Freeze- drying of equine sperm and sperm chromatin structure during dried storage

University of Veterinary Medicine Hannover

Freeze- drying of equine sperm

and sperm chromatin structure during dried storage

Inaugural-Dissertation

to obtain the academic degree Doctor medicinae veterinariae

(Dr. med. vet.)

submitted by Katharina Narten

Minden

Hannover 2017

Academic supervision: Prof. Dr. Harald Sieme Clinic for Horses

Unit for Reproductive Medicine

Dr. Ir. Harriëtte Oldenhof Clinic for Horses

Unit for Reproductive Medicine

1. Referee: Prof. Dr. Harald Sieme

2. Referee: Prof. Dr. Dagmar Waberski

Day of the oral examination: 2017/05/11

A contribution from the Virtual Center for Reproductive Medicine, Lower Saxony

III

Meiner Familie

IV

V

Parts of this thesis were presented at the following congresses and published at the corresponding scientific journals.

OLDENHOF, H., NARTEN, K., BIGALK, J., WOLKERS, W.F., SIEME, H. (2017):

Preservation of sperm chromatin during dried storage. Chromatinintegrität gefriergetrockneter Spermien während der Lagerung.

50. Jahrestagung Physiologie und Pathologie der Fortpflanzung, gleichzeitig 42. Veterinär- Humanmedizinische Gemeinschaftstagung, München, 15.-17.02.2017

Reprod Dom Anim; 52 (Suppl. 1):38.

OLDENHOF H, ZHANG M, NARTEN K, BIGALK J, SYDYKOV B, WOLKERS WF, SIEME H (2016):

Freezing-induced uptake of trehalose by stallion sperm.

Proc. 7th International Symposium on Stallion Reproduction (ISSR), Illinois, USA.

J Equine Vet Sci 43, 73-74

VI CONTENTS

C

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

A

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. I

:

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. L

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 0,045^a

4 EDTA

4 Kaneko and Serikawa (2012)

0,038 and 0,058^a

4 EDTA- TRIS

5 Kawase et al.

(2005)

0,040 and 0,001^a

8 and 6^a

6 Kawase et al.

(2007, 2009)

0,37 and 0,001^a

13 and 6^a 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 3^a

Rabbit 11 Liu et al. (2004) 0,023 - 0,040

4 EGTA- TRIS- HCl buffer

Dog 12 Watanabe et al.

(2009)

0,37 and 0,001^a

- EGTA- TRIS- HCl buffer

Cat 13 Ringleb et al.

(2011)

0,16 4

Pig 14 García Campos et al. (2014)

0,015 - 0,005^a

24 and 6^a 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 0,13^a

19 and 3^a 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 0,001^a

14 and 3^a

19 Hara et al.

(2011)

0,37 and 0,001^a

14 and 3^a 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

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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

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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

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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

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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).

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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.

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3. M

M

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.

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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)

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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.

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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.

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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.

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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.

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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.

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Figure 3.5. Temperature profiles to which samples were exposed for freeze-drying. First, samples were frozen by placing in a −150°C freezer (A). Samples were kept in the freezer for a minimum of 30 min (i.e. for reaching −80°C). Then, samples were placed in the lyophilizer, and subjected to the protocol as shown in panel B. Here both the temperature (blue line) and pressure (grey line) profiles are presented versus time, as explained in detail in section 3.4. The freeze-drying protocol took in total 24 h.

B A

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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.

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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

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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.

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Figure 3.6. Original microscopic images as obtained with the ‘halo-test’ (A, C), as well as the same images after processing using imageJ software (B, D) for analysis of halo sizes. Examples are presented for sperm with intact (A, B) and damaged (C, D) DNA; with large and small halo’s, respectively. Images were converted into black/white, and a similar background threshold was set to automatically detect sperm with halos and determine the area they covered. This is illustrated in panel E, where sperm with halo’s are marked in yellow. See section 3.8 for a detailed description.

Scale bar represents 50 µm