sp-100 (ChemoMetec A/S, Allerød, Denmark). Semen
was diluted with pre-warmed (37°C) skim milk extender (INRA-82) to a concentration of
100×106 sperm mL−1. To remove the seminal plasma, diluted semen was centrifuged in 50
mL conical tubes at 600×g for 10 min, the supernatant was removed and the sperm pellet was
resuspended with fresh INRA-82 to a concentration of 100 or 200×106 sperm mL−1.
INRA-82 was prepared by mixing equal volumes of commercial 0.3% ultra-heat-
treated skim milk and glucose saline solution, according to VIDAMENT et al. (2000).
Glucose saline solution was prepared by dissolving the following components in 500 mL
water: 25 g glucose monohydrate, 1.5 g lactose monohydrate, 1.5 g raffinose pentahydrate,
0.25 g sodium citrate dihydrate, 0.41 g potassium citrate monohydrate, 4.76 g HEPES, 0.5 g
penicillin, 0.5 g gentamycin. The pH was 6.8−7.0 and the osmolality 300−330 mOsm.
MATERIAL AND METHODS
17
3.2 Sperm cryopreservation
For cryopreservation, 1 mL INRA-82 supplemented with two-fold the final
concentration of cryoprotective agents described in detail below was slowly added to an equal
volume of diluted semen (100×106 sperm mL−1 in INRA-82). This resulted in a final volume
of 2 mL, from which 500 µL was removed for pre-freeze measurements, while the remaining
1.5 mL was cooled down to 5°C, at ~0.1°C min−1 during 2 h. This was done by placing
samples in a beaker with room temperature water in a fridge set at 5°C. While maintaining
samples at 5°C in a cooling cabinet, 500 µL straws were filled with diluted semen, and placed
on racks. Straws were cooled at ~40°C min−1 by placing the racks in a polystyrene box filled
with liquid nitrogen such that the straws were 3 cm above the liquid level in the vapor phase
of liquid nitrogen. After 10 min, straws were plunged in liquid nitrogen and stored for at least
one day. Post-thaw analysis was done after incubating straws for 30 s in a 37°C water bath.
Two different cryopreservation studies were performed. In Experiment 1, six
ejaculates from different stallions (ages 3−10 years) were used for determining the optimal
sucrose (SUC) and albumin (BSA: bovine serum albumin) concentrations for sperm
cryosurvival. Therefore, sperm were frozen in INRA-82 supplemented with 2.5% (v/v)
clarified egg yolk (EY) and 0−200 mM sucrose (Carl Roth, Karlsruhe, Germany) or 0−10%
(w/v) BSA (fraction V, pH 7.0; Serva, Heidelberg, Germany). Five different concentrations
were tested, both for SUC and BSA. In addition, the optimal sucrose concentration (50 mM)
was tested in combination with 0−10% BSA. For comparison, also 2.5% (v/v) glycerol in
combination with 0−10% BSA was tested. In Figure 3.1 a schematic presentation is presented
about the study design of Experiment 1.
In Experiment 2, sperm cryopreservation was performed using different sugars, alone
as well as in combination with BSA. These combinations were also used later for freeze-
drying (Experiment 4). For Experiment 2, semen from nine different stallions (3−20 years)
was used. In total 8 different freezing formulations were tested and sperm characteristics were
analyzed both before and after freezing-and-thawing. The regular freezing extender for
cryopreservation was composed of INRA-82, supplemented with 2.5% EY and 2.5% GLY. In
addition, INRA-82 with EY without further supplements was tested, as well as supplemented
with glucose (GLU; 100 mM), sucrose (SUC; 50 mM) or trehalose (TRE; 50 mM) with(out)
MATERIAL AND METHODS
18
BSA (1.71%). Glucose is a reducing monosaccharide, whereas sucrose and trehalose are non-
reducing disaccharides. Concentrations that were tested were mass equivalents of 50 mM
SUC, which was 1.71% (w/v). Sugar/BSA mixtures were tested at a 1/1 (w/w) ratio, meaning
1.71 g of each per 100 mL. In Figure 3.2, a schematic presentation is presented about the
study design of Experiment 2.
Figure 3.1. Schematic presentation of Experiment 1, in which sperm were cryopreserved in INRA-
82 supplemented with egg yolk (EY), and various concentrations of sucrose (SUC) as well as
albumin (BSA), and glycerol (GLY). Six ejaculates from different stallions were tested, using a
split sample approach. Sperm was frozen in INRA-82 supplemented with 2.5% (v/v) clarified egg
yolk (EY) and 0−200 mM sucrose (SUC) or 0−10% (w/v) bovine serum albumin (BSA). Five
different concentrations were tested. In addition, the optimal sucrose concentration (50 mM) was
tested in combination with 0−10% BSA. For comparison, also 2.5% (v/v) glycerol (GLY) in
combination with 0−10% BSA was tested. Post-thaw analysis of sperm motility and membrane
integrity was done for all formulations/treatments (T1- T5) that were tested, whereas pre-freeze
analysis was done only for sperm diluted in INRA-82. See section 3.2 for a detailed description.
MATERIAL AND METHODS
19
Figure 3.2. Schematic presentation of Experiment 2, in which sperm were cryopreserved in INRA-
82 supplemented with 2.5% (v/v) egg yolk (EY) without further supplements as well as with 2.5%
(v/v) glycerol (GLY), 100 mM glucose (GLU), 50 mM sucrose (SUC) or 50 mM trehalose (TRE),
both alone as well as with BSA added at a 1/1 mass ratio (1.71 w-% each). Nine ejaculates from
different stallions were tested. Sperm motility and membrane integrity were evaluated both before
and after freezing-and-thawing; for all formulations/treatments (T1- T8) tested. See section 3.2 for
a detailed description.
MATERIAL AND METHODS
20
3.3 Hydrated storage of sperm
In a separate experiment, Experiment 3, sperm chromatin structure/stability was
studied during hydrated storage in various extenders. Therefore, semen from 6 different
stallions (3−16 years) was used, and diluted to 100×106 sperm mL−1 in INRA-82, or INRA-82
supplemented with sucrose (SUC; 50 mM) with and without BSA (1.71%). Diluted semen
was divided in five 1 mL aliquots for storage in an incubator set at 37°C, for different
durations up to 3 d. At defined time-points (0, 6, 24, 48, 72 h) samples were collected,
plunged in liquid nitrogen and stored for later analysis of sperm chromatin structure as
described in detail below. In Figure 3.3, a schematic presentation is shown on the study
design of Experiment 3.
Figure 3.3. Schematic presentation of Experiment 3, in which sperm chromatin structure was
evaluated during hydrated storage at 37°C. Ejaculates from 6 different stallions were tested. Sperm
were diluted in INRA-82 supplemented with 50 mM sucrose (SUC), 1.71 w-% BSA (1.71%) or the
combination of both at a 1/1 mass ratio (i.e. 1.71% each). Samples with different treatments (T1-
T4) that were analyzed at different time points were incubated as aliquots of the same solution in
an incubator at 37°C, and shock-frozen in liquid nitrogen after 0, 6, 24, 48, or 72 h incubation for
later analysis of sperm chromatin structure. See section 3.3 for a detailed description.
MATERIAL AND METHODS
21
3.4 Sperm freeze- drying
Freeze-drying studies were performed in Experiment 4. After collection, semen was
directly diluted with either INRA-82 or TRIS+ (10 mM TRIS-HCl, 1 mM EDTA, 150 mM
NaCl, pH 8) to a concentration of 100×106 sperm mL−1. Diluted semen was centrifuged at
600×g for 10 min, the supernatant was removed and sperm were resuspended in fresh medium
to 200×106 sperm mL−1. Such samples were diluted with an equal volume of medium
containing two-fold the desired final concentration of protectants. In total 8 different freeze-
drying formulations were tested, and sperm chromatin structure was studied before and after
freeze-drying and rehydration, as well as during dried storage at 37°C for up to 3 months.
Sperm was diluted in INRA-82 or TRIS+ without supplements or TRIS+ supplemented with
glucose (GLU; 100 mM), sucrose (SUC; 50 mM) or trehalose (TRE; 50 mM) with(out) BSA
(1.71%). Sugar concentrations were equal to 1.71% (w/v), and thus sugar/BSA mixtures were
tested using a 1/1 (w/w) ratio. In Figure 3.4, a schematic presentation is shown of Experiment
4.
After dilution, 500 µL samples (100×106 sperm mL−1 in INRA-82 or TRIS+ with or
without supplements) were transferred into freeze-drying vials (2R injection vials, Christ;
Landgraf Laborsysteme, Langenhagen, Germany), and cooled at ~10°C min−1 to −80°C via
placing in a −150°C freezer (see Figure 3.5A). Cooling rates were verified using a T-type
thermocouple (Fluke, Everett, WA, USA). Frozen samples were transferred to the
temperature-controlled shelves of a lyophilizer (Virtis Advantage Plus Benchtop freeze dryer;
SP scientific, Warminster, PA, USA) set at −10°C. Shelves were then cooled to −30°C and
held at this temperature for 1 h, after which primary drying was performed at a temperature of
−30°C and a pressure of 60 mTorr for 4 h. Then, the shelf temperature was increased to 20°C,
at 0.1°C min−1 while maintaining a pressure of 60 mTorr, after which secondary drying was
performed at a pressure of 10 mTorr for 6 h (see Figure 3.5B). After freeze-drying, samples
were closed immediately and stored in vacuum-sealed bags at 37°C for up to 3 months. At
defined time points samples were collected, rehydrated by adding 500 µl water, transferred to
1 mL-cryovials and stored in liquid nitrogen for later analysis of sperm chromatin structure.
MATERIAL AND METHODS
22
Figure 3.4. Schematic presentation of Experiment 4, in which sperm were freeze-dried in INRA-
82 or TRIS+ without supplements or supplemented with 100 mM glucose (GLU), 50 mM sucrose
(SUC) or trehalose (TRE) both alone as well as with BSA added at a 1/1 mass ratio (1.71 w-%
each). Six ejaculates from different stallions were tested. Freeze-dried samples were stored in
vacuum-sealed bags at 37°C for up to 3 months. At defined time points (pre-freeze, 0 d, 1 5d, 30 d,
90 d) different treated (T1- T8) samples were rehydrated and shock-frozen in liquid nitrogen for
later analysis of sperm chromatin structure. See section 3.4 for a detailed description.
MATERIAL AND METHODS
23
Fig
ure
3.5
. T
emp
erat
ure
pro
file
s to
whic
h s
ample
s w
ere
expose
d f
or
free
ze-d
ryin
g.
Fir
st,
sam
ple
s w
ere
froze
n b
y p
laci
ng i
n a
−1
50
°C f
reez
er
(A).
Sam
ple
s w
ere
kep
t in
th
e fr
eeze
r fo
r a
min
imum
of
30 m
in (
i.e.
for
reac
hin
g −
80°C
). T
hen
, sa
mp
les
wer
e pla
ced
in
th
e ly
op
hil
izer
, an
d
subje
cted
to
th
e p
roto
col
as s
ho
wn i
n p
anel
B.
Her
e both
the
tem
per
ature
(blu
e li
ne)
and p
ress
ure
(gre
y l
ine)
pro
file
s ar
e p
rese
nte
d v
ersu
s ti
me,
as e
xpla
ined
in
det
ail
in s
ecti
on
3.4
. T
he
free
ze-d
ryin
g p
roto
col
took i
n t
ota
l 24 h
.
B
A
MATERIAL AND METHODS
24
3.5 Computer assisted sperm analysis of motility (CASA)
Computer assisted sperm analysis (CASA; Spermvision; Minitüb, Tiefenbach,
Germany) was used for assessment of sperm motility. The setup that was used included a
microscope with a temperature controlled stage (37°C) and camera for collecting images at 60
frames s−1. Software settings for motility analyses were according to the instructions provided
by the manufacturer. Sperm motility characteristics were calculated as mean values from 8
microscopic fields. After removal of 10 µL for use for flow cytometry, 500 µL samples in
microtubes (50×106 sperm mL−1) were incubated for 10 min at 37°C in a heating block.
CASA measurements were performed while maintaining samples at 37°C, after loading 3 µL
aliquots into a chamber of a Leja 20 micron four chamber slide (Leja Products BV, Nieuw
Vennep, Netherlands).
3.6 Flow cytometric analysis of membrane integrity (FCM)
Plasma membrane integrity was determined by flow cytometric analysis of sperm
stained with propidium iodide (PI) and SYBR-14. All plasma membranes are permeable to
SYBR-14, which exhibits green fluorescence upon binding to DNA, whereas PI can only
enter sperm with damaged plasma membranes and shows red fluorescence upon replacing
SYBR-14. Ten µL sperm sample (50×106 sperm mL−1) was diluted in 487 µL HEPES-
buffered saline solution (HBS; 20 mM HEPES pH 7.4, 137 mM NaCl, 10 mM glucose 2.5
mM KOH) supplemented with 2 µL 0.75 µM PI and 1 µL 0.5 µM SYBR-14. This resulted in
1×106 cells mL−1, 3 µM PI and 1 nM SYBR-14. Samples were incubated for 10 min at room
temperature, in darkness, after which they were analyzed using a flow cytometer (FCM; Cell
Lab Quanta SC MPL, Beckham-Coulter, Fullerton, CA, USA). A sheath fluid rate of 30 µL
min−1 was used, resulting in 200−500 counts s−1. Sperm was selected based on their side
scatter and electronic volume properties and a minimum of 5000 sperm were measured. The
percentage of PI-negative/SYBR-14-positive sperm was determined in plots of green
fluorescence versus red fluorescence of particles.
MATERIAL AND METHODS
25
3.7 Sperm chromatin structure assay (SCSA)
The sperm chromatin structure assay (SCSA), as described by EVENSON et al.
(1980), was used to evaluate chromatin integrity. In this assay, sperm is treated with acid and
detergent after which the level of induced DNA denaturation is determined (EVENSON and
JOST 2000). Sperm samples prepared and/or treated as described above were used, which had
a concentration of 100×106 sperm mL−1 and were shock frozen and stored in liquid nitrogen
until analysis. After thawing in a 37°C water bath, 10 µL sample was diluted with 490 µl
TNE buffer (0.15 M NaCl, 0.01 M TRIS-HCL, 1 mM disodium EDTA, pH 1.2). Then, from
this aliquot 200 µL was taken and 400 µL acid solution (0.08 M HCL, 0.15 M NaCl, 0.1%
Triton X-100, pH 1.2) was added, while maintaining samples in darkness, after which
samples were vortexed 30 s. To stop the denaturation reaction 1.2 mL acridine orange
(Polysciences, Warrington, PA, USA) staining solution (0.15 M NaCl, 0.0037 M citric acid,
0.126 M Na2HPO2, 0.0011 M disodium EDTA, pH 6.0; containing 6 µg mL-1 acridine orange)
was added. Samples were placed on ice for 3 minutes and then 10000 cells were analyzed
with an average flow rate of 200−300 per s, using a FACScan flow cytometer (Becton-
Dickinson, Heidelberg, Germany). The DNA fragmentation index (DFI) was determined as
described by EVENSON et al. (2002).
3.8 Sperm chromatin dispersion test (SCD)
The sperm chromatin dispersion test (SCD) is described in detail by FERNANDEZ et
al. (2003), and is commercially available as ‘Halosperm kit’ (Halotech DNA SL, Madrid,
Spain)]. To ensure that sperm maintained on microscope slides during the procedure, agarose-
coated slides were prepared. Slides were cleaned, and a 50−100 µL droplet of a 0.5% (w/v)
agarose solution was added per slide after which a second slide was used for preparing a thin
film. Slides were dried overnight at 37°C and stored at room temperature until use. Sperm
were diluted to 20×106 sperm mL−1 in PBS, and then 25 µL of this solution was added to 800
µL 1% agarose (w/v, prepared in PBS) which was kept melted at 37°C. Two 14 µL drops of
sperm in agarose were added per agarose-coated slide, which was placed on a block of 37°C,
and directly covered with coverslips (10×10 mm). For solidification of the agarose, the slides
MATERIAL AND METHODS
26
were placed on a pre-cooled shelf at 4°C for 5 min, after which the coverslips were carefully
removed. Slides were placed in horizontal position, and 1 mL acid solution (0.08 N HCl) was
added per slide. After 7 min incubation, the solution was removed and 1 mL lysis solution
(2.5 mL NaCl, 0.1 M Na2EDTA, 10 mM TRIS, 0.1% Triton-X100, 25 mM DTT) was added
per silde. DTT was added to the solution just before use, and lysis solution was kept at 4°C.
Samples were incubated with lysis solution for 30 min, after which they were washed for 2
min in distilled water. Specimens were dehydrated by passing through a graded ethanol series;
70%, 90%, and 100% (v/v) ethanol, 2 min each (in staining jars). Slides were air-dried and
specimens were stained using 1 mL Wright staining solution. After 15 min, slides were
washed under tap water followed by air-drying. Slides were examined using light microscopy,
at a 10×20 magnification. Sperm with intact chromatin had a purple ‘halo’, whereas sperm
with damaged chromatin had a smaller or no halo and a less pink nucleus. For the
quantification ~40 sperm per sample were analyzed. In Figure 3.6 microscopic images are
shown.
MATERIAL AND METHODS
27
Fig
ure
3.6
. O
rigin
al m
icro
sco
pic
im
ages
as
obta
ined
wit
h t
he
‘hal
o-t
est’
(A
, C
), a
s w
ell
as t
he
sam
e im
ages
aft
er p
roce
ssin
g u
sin
g i
mag
eJ
soft
war
e (B
, D
) fo
r an
alysi
s o
f h
alo s
izes
. E
xam
ple
s ar
e pre
sente
d f
or
sper
m w
ith i
nta
ct (
A,
B)
and
dam
aged
(C
, D
) D
NA
; w
ith
lar
ge
and
sm
all
hal
o’s
, re
spec
tivel
y.
Images
wer
e co
nver
ted
into
bla
ck/w
hit
e, a
nd a
sim
ilar
bac
kgro
und t
hre
shold
was
set
to
au
tom
atic
ally
det
ect
sper
m w
ith
hal
os
and d
eter
min
e th
e ar
ea t
hey
cover
ed.
This
is
illu
stra
ted i
n p
anel
E,
wher
e sp
erm
wit
h h
alo
’s a
re m
arked
in
yel
low
. S
ee s
ecti
on
3.8
for
a
det
aile
d d
escr
ipti
on
.
Sca
le b
ar r
epre
sents
50 µ
m
MATERIAL AND METHODS
28
3.9 Single cell gel electrophoresis (SCGE)
Single cell gel electrophoresis (SCGE) also known as the ‘Comet Assay’ was
performed as described by LINFOR et al. (2002), with minor modifications. Agarose-coated
slides and embedding of sperm in a thin layer of agarose on slides were done as described
above for the SCD test. During the procedure, all solutions were kept cool and incubations
were done at 4°C protected from light. First, 1 mL lysis solution (see above) was added per
slide. After 30 min incubation, the solution was replaced by alkaline solution (300 mM
NaOH, 1 mM EDTA, pH>13) and slides were incubated for another 30 min. Then, alkaline
electrophoresis was performed during 20 min, using 20 V and 300 mA. After electrophoresis,
slides were washed in water and passed through a graded ethanol series (70%, 90% and
100%; 2 min each) after which slides were air-dried. To visualize DNA, 5−10 µL Hoechst
staining solution (150 µg mL−1) was added, a cover with slip was added, and specimens were
sealed using nail polish. Slides were examined using fluorescence microscopy at a 10×20
magnification. The extent of sperm DNA fragmentation followed from the length of the
‘comet-tail’ and fluorescence intensities of the nucleus versus comet-tail. More DNA damage
was evident as a longer comet tail and relatively lower fluorescence in the nucleus. For
detailed analysis, ‘Komet’ software was used (Andor Technology Ltd, Belfast, UK) on a
minimum of 40 sperm per sample. In Figure 3.7 microscopic images are shown.
MATERIAL AND METHODS
29
Figure 3.7. Original fluorescence microscopic images as obtained with the ‘comet-assay’ (A− C),
as well as an example during analysis on ‘comets’ using specialized software (D) for analysis of
comet tail lengths and relative DNA contents in the head and tail. Examples are shown for sperm
exhibiting different degrees of DNA damage (A− C), with more damage being visualized as longer
comet tail lengths and increased fluorescence intensities in the tail as compared to the head. With
the software, each comet should be enclosed within a box (ROI: region of interest) which includes
different regions as indicated in panel D (background, comet head and tail) and explained in detail
in section 3.9.
Scale bar represents 50 µm
MATERIAL AND METHODS
30
3.10 Statistical analysis
Various experiments were carried out for each stallion with pre – and post-treatment
measurements. Normal distribution of the model residuals was confirmed by Kolmogorov-
Smirnov-Test and visual assessment of q-q - plots. All data were included into a descriptive
analysis with calculation of the arithmetic mean and standard deviation.
Differences between the measured time points after cryopreservation and freeze-
drying as well as differences between the formulations were tested using two-way analysis of
variance for repeated measurements and tukey post hoc test for multiple pairwise
comparisons. Analyses were carried out with the statistical software SAS, version 9.3 (SAS
Institute, Cary, NC, USA). For the analysis of the linear models, the MIXED procedure was
used. Differences were taken to be statistically significant when p<0.05.
RESULTS
31
4. RESULTS
4.1 Sperm cryopreservation using various sugars and albumin
Sperm motility and membrane integrity were determined after cryopreservation with
different formulations. Prior to cryopreservation, the percentages of membrane intact and
motile sperm were about 80%. After cryopreservation, for all formulations tested, percentages
were significantly lower (i.e. below 40%, p<0.0001). In case of using sucrose as
cryoprotective agent, percentages of membrane intact sperm were found to be highest (23±
11%) when using 1.71% (equals 50 mM) sucrose. At higher concentrations, percentages
decreased in a dose-dependent manner. If using albumin as only cryoprotective agent,
percentages were highest (27±9%) when using 1% BSA. If BSA was added to freezing
extender containing 1.71% sucrose, cryosurvival was higher as compared to using sucrose or
albumin alone. These differences, however, were not significant. The highest percentages of
membrane intact and motile sperm were found when using sucrose/albumin mixtures at a 1/1
(w/w) ratio. Percentages of membrane intact sperm were highest when using a combination of
the permeating cryoprotectant glycerol and ~2% BSA (38±15%). This was significantly
higher compared to using a formulation consisting of only BSA (p<0.02).
Freezing extenders for cryopreservation of stallion sperm typically contain skim milk,
egg yolk, and glycerol as protective agents. We tested if, in addition to sucrose, glucose and
trehalose (alone as well as in combination with BSA) also had cryoprotective properties.
Diluting sperm in skim milk extender supplemented with 100 mM glucose or 50 mM sucrose
or trehalose with/out BSA (at a 1/1 mass ratio, 1.71% each) did not significantly affect sperm
motility nor membrane integrity pre-freeze, whereas motility was significantly decreased after
diluting in extender supplemented with 2.5% glycerol compared with the other formulations
(Figure 4.2C). After cryopreservation, percentages of motile and membrane intact sperm were
slightly (~5%, not significant) higher if freezing extenders containing sugars (glucose, sucrose
or trehalose) were supplemented with BSA.
RESULTS
32
Fig
ure
4.1
. P
erce
nta
ges
of
mem
bra
ne
inta
ct (
A,
C)
and m
oti
le (
B,
D)
sper
m,
det
erm
ined
bef
ore
(A
, B
; o
range
bar
s) a
nd
aft
er c
ryo
pre
serv
atio
n
(C, D
), i
n I
NR
A-8
2 s
upple
men
ted w
ith
2.5
% c
lari
fied
egg
yo
lk a
nd
0−
20
0 m
M s
ucr
ose
(re
d s
ym
bols
) or
0−
10%
BS
A (
blu
e sy
mbols
), a
s w
ell
as
50 m
M s
ucr
ose
in
co
mb
inat
ion
wit
h 0
−10%
BS
A (
gre
en s
ym
bols
). F
or
com
par
ison,
also
2.5
% g
lyce
rol
in c
om
bin
atio
n w
ith
0−
10
% B
SA
was
test
ed (p
urp
le sy
mb
ols
). P
re-f
reez
e an
alysi
s w
as done
only
fo
r sp
erm
dil
ute
d in
IN
RA
-82
, w
her
eas
post
-th
aw an
alysi
s w
as d
on
e fo
r al
l
form
ula
tio
ns
test
ed. M
ean
val
ues
± s
tand
ard d
evia
tions
are
show
n, det
erm
ined
usi
ng 6
eja
cula
tes
fro
m d
iffe
ren
t st
alli
on
s.
RESULTS
33
4.2 Sperm chromatin structure and stability during hydrated storage at
37°C
Sperm chromatin structure and stability were evaluated during hydrated storage at
37°C in INRA-82 without supplements as well as supplemented with sucrose and BSA.
Sperm chromatin structure was evaluated using SCSA, and both the DNA fragmentation
index and red fluorescence intensity were determined. Only small differences are seen
amongst the different formulations tested (Figure 4.3). In INRA-82, DFI-values clearly
increased during hydrated storage: from 11±3% at 0 h up to 50±20% after 6 h (p<0.0001) and
Figure 4.2. Percentages of motile (C, D) and membrane intact (A, B) sperm, determined both
before (red bars) and after (blue bars) cryopreservation using various freezing formulations. As
standard diluent for cryopreservation, INRA-82 supplemented with 2.5% egg yolk and 2.5%
glycerol was tested. Furthermore, INRA-82 with egg yolk without further supplements as well as
supplemented with 100 mM glucose, 50 mM sucrose or trehalose with(out) 1.71% BSA were
tested. Mean values ± standard deviations are presented, determined from 9 ejaculates of different
stallions.
RESULTS
34
86±10% after 48 h (p<0.0001) (Figure 4.3A). Changes in chromatin structure were also
evident as significant (the difference between 0h and 6h is not significant, all other are
significant) changes in the red fluorescence intensity of acridine orange stained acid-
denatured sperm (Figure 4.3C, D). For sperm diluted and stored in INRA-82, fluorescence
intensities increased from 189±16 at 0 h to 216±17 and 307±30 at 6 and 48 h, respectively
(Figure 4.3D). DFI-values were significantly lower for sperm stored in INRA-82
supplemented with sucrose and BSA compared to storage in INRA-82 alone after 48h
(74±8% versus 86±10% at 48 h; p=0.0001) (Figure 4.3B).
Figure 4.3. Sperm chromatin structure was evaluated during hydrated storage at 37°C in various
extenders. As a measure for chromatin intactness, DNA fragmentation index values (A, B) as well
as red fluorescence intensities (C, D) were determined after acid/lysis treatment and staining with
acridine orange. As extenders were tested: INRA- 82 without supplements (blue symbols), INRA-
82 supplemented with 50 mM sucrose (red symbols) or 1.71% BSA (green symbols), as well as
INRA-82 supplemented with both 50 mM sucrose and 1.71% BSA (purple symbols). Five different
durations of storage (0, 6, 24, 48, 72 h) were tested. Mean values ± standard deviations are shown,
determined using 6 ejaculates from different stallions. Statistically significant differences (p<0.05)
are indicated with an asterisk.
RESULTS
35
To evaluate chromatin structure and DNA stability/damage, in addition to SCSA
analysis, the sperm chromatin dispersion test (SCD) as well as single cell gel electrophoresis
(SCGE) were performed.
In Figure 4.4, representative images are shown illustrating sperm DNA damage directly after
dilution in INRA-82 and storage at 37°C for 6 and 72 h. In green versus red fluorescence
plots, as obtained with SCSA, it can be seen that sperm exhibiting increased red fluorescence
increased during storage for up to 72 h at 37°C. The increase in DNA damage during hydrated
storage was also visualized sperm via the sperm chromatin dispersion test (SCD). In figure
4.4 D−F it can be seen that freshly diluted sperm (0 h) exhibit large ‘halos’. The halo size and
nuclear staining decreases during 72 h storage at 37°C. With ‘single cell gel electrophoresis’
(SCGE; Figure 4.4G−I), DNA fragmentation during hydrated storage is visualized as an
increase in the comet tail length and tail fluorescence intensity at expense of the head
fluorescence (i.e. DNA contents).
DFI-values, halo sizes, and comet lengths as well as head/tail DNA contents (i.e.
relative Hoechst fluorescence intensities) were determined for sperm during hydrated storage
at 37°C, with the results shown in Figure 4.5. DFI-values increased significantly from 10±1%
directly after dilution to 77±7 and 89±11% after 24 and 72 h at 37°C, respectively (p<0.0001).
This coincided with a decrease in the halo size from 178±97 after dilution to 93±64 µm2 after
72 h (p=0.0019). The tail length assessed with SCGE increased from 130±25 to 181±18 µm
(p=0.0045), while the relative head DNA content decreased from 53±7 to 32±6% (p=0.0093).
Thus, DFI-values as well as comet tail lengths increased with increasing storage time,
whereas the halo-area decreased. This is also illustrated in Figure 4.6, where is shown that
DFI-values negatively correlate with halo-areas, while comet tail lengths positively correlate
with DFI-values. In agreement with this, halo size areas negatively correlate with comet tail
lengths.
RESULTS
36
Figure 4.4. Images illustrating differences in sperm chromatin structure after induced damage (i.e.
hydrated storage at 37°C up to 3 days in INRA-82) determined using different assays. Panels A−C
show flow cytometric green versus red fluorescence scatter plots, obtained using the ‘sperm
chromatin structure assay (SCSA)’. In panels D−F, light microscopic images are shown obtained
via the ‘sperm chromatin dispersion test (SCD) or halo-test’. In panels G−I, fluorescence
microscopic images are shown obtained via ‘single cell gel electrophoresis (SCGE) or comet-
assay’.
RESULTS
37
Figure 4.5. Different methods were used on the same samples to evaluate sperm chromatin
structure during hydrated storage in INRA-82 for up to 72 h at 37°C (0 h: red, 6 h: blue, 24 h:
green, 48 h: purple, 72 h: light blue symbols). DNA fragmentation index (DFI) values were derived
from flow cytometric data obtained using SCSA (A), whereas halo-sizes (i.e. area) were derived
from micrographs obtained with SCD (B). From microscopic specimens obtained with SCGE,
DNA contents (i.e. relative Hoechst fluorescence intensities) in the comet head and tail (C) as well
as tail lengths (D) were determined. With flow cytometric analysis, 10000 sperm were measured
per sample, whereas with microscopic observations 40 sperm were analyzed per sample. Mean
values ± standard deviations are shown, determined using 3 ejaculates from different stallions.
Values with different subscript letters differ significantly (p<0.05).
RESULTS
38
Fig
ure
4.6
. C
orr
elat
ion
plo
ts b
etw
een p
aram
eter
s det
erm
ined
usi
ng d
iffe
rent
assa
ys
on t
he
sam
e sa
mp
les
for
eval
uat
ing s
per
m c
hro
mat
in
stru
cture
an
d d
amag
e. D
FI-
val
ues
wer
e det
erm
ined
usi
ng S
CS
A,
hal
o-s
izes
usi
ng t
he
SC
D/h
alo
-tes
t, a
nd
co
met
tai
ls v
ia S
CG
E/c
om
et-a
ssay
. In
pan
el A
an
d B
, re
spec
tivel
y,
the
hal
o-a
rea
and c
om
et t
ail
length
are
plo
tted
ver
sus
DF
I-val
ues
. In
pan
el C
, th
e co
met
tai
l le
ngth
is
plo
tted
ver
sus
the
hal
o a
rea.
Dat
a fr
om
th
ree
ejac
ula
tes
of
dif
fere
nt
stal
lions
are
pre
sente
d.
Sem
en w
as d
ilute
d i
n I
NR
A-8
2 a
nd
sto
red
for
up
to
72
h a
t 3
7°C
to
induce
dif
fere
nt
deg
rees
of
DN
A d
amage.
Dat
a ad
apte
d f
rom
fig
ure
4.5
.
RESULTS
39
4.3 Sperm chromatin structure and stability after freeze- drying and dried
storage at 37°C
Sperm were freeze-dried in INRA-82 and TRIS+, as well as TRIS+ supplemented
with different sugars and BSA. Although no membrane intact sperm were recovered after
freeze-drying, sperm chromatin of freeze-dried sperm was found to be largely intact (DFI-
values: 5−8%). Chromatin structure and stability of sperm was evaluated before and after
freeze- drying as well as during dried storage at 37°C for up to three months. Storage was
performed at 37°C to accelerate aging, and reveal preservation differences amongst freeze-
drying formulations more clearly.
Sperm chromatin structure was evaluated using SCSA. In Figure 4.7, changes in
chromatin structure are seen in green versus red fluorescence intensity scatter plots. It was
found that the proportion of sperm outside the main population and red fluorescence increased
during storage for sperm freeze-dried. For sperm freeze-dried in TRIS+ without supplements,
is was found that all sperm exhibited a higher proportion of sperm with damaged DNA after 2
weeks dried storage at 37°C, as compared directly after freeze-drying. In contrast, at this time
point, a large fraction of sperm with intact DNA (i.e. in the main population similar as before
freeze-drying) was seen then for the other formulations tested. The fraction of damaged sperm
increased with longer storage duration.
In addition to SCSA, SCD and SCGE were performed to visualize differences in
sperm chromatin structure microscopically (Figure 4.8). Directly after freeze-drying, presence
of intact chromatin was evident as described above. No differences were seen amongst the
different freeze-drying formulations. With SCSA analysis, only a small population of cells
outside the main population was found, whereas SCD analysis revealed sperm with large
‘halos’. With SCGE, only short ‘comets’ were seen, which had a low relative tail fluorescence
intensity compared to the sperm head. After 90 d dried storage, with SCSA a high percentage
of cells outside the mean population was found whereas with SCD and SCGE sperm exhibited
small or no ‘halos’ and large ‘comets’, respectively.
Data obtained with SCSA were quantified, and percentages of cells outside the main
population (i.e. DFI-values) and red fluorescence intensities of rehydrated samples were
calculated for the different freeze-drying formulations during up to 3 months storage (Figure
RESULTS
40
4.9, table 9.1) After 2 weeks, DFI-values were (significantly) increased in case of freeze-
drying with INRA-82 (28±23%; p<0.0044) and TRIS+ (68±33%; p<0.0001), whereas they
maintained around 10±14% for all other formulations tested. Differences between
formulations were more pronounced after longer storage duration. Sperm freeze-dried in
TRIS+ without supplements exhibited DFI-values of 68±33 and 95±5% after 2 and 4 weeks,
respectively. Also, sperm freeze-dried in skim milk extender or TRIS+ supplemented with
glucose and albumin had DFI-values of 83−64% after one month. These DFI-values were
significantly higher with respect to values determined prior to and directly after freeze-drying.
In contrast, freeze-drying sperm in formulations supplemented with sucrose and BSA resulted
in minor changes in chromatin structure during 1 month storage at 37°C, and DFI values were
not significantly different from values determined before freeze-drying (DFI-values of
10±8%). Moreover, DFI-values of sperm freeze-dried using sucrose and BSA, after 90 d dried
storage at 37°C, were significantly lower as compared to when freeze-drying was done using
INRA82 or TRIS+ without supplements or TRIS+ supplemented with glucose and BSAND
This is also illustrated as changes in red fluorescence intensities: whereas red fluorescence
intensities of samples with INRA-82 and TRIS+ increased drastically during storage (from
171±14 at after 2 weeks to 315±38 after 3 months for INRA-82), fluorescence intensities
remained low for sperm freeze-dried in TRIS+ supplemented with sucrose and BSA (143±16
after 2 weeks, 174±16 after 3 months; p<0.005).
RESULTS
41
Figure 4.7. Representative green (FL-1) versus red fluorescence (FL-3) intensity scatter plots,
obtained by flow cytometric analysis with SCSA; performed before (A−D) and after freeze-drying
(E−T) of stallion sperm, as well as after dried storage at 37°C for 15 d (I−L), 30 d (M−P) and 90 d
(Q−T). Different formulations were tested for freeze-drying, including: INRA-82 (A, E, I, M, Q),
TRIS+ (B, F, J, N, R) and TRIS+ supplemented with 1.71% sucrose (C, G, K, O, S) and
sucrose/BSA (1/1 mass ratio: 1.71% each).
RESULTS
42
Fig
ure
4.8
. Im
ages
ill
ust
rati
ng d
iffe
ren
ces
in c
hro
mat
in s
tru
ctu
re i
n f
reez
e-d
ried
sta
llio
n s
per
m.
In a
ddit
ion t
o g
reen
ver
sus
red f
luore
scen
ce
scat
ter
plo
ts o
bta
ined
usi
ng S
CS
A (
A,
B,
C,
J, K
, L
), m
icro
scopic
im
ages
obta
ined
via
the
SC
D o
r hal
o-t
est
(D,
E,
F,
M,
N,
O)
and
SC
GE
or
com
et-a
ssay
(G
, H
, I,
P,
Q,
R)
wer
e co
llec
ted,
both
dir
ectl
y a
fter
fre
eze-d
ryin
g (
A−
I) a
s w
ell
as a
fter
90
d d
ried
sto
rage
at 3
7°C
(J−
R).
Var
ious
form
ula
tio
ns
wer
e te
sted
for
free
ze-d
ryin
g,
nam
ely I
NR
A-8
2 (
A,
D,
G,
J, M
, P
), T
RIS
+ (
B,
E, H
, K
, N
, Q
), S
UC
/BS
A a
nd
TR
IS+
su
pple
men
ted
wit
h s
ucr
ose
/alb
um
in a
t a
1/1
mas
s ra
tio (
C,
F,
I, L
, O
, R
).
RESULTS
43
Fig
ure
4.9
. F
rom
gre
en v
ersu
s re
d f
luore
scen
ce S
CS
A s
catt
er p
lots
, per
centa
ges
of
cell
s outs
ide
the
mai
n p
opu
lati
on
(i.
e. D
FI
val
ues
; A
) an
d
mea
n r
ed f
luo
resc
ence
inte
nsi
ties
(B
) w
ere
der
ived
. T
his
was
done
on r
ehydra
ted s
ample
s th
at w
ere
store
d i
n t
he
dri
ed s
tate
at
37
°C f
or
dif
fere
nt
dura
tions.
Sta
llio
n s
per
m w
as s
ubje
cted
to f
reez
e-d
ryin
g i
n I
NR
A-8
2 (
dar
k b
lue)
as
wel
l as
TR
IS+
wit
ho
ut
sup
ple
men
ts (
red
) or
sup
ple
men
ted
wit
h 1
00 m
M g
luco
se (
gre
en),
50 m
M s
ucr
ose
(li
gh
t blu
e) o
r tr
ehal
ose
(yel
low
), o
r co
mb
inat
ions
of
thes
e su
gar
s w
ith a
lbum
in a
t a
1/1
wei
ght
rati
o (
glu
cose
/BS
A:
pu
rple
, su
cro
se/B
SA
: ora
nge,
tre
hal
ose
/BS
A:
pin
k s
ym
bols
). M
ean v
alues
± s
tan
dar
d d
evia
tio
ns
are
sho
wn
, d
eter
min
ed
usi
ng 6
eja
cula
tes
fro
m d
iffe
rent
stal
lions.
Sta
tist
ical
ly s
ignif
ican
t dif
fere
nce
s (p
<0.0
5)
are
indic
ated
wit
h a
n a
ster
isk (
see
also
Tab
le 9
.1.)
RESULTS
44
Figure 4.10. Samples directly after freeze-drying of one stallion. All eight formulations tested are
shown. From left to the right: INRA-82, TRIS+, TRIS+/ GLU, TRIS+/ GLU/ BSA, TRIS+/ SUC,
TRIS+/ SUC/ BSA, TRIS+/ TRE, TRIS+/ TRE/ BSA. Browning reactions are seen of formulations
with glucose alone as well as with BSA. Well forming cakes are seen when using TRIS+/ SUC/
BSA as well as TRIS+/ TRE/ BSA.
DISCUSSION AND CONCLUSIONS
45
5. DISCUSSION AND CONCLUSIONS
5.1 Chromatin integrity of stallion sperm after freeze-drying and dried
storage
In the equine breeding industry, the importance of gamete and embryo preservation
for export and storage has drastically increased. Cryopreservation is typically used, which
involves use of liquid nitrogen and special containers. Freeze-drying might be an attractive
alternative preservation method for sperm, since it would allow for storage at ambient
conditions and easy handling. If genetic integrity is preserved after drying, non-viable sperm
can be used for fertilization via ICSI (KUSAKABE et al. 2001; CHOI et al. 2011).
The current study was undertaken to investigate stallion sperm chromatin structure
and stability after freeze-drying. Special emphasis was on use of formulations containing
sugars (glucose, sucrose, trehalose) and proteins (BSA, INRA-82), to preserve chromatin
structure and prevent DNA damage during dried storage. In addition to evaluation of
chromatin stability of diluted and dried sperm, sperm survival after cryopreservation was
studied. Storage was done at 37°C to accelerate aging. Chromatin structure and DNA damage
was evaluated using various assays, including SCSA, ‘halo’ and ‘comet’ tests, on hydrated as
well as dried samples. Hydrated samples were stored at 37°C for up to 3 days while dried
samples were stored for up to 3 months at 37°C. It was found that chromatin structure rapidly
degraded during hydrated storage for 3 d. Chromatin structure was preserved after freeze-
drying. Moreover, if freeze-drying was done using formulations composed of TRIS-buffered
saline with EDTA, disaccharides (i.e. sucose, trehalose) and BSA, chromatin structure could
be preserved during dried storage for up to 3 months. It is suggested that these compounds
have a role in formation of a glassy matrix and water replacement for protecting sperm
chromatin during dried storage (OLDENHOF et al. 2013; ZHANG et al. 2016).
Freeze drying includes multiple processes, including cooling, freezing, sublimation
and secondary drying; which all may provoke cellular damage. During cooling at suprazero
temperatures (i.e. before ice formation) biomolecules may undergo conformational changes
and cells are exposed to cold shock (FANGET and FRANCON 1996). Upon freezing and ice
formation, cells are subjected to additional stresses (MERYMAN et al. 1977). Intracellular
DISCUSSION AND CONCLUSIONS
46
and/or extracellular ice formation may result in mechanical forces resulting in rupture of
cellular membranes. In addition, the concomitant increase of the solute concentration in the
unfrozen fraction exposes cells to hyperosmotic stress and dehydration. Also medium pH
changes (ARAKAWA et al. 1990) may affect structure and protein function. During
sublimation with freeze-drying and secondary drying, water in the form of ice is removed as
well as water surrounding biomolecules. This may result in collapse of samples, fusion of
membranes, lipid phase separation and changes in protein structure. If dried, stability in the
dried state is affected by the temperature at which samples are stored (i.e. with respect to the
glass transition temperature of the sample) as well as the sample moisture content (GREIFF
and RIGHTSEL 1969). Also, presence and accumulation of reactive oxygen species may give
rise to structural changes during storage (HECKLY and QUAY 1983). Mobility in the glassy
state, and hence rate at which damaging reactions take place, is affected by the glass transition
temperature and moisture content.
For freeze-drying of sperm sugars were tested as protectants, since these will facilitate
formation of a glassy state upon dehydration. Moreover, addition of albumin is known to
increase the glass transition temperature and hydrogen bonding interactions in the glassy
state, likely resulting in a more stable glass at lower temperatures (SYDYKOV et al. 2017).
Freeze-drying of sperm was done in INRA-82 for comparison. The monosaccharide glucose
and disaccharides sucrose and trehalose were tested, both alone and in combination with
albumin. These were added to a buffered saline medium with chelator (TRIS+), as previously
described (KESKINTEPE and EROGLU 2015). Glucose has a low glass transition
temperature, which explains the observed collapse of the freeze-dried samples. Furthermore,
if combined with albumin this reducing sugar may be involved in damaging Amadori and
Maillard reactions (EDEAS et al. 2010). INRA-82 forms a good glass, however, also shows
‘browning’ reactions because of presence of glucose and milk proteins. Non-reducing
disaccharides like sucrose and trehalose do not facilitate such reactions. Moreover, the glass
transition temperature of formulations containing disaccharides is higher and specimens are
likely in a stable glassy state during storage at room temperature. Pure dry glucose, sucrose
and trehalose glasses have a glass transition of 30±2, 58±1, 108±3°C, respectively
(SYDYKOV et al. 2017). The glass transition temperature is lower with higher sample
moisture contents and is increased with addition of albumin. In addition to glass formation, in
DISCUSSION AND CONCLUSIONS
47
the absence of water, sugar hydroxyl groups may replace hydrogen bonds which normally
exist between water and the phospholipid polar head groups of membrane lipids (CROWE et
al. 1992; CROWE et al. 2001; OLDENHOF et al. 2013). High sugar concentrations likely
form a ‘denser’ glassy matrix for preservation, however, they expose cells to more severe
osmotic stress (OLDENHOF et al. 2013). The sugar concentrations employed in this study
(50−100 mM) did not result in decreased numbers of membrane intact sperm upon addition,
and had cryoprotective properties. No differences in cryoprotective properties between
trehalose and sucrose were found, nor in their protective effects during dried storage (if they
were used alone). SITAULA et al. (2009) reported that loading of bovine sperm with
intracellular sugars resulted in an increased survival after drying. Also for other cells, loading
with and/or presence of trehalose resulted in increased survival upon evaporative drying down
to water content not lower as 0.3 g H2O per g dry weight (CROWE et al. 2005; MCGINNIS et
al. 2005; LI et al. 2007). Beneficial effects of trehalose may be related to its action as an
osmolyte forming a protective milieu around biomolecules. Various drying techniques have
been employed (ELMOAZZEN et al. 2009).
In addition to sugars, chelators and antioxidants may improve stability during
preservation (SITAULA et al. 2009). In the present study albumin was added for which
antioxidant properties are described (MARTINS et al. 2007; LEWIS et al. 2010;
TSUKAMOTO et al. 2012). Whereas addition of BSA to sucrose only showed minor
protective properties during hydrated storage, with long term storage in the dried state
positive effects on preserving chromatin structure were found. Antioxidants may counteract
formation of reactive oxygen species (SARIOZKAN et al. 2013) and damaging reactions
affecting membrane integrity (SITAULA et al. 2009).
The storage temperature drastically affects sperm preservation in the dried state
(WAKAYAMA and YANAGIMACHI 1998; KANEKO and NAKAGATA 2005; HOCHI et
al. 2008; KANEKO and SERIKAWA 2012). KANEKO and NAKAGATA (2005) reported
that sperm can be stored in the dried state at 4°C for up to 17 months, without damage to
chromatin structure. In the current study, samples were stored at 37°C to accelerate aging, and
mimic storage under suboptimal conditions, to enhance possible differences between
formulations used for storage in the dried state. Similar to findings of others (KUSAKABE et
al. 2001), it was found that chromatin structure was not affected by freeze-drying itself, and
DISCUSSION AND CONCLUSIONS
48
no differences were found between the formulations tested. Interestingly, clear differences
amongst freeze-drying formulations arose already after 15−30 d storage at 37°C. DFI-values
particularly increased when using INRA-82, TRIS+ without supplements or with presence of
both glucose and BSA. Damage here can be explained by absence of compounds that
facilitate formation of a protective glassy state, or presence of reducing sugars in combination
with proteins facilitating damaging Amadori and Maillard reactions. Such damage may not
occur in the presence on non-reducing disaccharides. It should be noted that damaging
reactions will be slowed in case of storage at lower temperature (4°C or −20°C). Preservation
at 4°C will be economically advantageous for long-term storage and transport compared to
preservation at subzero temperatures, since it omits the need of liquid nitrogen and/or special
containers.
5.2 Comparison of different assays for evaluating sperm chromatin
structure, and their application for evaluation of different characteristics of
freeze-dried sperm
Freeze-dried sperm were not viable and motile after rehydration. Their chromatin
structure, however, was not affected as revealed using SCSA. DFI-values remained similar as
before freeze-drying, and did not change during storage for up to 3 months if freeze-drying
was done using formulation consisting of TRIS+, trehalose/sucrose and BSA. As stated
above, it can be expected that individual sperm from such preparations can be used for in
vitro fertilization via ICSI, for artificially passing the zona pellucida and plasma membrane of
the oocyte (CHOI et al. 2011).
In an attempt to evaluate in more detail whether sperm chromatin structure exhibited
different characteristics amongst treatments, various assays for evaluation of DNA damage
were employed. To validate the different methods, the same samples were tested and derived
parameters were correlated. The assays used included SCSA (EVENSON and JOST 1994;
LOVE 2005), which is the standard method for evaluation of sperm chromatin intactness.
With this assays damage is expressed as DFI-values. In addition, the SCD-test was used
(FERNANDEZ et al. 2003) for evaluation of halo sizes, and SCGE (LINFOR and MEYERS
DISCUSSION AND CONCLUSIONS
49
2002; GYORI et al. 2014) for evaluation of comet characteristics (i.e. tail lengths and
head/tail DNA contents).
The sperm chromatin structure assay was first described by EVENSON et al. (1980).
In their paper, a relationship was found between sperm DNA integrity (i.e. DFI-values) and
fertility. The procedure involved heating of the sample to denature DNA, followed by
acridine orange staining and flow cytometric analysis of green intact double stranded and red
single strand damaged DNA. Later, instead of heating, acid treatment was used to denature
DNA. The advantage of such flow cytometric approaches is that many events can be
analyzed, like 10000 sperm per sample. In case of microscopic analysis of individual sperm,
as done with SCD and/or SCGE, typically only a limited number of sperm (50−300 per
sample) is analyzed. Therefore, these approaches lack statistical power for diagnosis and
prognosis (LINFOR and MEYERS 2002; FERNANDEZ et al. 2003; EVENSON 2016). With
analyzing diluted samples stored at 37°C, it was found that parameters derived with these
different methods (i.e. DFI-value, halo-size, comet tail length and head/tail DNA content) all
correlated with the extent of induced damage, and (weak) correlations were found if plotted
against each other. Whereas DFI-values exhibited more clear differences in the range were
damage seemed to increase exponentially with exposure to damaging conditions. With these
conditions, halo-sizes and comet tail lengths as well as head/tail DNA contents amongst
treatments showed smaller differences. Moreover, for the two latter, variation amongst
assessments done at different days/experimental runs was larger. Issues with handling and
procedures for data analysis with these methods have been described before. For example,
sperm with small/compact halos have been described to represent intact chromatin (LOPEZ-
FERNANDEZ et al. 2007; CORTES-GUTIERREZ et al. 2009) as well as increased DNA
fragmentation (FERNANDEZ et al. 2003). These contradictive results may be explained by
application of different DNA denaturation and staining procedures. Since the use of different
assays on the same samples it can be claimed that decreased sperm chromatin integrity is
evident as increased DFI-values with SCSA, decreased halo-sizes with SCD and increased
comet tail lengths with SCGE. If freeze-dried samples were analyzed, the microscopic
approaches revealed similar tendencies with respect to chromatin integrity as revealed with
SCSA. These methods are a nice visualization of differences in chromatin intactness and
DNA damage in individual cells.
DISCUSSION AND CONCLUSIONS
50
5.3 Conclusions
It was found that sperm chromatin structure was intact after freeze drying and
remained intact during dried storage at 37°C for up to 3 months after freeze-drying with a
TRIS-buffered formulation supplemented with non-reducing sucrose or trehalose and BSA. In
contrast with this, chromatin degradation took place within 3 d if diluted/hydrated samples
were stored at such conditions. Degradation will be slower if storage is done at lower
temperatures (e.g. at room temperature or 4°C), which would be an attractive alternative for
storage and shipping of sperm for use for ISCI.
Different assays were tested for evaluation of sperm chromatin structure, and it was
found that all can be used to quantify DNA damage. Decreased chromatin integrity correlated
with increased DFI-values with SCSA, decreased halo-sizes with SCD, and increased comet
tail lengths with SCGE in a dose dependent matter. SCSA, with flow cytometric analysis of
samples, seemed to be the most reproducible approach for deriving similar values denoting
damage amongst experimental days. The SCD test and SCGE, for which cells are embedded
in agarose on microscope slides, are relatively simple and inexpensive. However, due to
handling issues (e.g. thickness of agarose samples) they exhibit larger variation and therefore
the need to analyze samples for comparison in parallel. Furthermore, with these methods,
there are no reference values correlating with fertility.
Taken together, it is concluded that stallion sperm chromatin structure after freeze-
drying and dried storage can be preserved if using formulations supplemented with non-
reducing disaccharides and albumin. Furthermore, storage at accelerated aging conditions
(e.g. 37°C) can be useful for evaluation of differences amongst formulations. For evaluation
of chromatin integrity, the ‘sperm chromatin structure assay’ is most robust for quantification
of small differences amongst samples. For visualization of DNA damage in individual cells,
however, additional/alternative approaches (e.g. SCD and SCGE) may be useful.
SUMMARY
51
6. SUMMARY
Katharina Narten (2017):
Freeze- drying of equine sperm and sperm chromatin structure during
dried storage
Freeze drying of sperm does not result in viable motile sperm. However, if chromatin
structure is preserved, sperm can be used for intracytoplasmic sperm injection (ISCI) and
fertilization of an oocyte. Moreover, storage and transport in the dried state at ambient
conditions (i.e. room temperature), 4°C or even −20°C, is advantageous since it would
eliminate the need of liquid nitrogen and special containers. For freeze drying, protective
agents are needed that preserve biomolecular structure during both freezing and drying.
Sugars, are often used for freeze-drying; since they have good glass forming properties and
the ability to replace water in the dried state.
The aim of the current study was to evaluate stallion sperm chromatin structure after
freeze drying and during dried storage. Special emphasis was on the use of formulations
containing sugars (glucose, sucrose, trehalose) and proteins (BSA, INRA-82), as well as
different assays for evaluating sperm chromatin structure and DNA damage. Sperm survival
was studied after cryopreservation as well as freeze drying. Storage was done at 37°C to
accelerate aging, and chromatin structure was evaluated after hydrated and dried storage for
up to 3 days and 3 months, respectively. It was found that chromatin structure rapidly
degraded during hydrated storage during 3 d. Whereas, after freeze drying, chromatin
structure was preserved. Especially using formulations composed of TRIS-buffered saline
with chelator (EDTA), disaccharides (i.e. sucose, trehalose) and albumin (e.g. sucrose/BSA
mixtures at a 1/1 mass ratio; 1.71% each), chromatin structure could be preserved during
dried storage for up to 3 months. These compounds may play a role in formation of a glassy
matrix and water replacement for protecting sperm chromatin during dried storage.
DNA damage was analyzed using the sperm chromatin structure assay (SCSA), which
involves acid denaturation and flow cytometric analysis of acridine orange stained sperm for
SUMMARY
52
discriminating between intact and damaged DNA. Furthermore, the sperm chromatin
dispersion test (SCD), and single cell gel electrophoresis (SCGE) were performed. It was
found that all assays can be used to quantify DNA damage. In dependence to the storage time
at 37°C, the decreased chromatin integrity correlated with increased DFI-values with SCSA,
decreased halo-sizes with SCD, and increased comet tail lengths with SCGE.
ZUSAMMENFASSUNG
53
7. ZUSAMMENFASSUNG
Katharina Narten (2017):
Gefriertrocknung von Hengstsperma und Spermienchromatinstruktur
während der Lagerung
Nach der Gefriertrocknung von Sperma sind die Spermien devital und nicht mehr
motil. Dennoch kann die Chromatinstruktur erhalten und die Spermien via
intrazytoplasmatischer Spermieninjektion in eine Oozyte übertragen werden und zur
erfolgreichen Befruchtung führen. Darüber hinaus ist die Lagerung und der Transport im
gefriergetrockneten Zustand bei Umgebungstemperaturen, 4°C oder auch −20°C vorteilhaft,
da kein Stickstoff und kein teures Aufbewahrungs-/Transportbehältnis benötigt wird. Für die
erfolgreiche Gefriertrocknung werden protektive Agenzien, welche die biomolekulare
Struktur während des Einfrierens und des Trocknens schützen, benötigt. Zucker werden
häufig für die Gefriertrocknung verwendet, da sie als gute „Glasbildner" bekannt sind und im
getrockneten Zustand Wassermoleküle ersetzen können.
Das Ziel dieser Arbeit war es, die Spermienchromatinstruktur und -stabilität nach dem
Gefriertrocknen und der Trockenlagerung zu bestimmen. Besonderer Schwerpunkt lag dabei
im Vergleich verschiedener Formulierungen, die Zucker (Glukose, Saccharose, Trehalose)
und Proteine (BSA, INRA-82) beinhalteten. Zudem wurden unterschiedliche
Analyseverfahren zur Bestimmung der Chromatinstruktur und DNA- Schädigung verglichen.
Das Überleben der Spermien wurde nach dem Einfrieren sowie nach der Gefriertrocknung
ermittelt. Um die Alterung zu beschleunigen, wurde die Lagerung bei 37°C durchgeführt und
die Chromatinstruktur nach drei Tagen bei hydratisierter Lagerung und nach drei Monaten
Trockenlagerung ermittelt. Es wurde festgestellt, dass im Verlauf der dreitägigen
hydratisierten Lagerung die Chromatinstruktur schnell degradiert, wohingegen nach
Gefriertrocknung die Chromatinstruktur intakt bleibt. Vor allem bei Verwendung von
Formulierungen aus TRIS+ gepufferten Salzlösungen mit chelatbildenden Verbindungen
(EDTA), Disacchariden (d.h. Saccharose, Trehalose) und Albumin (z.B. Saccharose/ BSA
ZUSAMMENFASSUNG
54
Mixturen in einem 1/1 Massenverhältnis, jedes bei 1.71%) konnte die
Spermienchromatinstruktur während der Trockenlagerung bis zu drei Monaten erhalten
werden. Diese Formulierungskomponenten beeinflussen wahrscheinlich die Bildung einer
„glasartigen“ Zellmatrix und den Wasseraustausch und ermöglichen dadurch einen Schutz der
Spermienchromatinstruktur während der Trockenlagerung.
Die DNA- Integrität wurde mit Hilfe des Spermien-Chromatin-Struktur-Assay
(SCSA) ermittelt; dieses Verfahren beruht auf Säuredenaturierung und flowzytometrischer
Analyse Acridine Orange–gefärbter Spermien zur Unterscheidung intakter und geschädigter
DNA. Außerdem wurde der Spermien-Chromatin-Dispersions-Test (SCD) und die Einzel-
Zell-Gel-Elektrophorese (Single cell gel electrophoresis, SCGE) durchgeführt. Dabei wurde
herausgefunden, dass alle drei Verfahren zur Quantifizierung von DNA- Schädigung
verwendet werden können. Hierbei korrelierte in Abhängigkeit von der Lagerungsdauer bei
37°C eine reduzierte DNA-Integrität mit einem Anstieg des DNA-Fragmentations-Indexes
ermittelt mittels SCSA, sowie reduzierten Halo- Größen (SCD) und einem Anstieg der
Cometen-Schwanz Länge (SCGE).
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APPENDIX
76
9. APPENDIX
APPENDIX
77
9.1. Supplemental table
Table 9.1. Pr> |t| and Adj-P numbers of DFI- and FL3- values after freeze- drying. For detail
description see figure 3.4.
Pr >
|t|
Adj
PP
r >
|t|
Adj
PP
r >
|t|
Adj
PP
r >
|t|
Adj
PP
r >
|t|
Adj
P
E5-
T1
E5-
T2
0.2
501
0.9
353
0.0
010
0.0
203
<.0
001
0.0
002
0.1
342
0.7
846
0.4
683
0.9
953
E5-
T1
E5-
T3
0.0
215
0.2
693
0.0
030
0.0
541
0.0
066
0.1
059
<.0
001
<.0
001
0.5
238
0.9
979
E5-
T1
E5-
T4
0.3
097
0.9
663
0.0
092
0.1
397
0.0
252
0.3
021
0.0
207
0.2
624
0.2
613
0.9
426
E5-
T1
E5-
T5
0.0
401
0.4
152
0.0
014
0.0
280
0.0
068
0.1
090
<.0
001
<.0
001
0.0
009
0.0
191
E5-
T1
E5-
T6
0.2
443
0.9
312
0.1
894
0.8
777
0.0
094
0.1
424
<.0
001
<.0
001
<.0
001
0.0
019
E5-
T1
E5-
T7
0.0
268
0.3
164
0.0
002
0.0
039
0.0
286
0.3
311
<.0
001
<.0
001
0.0
004
0.0
078
E5-
T1
E5-
T8
0.0
989
0.6
900
0.0
860
0.6
455
0.0
176
0.2
325
<.0
001
<.0
001
0.0
070
0.1
108
E5-
T2
E5-
T3
0.2
239
0.9
146
0.6
925
0.9
999
<.0
001
<.0
001
<.0
001
<.0
001
0.1
772
0.8
613
E5-
T2
E5-
T4
0.8
906
10.0
00
0.4
124
0.9
901
<.0
001
<.0
001
0.0
004
0.0
076
0.6
853
0.9
999
E5-
T2
E5-
T5
0.3
421
0.9
767
0.8
992
10.0
00
<.0
001
<.0
001
<.0
001
<.0
001
0.0
001
0.0
026
E5-
T2
E5-
T6
0.9
883
10.0
00
0.0
310
0.3
499
<.0
001
<.0
001
<.0
001
<.0
001
<.0
001
0.0
002
E5-
T2
E5-
T7
0.2
614
0.9
427
0.5
433
0.9
984
<.0
001
<.0
001
<.0
001
<.0
001
<.0
001
0.0
010
E5-
T2
E5-
T8
0.6
022
0.9
994
0.0
774
0.6
118
<.0
001
<.0
001
<.0
001
<.0
001
0.0
010
0.0
195
E5-
T3
E5-
T4
0.1
774
0.8
615
0.6
692
0.9
998
0.5
860
0.9
992
<.0
001
<.0
001
0.0
828
0.6
332
E5-
T3
E5-
T5
0.7
849
10.0
00
0.7
878
10.0
00
0.9
891
10.0
00
0.9
943
10.0
00
0.0
054
0.0
895
E5-
T3
E5-
T6
0.2
294
0.9
193
0.0
730
0.5
930
0.8
877
10.0
00
0.9
123
10.0
00
0.0
005
0.0
110
E5-
T3
E5-
T7
0.9
236
10.0
00
0.3
182
0.9
694
0.5
483
0.9
986
0.9
824
10.0
00
0.0
022
0.0
411
E5-
T3
E5-
T8
0.4
812
0.9
961
0.1
643
0.8
417
0.6
932
0.9
999
0.8
123
10.0
00
0.0
327
0.3
630
E5-
T4
E5-
T5
0.2
782
0.9
522
0.4
873
0.9
964
0.5
954
0.9
994
<.0
001
<.0
001
<.0
001
0.0
008
E5-
T4
E5-
T6
0.8
791
10.0
00
0.1
651
0.8
429
0.6
862
0.9
999
<.0
001
<.0
001
<.0
001
<.0
001
E5-
T4
E5-
T7
0.2
090
0.9
001
0.1
578
0.8
307
0.9
552
10.0
00
<.0
001
<.0
001
<.0
001
0.0
003
E5-
T4
E5-
T8
0.5
107
0.9
974
0.3
291
0.9
730
0.8
801
10.0
00
<.0
001
<.0
001
0.0
003
0.0
066
E5-
T5
E5-
T6
0.3
495
0.9
787
0.0
412
0.4
226
0.8
986
10.0
00
0.9
066
10.0
00
0.3
989
0.9
883
E5-
T5
E5-
T7
0.8
594
10.0
00
0.4
634
0.9
950
0.5
574
0.9
988
0.9
766
10.0
00
0.7
383
10.0
00
E5-
T5
E5-
T8
0.6
648
0.9
998
0.0
996
0.6
922
0.7
033
0.9
999
0.8
068
10.0
00
0.4
627
0.9
949
E5-
T6
E5-
T7
0.2
675
0.9
463
0.0
071
0.1
122
0.6
455
0.9
997
0.9
299
10.0
00
0.6
083
0.9
995
E5-
T6
E5-
T8
0.6
123
0.9
995
0.6
712
0.9
999
0.7
998
10.0
00
0.8
987
10.0
00
0.1
193
0.7
490
E5-
T7
E5-
T8
0.5
422
0.9
984
0.0
202
0.2
576
0.8
361
10.0
00
0.8
295
10.0
00
0.2
878
0.9
570
90
dT
reatm
en
t vs
Treatm
en
t D
FI-
valu
es
befo
re F
D 0
d 1
5d
3
0d
APPENDIX
78
Pr >
|t|
Adj
PP
r >
|t|
Adj
PP
r >
|t|
Adj
PP
r >
|t|
Adj
PP
r >
|t|
Adj
P
E5-
T1
E5-
T2
0.1
139
0.7
345
<.0
001
0.0
010
<.0
001
<.0
001
<.0
001
<.0
001
<.0
001
0.0
002
E5-
T1
E5-
T3
0.0
063
0.1
022
0.0
041
0.0
701
0.0
725
0.5
910
0.0
014
0.0
278
0.0
007
0.0
150
E5-
T1
E5-
T4
0.2
800
0.9
531
0.0
052
0.0
870
0.3
136
0.9
678
0.3
193
0.9
698
0.0
030
0.0
533
E5-
T1
E5-
T5
0.0
377
0.3
988
0.0
010
0.0
208
0.0
528
0.4
932
0.0
004
0.0
093
0.0
023
0.0
426
E5-
T1
E5-
T6
0.0
944
0.6
751
0.1
303
0.7
758
0.0
664
0.5
633
0.0
007
0.0
150
0.0
002
0.0
042
E5-
T1
E5-
T7
0.0
070
0.1
112
<.0
001
0.0
011
0.0
700
0.5
801
0.0
003
0.0
072
0.0
003
0.0
075
E5-
T1
E5-
T8
0.0
331
0.3
654
0.0
581
0.5
220
0.1
043
0.7
068
0.0
007
0.0
152
0.0
004
0.0
091
E5-
T2
E5-
T3
0.2
078
0.8
989
0.1
198
0.7
501
<.0
001
<.0
001
<.0
001
<.0
001
<.0
001
<.0
001
E5-
T2
E5-
T4
0.6
035
0.9
994
0.0
996
0.6
923
<.0
001
<.0
001
<.0
001
<.0
001
0.0
555
0.5
083
E5-
T2
E5-
T5
0.5
934
0.9
993
0.2
816
0.9
540
<.0
001
<.0
001
<.0
001
<.0
001
<.0
001
<.0
001
E5-
T2
E5-
T6
0.9
225
10.0
00
0.0
036
0.0
631
<.0
001
<.0
001
<.0
001
<.0
001
<.0
001
<.0
001
E5-
T2
E5-
T7
0.2
217
0.9
125
0.9
684
10.0
00
<.0
001
<.0
001
<.0
001
<.0
001
<.0
001
<.0
001
E5-
T2
E5-
T8
0.5
539
0.9
987
0.0
103
0.1
530
<.0
001
<.0
001
<.0
001
<.0
001
<.0
001
<.0
001
E5-
T3
E5-
T4
0.0
793
0.6
193
0.9
234
10.0
00
0.4
125
0.9
901
0.0
194
0.2
495
<.0
001
<.0
001
E5-
T3
E5-
T5
0.4
615
0.9
948
0.6
194
0.9
996
0.8
791
10.0
00
0.6
756
0.9
999
0.6
806
0.9
999
E5-
T3
E5-
T6
0.2
438
0.9
308
0.1
359
0.7
882
0.9
659
10.0
00
0.8
108
10.0
00
0.6
401
0.9
997
E5-
T3
E5-
T7
0.9
690
10.0
00
0.1
290
0.7
726
0.9
865
10.0
00
0.6
098
0.9
995
0.7
957
10.0
00
E5-
T3
E5-
T8
0.4
973
0.9
969
0.2
719
0.9
488
0.8
552
10.0
00
0.8
146
10.0
00
0.8
518
10.0
00
E5-
T4
E5-
T5
0.2
950
0.9
603
0.5
537
0.9
987
0.3
325
0.9
740
0.0
069
0.1
094
<.0
001
<.0
001
E5-
T4
E5-
T6
0.5
379
0.9
983
0.1
617
0.8
374
0.3
889
0.9
867
0.0
108
0.1
588
<.0
001
<.0
001
E5-
T4
E5-
T7
0.0
857
0.6
443
0.1
075
0.7
166
0.4
031
0.9
889
0.0
054
0.0
895
<.0
001
<.0
001
E5-
T4
E5-
T8
0.2
696
0.9
475
0.3
150
0.9
682
0.5
227
0.9
979
0.0
109
0.1
603
<.0
001
<.0
001
E5-
T5
E5-
T6
0.6
620
0.9
998
0.0
503
0.4
789
0.9
130
10.0
00
0.8
575
10.0
00
0.3
813
0.9
854
E5-
T5
E5-
T7
0.4
852
0.9
963
0.2
992
0.9
621
0.8
925
10.0
00
0.9
265
10.0
00
0.5
035
0.9
971
E5-
T5
E5-
T8
0.9
535
10.0
00
0.1
147
0.7
369
0.7
381
10.0
00
0.8
537
10.0
00
0.5
502
0.9
986
E5-
T6
E5-
T7
0.2
594
0.9
414
0.0
040
0.0
692
0.9
793
10.0
00
0.7
859
10.0
00
0.8
343
10.0
00
E5-
T6
E5-
T8
0.6
204
0.9
996
0.6
842
0.9
999
0.8
218
10.0
00
0.9
961
10.0
00
0.7
785
10.0
00
E5-
T7
E5-
T8
0.5
221
0.9
978
0.0
114
0.1
656
0.8
420
10.0
00
0.7
821
10.0
00
0.9
425
10.0
00
9
0d
Treatm
en
t vs
Treatm
en
t
(FL
3-
valu
es)
b
efo
re F
D 0
d 1
5d
3
0d
APPENDIX
79
9.2. Sperm chromatin dispersion test and evaluation with ImageJ program
Steps after observation using light microscopy (10 x 20 magnification):
1. Open file (first picture hemocytometer/ counting chamber)
2. Select freehand lines (button) straight line
3. Analyze set scale distance of pixel: 170; known distance: 50; pixel aspect
ratio: 1.0; unit of length: µm
4. Image type 16- bit
5. Analyze tools ROI manager add (straight line 0704- 0897) measure
(Area: 14.792; Mean: 209.942; Min: 196; Max: 214; Angle: 0; Length 50)
6. Open file (second picture with sperms)
7. Image type 16- bit
8. Image adjust threshold set constant (170), click apply
9. Image type 16- bit (ones again)
10. ROI manager hook at show all and labels click number 1 at the straight line
11. Analyze set scale distance of pixel: 170; known distance: 50; pixel aspect
ratio: 1.0; unit of length: µm
12. Select tracing tool (button) select cells and add (t)
13. Select all cells in ROI manager (shift), click measure
14. File save as Jpeg/ Tiff
80
10. DANKSAGUNG
Mein erster Dank gilt meinem Doktorvater, Herrn Prof. Dr. Harald Sieme, für die
Überlassung des interessanten Dissertationsthemas und seiner fortwährenden Unterstützung,
sowie das Vertrauen in mich.
Ebenfalls bedanken möchte ich mich ganz herzlich bei Dr. Ir. Harriëtte Oldenhof für die
gewissenhafte und umsichtige Betreuung während der Versuche sowie des Schreibens an der
Dissertation. Auf der einen Seite in Form fachlicher Anregungen und konstruktiver Kritik und
auf der anderen Seite mit ihren immer motivierenden und aufmunternden Worten, half sie mir
zu jeder Zeit, zu später Abendstunde oder am Wochenende.
Weiterhin möchte ich Dr. Axel Brockmann sowie allen Mitarbeitern des Landgestütes Celle
für die Zusammenarbeit danken. Ein spezieller Dank geht dabei an Dr. Gunilla Martinsson, an
die Kollegen der Stationen Adelheidsdorf und Celle und nicht zu vergessen an meine
Mitdoktoranden für die tolle Zeit. Ich finde wir waren für eineinhalb Jahre ein super Team,
das sich sehen lassen konnte.
Danken möchte ich zudem Prof. Dr. Ir. Willem F. Wolkers von der Leibniz Universität
Hannover, dass ich meine Gefriertrocknungsversuche bei ihm im Institut für
Mehrphasenprozesse machen durfte. Außerdem danke ich Judith Bigalk für die unermüdliche
Hilfe bei den Versuchen und den unzähligen SCSA Messungen. Ein weiterer Dank geht an
Dr. Karl Rohn für die Hilfe bei statistischen Fragen und Problemen.
Zu guter Letzt geht mein Dank an meine Freunde und Familie, dabei vor allem an meine
Eltern, meine Schwester und meinen Freund. Ohne eure Unterstützung, die aufmunternden
Worte und den stetigen Zusammenhalt wäre ich heute nicht da wo ich jetzt bin.