university of veterinary medicine hannover · plasma membrane can be evaluated as sperm swelling in...

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

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.


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)


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.


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.


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.


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.


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.


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

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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

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t in

th

e fr

eeze

r fo

r a

min

imum

of

30 m

in (

i.e.

for

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). T

hen

, sa

mp

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e pla

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op

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subje

cted

to

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roto

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ho

wn i

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anel

B.

Her

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(blu

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(gre

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ail

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3.4

. T

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free

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took i

n t

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l 24 h

.

B

A


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.


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


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.


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

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Images

wer

e co

nver

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ck/w

hit

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nd a

sim

ilar

bac

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und t

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shold

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to

au

tom

atic

ally

det

ect

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m w

ith

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os

and d

eter

min

e th

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ea t

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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


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.


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


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


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


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


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


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.


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

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0.5

539

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T3

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0.0

793

0.6

193

0.9

234

10.0

00

0.4

125

0.9

901

0.0

194

0.2

495

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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

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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

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094

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E5-

T4

E5-

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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

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E5-

T4

E5-

T7

0.0

857

0.6

443

0.1

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0.7

166

0.4

031

0.9

889

0.0

054

0.0

895

<.0

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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

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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

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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.

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