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Biomedical Science

Faculty of Health and Society Malmö University

SE-205 06 Malmö Sweden

Master programme in Biomedical Surface Science http://edu.mah.se/en/Program/VABSE

Master degree thesis, 30 ECTS Examensarbete, 30 hp

Effect of Dextrans on Cryopreservation

of Human Spermatozoa

Fie Barbara Nordskov Harder

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AUTHOR: Fie Barbara Nordskov Harder ABSTRACT

Cryopreservation is a process where a sample, e.g. cells or tissue, is preserved by cooling to sub-zero temperatures, usually -196°C. Upon freezing these materials, ice crystals form, which eventually results in cell death. In order to reduce the formation of ice crystals, cryoprotectants are added to the storage solution. Current cryoprotective agents have several weaknesses, making the development of cryoprotective agents with advanced properties of great interest. In this study, two possible non-penetrating cryoprotectants termed Dextran Sulphate Sodium Salt 80 (DSSS 80) and Dextran Sulphate Sodium Salt (DSSS 140), were developed. The cryopreservative effect of DSSS 80 and DSSS 140 was studied on human spermatozoa regarding cryorecovery, using a simple cryopreservation procedure. Moreover, the new compound’s cryoprotective properties were compared to conventional types of dextrans, which in previous studies have proven effective. The percentage of sperm motility was assayed before freezing by the optical microscopic method using a haemocytometer as counter chamber. After thawing, the cells were stained with trypan blue and post-thaw motility was determined. It was found that all sperm cells lost their motility, regardless cryoprotectant, concentration or in the absence of dextrans, which indicated that the applied cryopreservation was unfitting. Thus, since the method used in study was not optimal for cryopreservation of sperm cells, it was not possible to determine whether DSSS 80 or DSSS 140 display cryoprotective properties or not, nor if they are superior to conventional types of cryoprotectants.

Keywords: Dextran, Dextran Sulphate Sodium Salt, Synthesis, Cryopreservation, Sperm Motility

This master thesis has been defended on August 21, 2018 at the Faculty of Health and Society, Malmö University.

Opponent: Zoltan Blum

Biomedical Laboratory Science

and Technology Faculty of Health and Society Malmö University

Examiner: Prof. Thomas Arnebrant

Biomedical Laboratory Science and Technology Faculty of Health and Society

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TABLE OF CONTENT

1. INTRODUCTION……….5

1.1. Cryopreservation..……….……….……..5

1.2. The significance of cryoprotective agents……….……….……..6

1.3. Dextran as cryoprotectants……….………8

1.4. What is dextran?...8

1.5. Derivatives of Dextran……….…..…9

1.6. Aim of the study……….…..10

2. MATERIALS and METHODS………11

2.1. Materials/Reagents……….……..11

2.2. Methods……….………11

2.2.1. Synthesis of Dextran Sulphate Sodium Salt 80 and 140………..11

2.2.2. Molecular Weight Determination and Sulphur Content analysis...12

2.2.3. Preparation of spermatozoa and plasma……….…..12

2.2.4. Determination of motility………...13

2.2.5. Cryopreservation procedure……….……….13

2.2.6. Data processing………..……….……….………..14

3. RESULTS……….………….14

3.1. Characterisation of DSSS 80 and DSSS 140……….…………..14

3.2. Effect of dextrans on cryopreservation of spermatozoa………….….………….15

3.2.1. Cryoprotective potential of DSSS 80 and DSSS140………..15

3.2.2. Cryopreservation for 24 hours………..16

3.2.3. Verification of cryopreservation procedure………..……..16

4. DISCUSSION……….…………..….18

4.1. Synthesis and characterisation of DSSS 80 and DSSS 140….……...18

4.1.1. Sulphur Content………18

4.1.2. Molecular Weight………19

4.2. Effect of dextrans on cryopreservation of spermatozoa….………..20

5. CONCLUSIONS AND PERSPECTIVES ………23

6. Acknowledgements………...24

7. References………25

8. Appendix 1……….………..28

9. Appendix 2………..….29

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ABBREVIATIONS

Abbreviation Explanation

ATP-ase Adenosine triphosphatase

cAMP Cyclic adenosine monophosphate

Da Dalton

DMEM Dulbecco’s Modified Eagles Medium

DMSO Dimethyl Sulfoxide

DSSS 80 Dextran Sulphate Sodium Salt 80

DSSS 140 Dextran Sulphate Sodium Salt 140

HPLC High Performance Liquid

Chromatography

IEX Ion Exchange Chromatography

kDa Kilo Dalton

MDa Mega Dalton

PKC pK Chemicals

SEC Size Exclusion Chromatography

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1.

INTRODUCTION

1.1. Cryopreservation

Cryopreservation is a process where a sample, e.g. cells or tissue, is preserved by cooling to sub-zero temperatures, usually -196°C[1,2]. At this low temperature, any biological activity, including the biochemical reactions that cause cell death, is efficiently stopped [1-3]. Once the sample is thawed, the procedure allows the cells to be restored to the same living state and moreover remain its original functions [1,4,5].

Consistently, cryopreservation

techniques are being used for long-term preservation of aqueous materials such as cells and tissues from plants, animals and humans. It is well established that upon freezing these materials, ice crystals are formed, which result in uneven concentrations of solutes and contaminants excluded by water molecules. This is referred to as “freeze concentration “ [3]. In an attempt to dilute the solutes, water rushes out of the cell, as pictured in figure 1. This event results in cell shrinkage and eventually death. But with addition of cryoprotectants to the storage solution, the formation of ice crystals can be reduced and controlled. This helps to equalize the imbalance of solutes, thus preventing water loss and cell damage [1-3].

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In conventional cryopreservation techniques, the sample is harvested, placed in a storage solution, and then preserved by freezing [1-3]. When the sample is to be used, it is thawed, and for cells taken from human donor sources, the samples are brought back to the normal human body temperature (37°C), and afterwards placed in a cell culture medium [1,2,4].

Through cryopreservation protocols, the cells are additionally subject to a multitude of stresses throughout the process of cell harvesting, freezing, and thawing. These stresses can cause irreversible damage to the cell, and ultimately cause cell death. For cryopreservation to be useful, the preserved sample should retain its integrity and viability to a reasonable level at the time of harvest [2,3].

Hence, the process of preserving the sample should naturally not cause damage or destroy the cells or tissue in the matter of morphology and viability. Cryoprotectant solutions are typically used to prevent damage caused by freezing both during the cooling and thawing process. Consequently, the use of cryoprotectants is of critical matter.

1.2. The significance of cryoprotective agents

The first efficient cryopreservation study was described by Polge et al. [7], as the

investigators reported the cryoprotective property of glycerol on fowl sperm. Next, Lovelock et al. [8], discovered that dimethyl sulfoxide (DMSO) could serve as a

successful cryoprotectant for red blood cells. Nonetheless, these two penetrating cryoprotective agents have certain disadvantages; glycerol is a relatively weaker cryoprotectant, and DMSO displays both high cytotoxicity and affects differentiation on cardiac myocytes, neuron-like cells and granulocytes [1,9]. Thus, DMSO needs to be removed immediately after thawing.

It has been suggested that the protective action of glycerol is due to the formation of hydrogen-bonds between glycerol’s OH-groups and the oxygen atoms of the phospholipid hydrophilic head groups of the cell membrane. The structural formula of phospholipids is illustrated in figure 2. Hereby a protective layer over the cell membrane is formed, which serves as a cushion for protection against

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In general, the effectiveness of cryoprotectants is dependent on their ability to penetrate the cell membrane, cytotoxicity and the molecular ratio of the cryoprotectant to the endogenous solutes in-and outside of the cell [2,3]. A second class of cryoprotectants is the non-penetrating solutes, which includes sugars and higher molecular weight compounds like hydroxyethyl starch, polyethylene glycols and dextrans [3,11].It is believed that this class of cryoprotectants does not display sufficient protective properties in the absence of a penetrating cryoprotectant, such as glycerol and DMSO. But instead the non-penetrating cryoprotectants increase the effectiveness of e.g. glycerol and DMSO, and allows use of lower concentrations of the penetrating cryoprotectants [1,5].

Figure 3: Structure of the cell membrane bilayer, consisting of two inverted phospholipid layers, in which the non-penetrating cryoprotectants can form

hydrogen bonds with the extracellular side [12].

The mechanism of the non-permeating cryoprotectants is, like the penetrating, that they can form hydrogen bonds with the polar head groups of the phospholipids on the extracellular side. An illustration of the cell membrane bilayer is shown in figure 3. Thus, they prevent fusion events of juxta-posed membranes, stabilize the cell membrane and inhibit progressive ice formation [1,5,11,13].

Due to the absence of efficient substitutes to DMSO, this cryoprotectant remains habitually used as the preferred cryoprotective agent in numerous biological applications [2,3]. Hence, the development of new and more effective cryoprotective agents is of great importance.

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A recent study made by Matsumura et al. [14], where the research group developed a non-penetrating polymer-based cryoprotectant, carboxylated poly-L-lysine (COOH-PLL), was published. This cryoprotectant showed brilliant cryoprotective abilities even after cryopreservation for 24 months. This cryoprotectant has both positive and negative charges on the polymer chain (a polyampholyte). They can be both neutral in charge or have a net charge. Polyampholytes have found a use in various biomedical applications. Beside from the development of COOH-PLL, the investigators also found an alternative cryoprotectant that showed excellent cryoprotective property on various cell types; Dextran-based polyampholytes.

1.3. Dextran as cryoprotectant

The use of dextran to cryopreserve cell lines, stem cell preparations and other biological samples, is a well described topic in the patent of Pharmacosmos

“Cryoprotecting agent, cryoprotecting and cryopreserved compositions, uses

thereof, and methods of cryopreservation” [1]. In this patent, it is additionally

described that dextran can be used as a cryoprotectant in combination with DMSO or glycerol. The most common type of dextran used for cryopreservation is Dextran 40, but also other types such as Dextran 10, 70 and 500 kDa are used [1-3]. When looking further into literature, a cryopreservation study made with goat cauda epididymal spermatozoa showed that Dextrans of 10, 40, 73, 173, 252, 500 and 2000 kDa offered maximum cryorecovery of forward motility, but the optimum concentration varied for each specific type[5]. Thus, it appears that each dextran offers characteristic cryoprotective profiles, if the right concentration is found.

1.4. What is Dextran?

Dextran is prepared by fermentation of sugar [15]. Most manufacturers use the Leuconostoc mesenteroides NRRL B-512(F) or B-512 strain for the fermentation

[15,16]. The organism, Leuconostoc mesenteroides, is a member of the

Lactobacillaceae family, and classifies as a gram-positive facultative anaerobe. Most manufacturers of dextran use a process based on the batch-wise culture of

Leuconostoc in the presence of sucrose. Although many sugars, e.g. glucose, can

serve as energy source for the growth of bacteria, only sucrose acts to induce dextransucrase production. Besides from dextran and lactic acid, the bacteria also produces carbon dioxide, ethanol, mannitol and acetic acid [11]. After harvest and purification, the native dextran obtained is hydrolysed in dilute acid and separated into the desired molecular weight [11,15].

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Figure 4: Chemical structure of Dextran [17].

Dextran is a branched glucan, a polysaccharide consisting of many glucose molecules. Dextrans can be composed of chains of variable lengths from 1000 Da-500 MDa. The straight chain is made up of α-1,6 glycosidic linkages between the glucose molecules, as shown in figure 4. The side branches are mainly α-1,3 linkages. The α-1,6 glycosidic linkage is made up by a primary and secondary hydroxyl group. It is a non-ionic compound, appearing as a white powder [15]. Dextran is highly water-soluble (50w/v), and pH-neutral. Beside use in cryopreservation, dextran is widely used as an antithrombotic, blood volume expander, vaccine adjuvant, lubricant in eye care liquids and in drug delivery systems [11,15]. Dextran 70 is stated as one of the most important medications needed in health system on WHO model list of Essential Medicines [18].

1.5. Derivatives of Dextran

Beside from the production of Dextrans of various molecular weight, the product can additional be converted into different derivatives that are manufactured for different purposes. Dextran Sulphate is a polyanionic derivative of dextran, which is presented in figure 5. It is a white, sodium salt powder, which is stabilized by a small amount of phosphate buffer [11,15,19]. Dextran Sulphate is freely soluble in water and forms a stable, clear solution, which can be further sterilized by filtration.

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Figure 5: Molecular structure of Dextran Sulphate Sodium Salt [20].

Due to the high purity and reproducible quality, Dextran Sulphate is widely used in many applications in molecular biology and in the health-care sector [11,15]. Dextran Sulphate displays following properties: accelerates hybridization rates, anti-coagulant and anti-viral properties. Moreover, the product is used in cosmetic applications, it can interact with lipoproteins, enzymes and cells and stabilize proteins [11,15].

1.6. Aim of the study

The aim of this master thesis work was to synthesize two novel types of non-penetrating cryoprotective agents and study their effect on cryopreservation. Moreover, the aim was to analyse if there was a correlation between concentration of the two new compounds and cryorecovery of the cells. The new types of cryoprotectants were developed from Dextran 40 and 70, and termed Dextran Sulphate Sodium Salt 80 (DSSS 80) and Dextran Sulphate Sodium Salt 140 (DSSS 140). Because Dextran 40 is one of the most commonly used dextrans in cryopreservation, this product was chosen for further development. Moreover, since Dextran 70 is on WHO model list of Essential Medicines, this variation was also selected. Due to the fact that sulphur is highly hydrophilic, the hypothesis was that a Dextran Sulphate could increase water affinity. By increasing water affinity, the environmental stresses on the cell caused by freezing and thawing during cryopreservation could be further stabilized, which in theory would make DSSS 80 and DSSS 140 advantageous cryoprotectants.

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Both sperm and embryo cryopreservation have become regular procedures in human assisted reproduction [21,22]. But also, oocyte cryopreservation is starting to be introduced into clinical practice. Embryo cryopreservation has decreased the number of fresh embryo transfers and maximized the effectiveness of the IVF cycle [21,22]. Data shows that women who had transfers of embryos obtained 8% additional births when using their cryopreserved embryos [21,22]. Therefore, cryopreservation is of great importance for treating e.g. infertility.

Subsequently, human spermatozoa were selected as subject to evaluate the effect of DSSS 80 and DSSS 140 on cryopreservation, by analysing the cryorecovery of the cells. Moreover, Dextran 40 and 70 were included in the study, for better comparison and validation of the potential superior cryoprotective properties of DSSS 80 and DSSS 140.

2.

MATERIALS and METHODS

Human spermatozoa were provided from 2 healthy male volunteers. The two participants were selected based on age, number of children, health etc. The men were to sign an informational letter (Appendix 1) before participating in this study. This was done to confirm that the participants were well informed of the terms and conditions of contributing to this medical study. Also, the samples were treated anonymously in order to ensure the integrity of the participants.

2.1. Materials/Reagents

All materials used for the synthesis of DSSS 80 and DSSS 140 were provided by pK Chemicals A/S (PKC), Koege, Denmark. Dextran 40 and Dextran 70 were likewise provided by PKC. Cell culture medium, haemocytometers, cryopreservation tubes and trypan blue were purchased from Sigma-Aldrich, Soeborg, Denmark.

2.2. Methods

2.2.1. Synthesis of Dextran Sulphate Sodium Salt 80 and 140

The development and synthesis of DSSS 80 and 140 were made based on internal protocols of current products from PKC. As the development of novel products for PKC has the potential for prospective marketing and selling, the development protocols of DSSS 80 and DSSS 140 are confidential. Therefore, the development and synthesis of DSSS 80 and DSSS 140 will not be described in detail.

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Figure 6: A) The formation of sulfonating species (Vilsmeier adduct) from the

reaction between formamide and chlorosulfonic acid. B) The reaction between dextran and the sulfonating species [23].

In brief, the first step in the process is the reduction of aldehydes on dextran with sodium borohydride. Next, the functionalization of dextran to produce DSSS can be described as two processes, which is demonstrated in figure 6; the formation of sulfonating species and its reaction with hydroxyl moieties on dextran, which forms dextran sulphate. The formation of sodium salt is made by stripping the protons from the product with sodium hydroxide, and purified by multiple precipitations.

2.2.2. Molecular Weight Determination and Sulphur Content analysis The molecular weight of the polymers was determined by high performance liquid chromatography (HPLC; Agilent Technologies 1260 Infinity). The specific type of HPLC, used for determination of the molecular weight of DSSS 80 and DSSS 140 in this study, was size-exclusion chromatography (SEC). The Sulphur Content of the DSSS 80 and DSSS 140 was determined using ion-exchange chromatography (IEX; Agilent Bio EIX 5190-2431). The analyses were made by Christian Sörensen, Rie Meyer Scheuermann and Mona Nielsen, laboratory technicians at PKC, Haarlev, Denmark.

2.2.3. Preparation of spermatozoa and plasma

The semen was prepared in accordance with the procedure described by Mandal et

al. [24] (Appendix 2). Human spermatozoa were sedimented by gentle hand-centrifugation for approximately 5 min at room temperature (23 ± 2), and the supernatant/plasma was removed for further use. The cells were then dispersed in Dulbecco’s Modified Eagles Medium (DMEM) without DMSO.

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The spermatozoa in the cell preparation were highly motile and healthy as judged by optical microscope. The number of spermatozoa in the sample was determined using a haemocytometer.

2.2.4. Determination of Motility

The percentage of sperm motility was assayed before freezing and after thawing by the optical microscopic method using a haemocytometer as counter chamber. The motility was analysed in the presence of seminal plasma in order to eliminate sperm adhesion to glass [25]. Spermatozoa (0.5 x 106 cells) were incubated with seminal plasma at room temperature for 1 min in a volume of 0.5ml DMEM. Sperm suspension was then injected into the haemocytometer. The number of cells with forward motility (cells that moved in circles were excluded) and the total number of cells were counted under an optical microscope at 40x magnifications. Following, the percentage of forward motility and total number of cells were calculated. The percentage recovery of motility was calculated by relating the motility percentages before freezing and after thawing. The percentage recovery of motility was !"#$%$#& ()*#+, #-).$/0)!"#$%$#& (2+*",+ *,++3+) x 100.

2.2.5. Cryopreservation procedure

Freezing. Spermatozoa were cryopreserved using the methods reported by Kundu

et al. [5] and Rajan et al. [26], with adjustments regarding the equipment, which was

present in the laboratory (Appendix 2).

Cryoprotectant solutions of Dextran 40, Dextran 70, DSSS 80 and DSSS 140 were prepared by dissolving the compounds in DMEM. The concentrations were of 1.25, 2.50 and 5.00 mmol/L, which are listed in table 1. Cryoprotectant concentrations were chosen based on optimum concentrations for various types of dextrans for cryoprotection of sperm motility reported by Kundu et al. [5]. 1 ml of each specified cryopreservative solution was transferred into a cryogenic tube with screw cap.

Table 1. The various concentrations of cryoprotective agents used for cryopreservation of spermatozoa.

Cryoprotective

agent Concentrations

Dextran 40 1.25 mmol/L 2.50 mmol/L 5.00 mmol/L Dextran 70 1.25 mmol/L 2.50 mmol/L 5.00 mmol/L

DSSS 80 1.25 mmol/L 2.50 mmol/L 5.00 mmol/L

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The sperm preparations were subsequently added to the cryopreservative solution, so that the final concentration of cells was 80 x106 cells/ml. The samples were made in triplicates. The cryogenic tubes were then stored at -80°C without controlling the cooling rate.

Thawing.After 1 week, the tubes were plunged into a 37°C thermostatic waterbath

for 2-3 min, and the cells were dispersed in DMEM. Next, the pellet was collected by centrifugation, subsequently the pellet was re-suspended in plasma and medium. The cells were then stained with trypan blue, and post-thaw motility was determined.

2.6. Data processing

The results (the percentage of cryorecovery of sperm motility) were expressed as means ± standard deviation (SD). All samples for the experiments were conducted in triplicate.

3. RESULTS

This study meant to develop two novel types of dextran sulphates and to study their cryoprotective potential on human spermatozoa, using a simple sperm cryopreservation procedure. Moreover, the objective was to make a comparison of four cryoprotective agents, and to investigate if there was any correlation between increasing concentrations of dextrans and recovery of motility.

3.1. Characterization of DSSS 80 and DSSS 140

The synthesis of DSSS 80 and DSSS 140 was performed 4 times for each compound. The aim was to produce the compounds, so that they were in accordance with the internal specifications at PKC. The two key parameters were the sulphur content and the molecular weight. Due to time limitation, it was not possible to synthesise DSSS 80 and DSSS 140, so that they were completely in accordance with the specifications. Therefore, the dextran sulphates which were chosen to be used in this study, were the ones that came closest to the above-mentioned parameters. A summary of the characteristics of the compounds is provided in Table 2 and 3.

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Table 2. Characteristics of DSSS 80.

Analysis Result Tolerance Difference

Sulphur content (%) 23.1 16-19 + 4.1 Molecular weight (Dalton) 223.789 80.000 + 143789 Table 3. Characteristics of DSSS 140.

Analysis Result Tolerance Difference

Sulphur content (%) 28.1 16-19 + 9.1 Molecular weight (Dalton) 64.139 140.000 - 75861

As it can be seen from Table 2 and 3, the sulphur content is not in agreement with the tolerance level, neither for DSSS 80 or DSSS 140, which is 4.1% and 9,1% higher, respectively.

The molecular weight of the two compounds is not in agreement with their respective tolerance level, which for DSSS 80 is 2.8 times higher than the limit. For DSSS 140, it can be seen (Table 3) that the molecular weight is 2.2 times lower than the tolerance level.

3.2. Effect of dextrans on cryopreservation of spermatozoa

3.2.1. Cryoprotecting potential of DSSS 80 and DSSS 140

This study investigated the cryoprotecting potential of two new types of dextran sulphates of different molecular weights, 80 and 140 kDa. Moreover, the new compounds’ cryoprotective capacity was related to two other conventionally used types of dextran with molecular weights of 40 and 70 kDa, using a simple sperm cryopreservation procedure. As it can be seen from Table 4, both forward and total motility were lost completely after freezing and thawing regardless which cryoprotectant was used. Neither does the concentration of cryoprotectant in the storage solution appear to have influence on the lost post-thaw motility. Likewise, were both forward and total motility lost when the cells were cryopreserved in the absence of dextran, as it is shown in Table 5.

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Table 4. Recovery of sperm motility at various concentrations for different molecular masses of dextran and DSSS used as cryoprotectant in the cryopreservation of human spermatozoa.

Cryoprotective agent Pre-freeze forward motility (%) Post-thaw motility day 7 (mean %) 1.25 mmol/L Post-thaw motility day 7 (mean %) 2.5 mmol/L Post-thaw motility day 7 (mean %) 5.00 mmol/L Dextran 40 45 0 ±0 0 ±0 0 ±0 Dextran 70 45 0 ±0 0 ±0 0 ±0 DSSS 80 45 0 ±0 0 ±0 0 ±0 DSSS 140 45 0 ±0 0 ±0 0 ±0

Table 5.Recovery of sperm motility in the absence of dextrans.

Pre-freeze motility (%) Post-thaw motility day

7 (mean %) Controls (absence of

cryoprotectant) 45 0 ±0

3.2.2. Cryopreservation for 24 hours

In order to clarify, whether the lost post-thaw motility was due to the cells being frozen for too many days, a similar experiment, using the same procedures, was conducted. However, the cells were only cryopreserved for 24h. Like the previous experiment, both forward and total motility were totally lost regardless type of cryoprotective agent, concentration or in the absence of dextrans. Consequently, as the results are similar to those shown in Table 4 and 5, the results of this experiment are not presented in this section, but can be seen in Appendix 3.

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3.2.3. Verification of cryopreservation procedure

A third experiment was carried out, aiming to verify, that the lost post-thaw motility could be due to the cryopreservation procedure used in this study. This experiment was likewise conducted in triplicate, using only Dextran 40 as cryoprotective agent, at a 2.50 mmol/l concentration, which according to the study made by Kundu et al. [5] is the optimum concentration for Dextran 40 for cryoprotection of sperm motility. Instead of preserving the samples in a freezer at -80°C, one sample was placed in the LAF-bench in the laboratory at room temperature, another sample was placed in the refrigerator at 5°C, and the last sample was placed in a freezer at -9°C. The motility of the samples were then analysed every hour.

Figure 7: Human spermatozoa subjected to experiment of total motility loss over

time at three different temperatures. Data are expressed as the mean ± SD for three

samples each.

From figure 7 it is shown that the percentage of total motility, for the samples at room temperature, remains constant until 2h. After 2h. at room temperature, the motility starts to decrease, and after 5h. the total motility and viability is completely lost. -10 0 10 20 30 40 50 60 70 80 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 % Total motility /Viable ce lls Time (h.)

Room temp. Refrigiator

Freezer Lineær (Room temp.)

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Regarding the cells in the refrigerator, the total motility starts to decrease already within the first hour. After 3h. the viability of the cells in the refrigerator was entirely lost. Observing the cells in the freezer, the viability of the cells was completely lost already within the first hour. In other studies, [5,13] where spermatozoa were subjected to cryopreservation, the cells were placed in a computer-controlled bio-freezer, which gradually decreases the temperature over several hours. Thus, it was assumed that the issue with the results showing lost sperm viability in this study, was due to the applied freezing protocol. Because the available equipment in this study did not include a computer-controlled bio-freezer, it was decided to end the experimental part.

4. DISCUSSION

During this study, two novel cryoprotective agents were synthesized. They were analysed for determination of sulphur content and molecular weight. Moreover, their efficiency as cryoprotectant agents was tested in relation to cryorecovery on human spermatozoa. As well, their cryoprotective potential were compared to other conventional types of agents.

4.1. Synthesis and characterization of DSSS 80 and DSSS 140

4.1.1. Sulphur content

DSSS 80 and DSSS 140 were prepared several times each. The compounds chosen for this study, were those whose values of sulphur content and molecular weight came closest to their respective tolerance level. Moreover, the appearance and texture of the two compounds influenced the choice of use for cryopreservation in this study. The analyses showed (Table 2 and 3) that the sulphur content of the two compounds was above the tolerance level, 4.1% (DSSS 80) and 9.1% (DSSS 140). Temperature is a factor that has an influence on the reaction of sulphur to dextran. Hence, an explanation for the high content of sulphur could be due to high temperatures during the addition of reduced dextran to the chlorosulfonic acid mixture. To high temperatures during this reaction could lead to an over addition of sulphate-groups to the dextran. As this process reacts exothermically, it can be a challenge to control the reaction-temperature. Thus, an improved temperature control during the synthesis could yield the required sulphur content.

As mentioned in the previous section, sulphur is highly hydrophilic. This fact would in theory make dextran sulphate an advantageous cryoprotectant, because of the possible increased water affinity, which would further stabilize the cell membrane and aid in controlling the formation of ice crystals. If the tolerance level of sulphur content of 16-19% is respected, there is approximately two sulfonating species for each glucose monomer. This amount of sulphur provides optimum conditions for interactions with cells and stabilisation of proteins [11].

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Thus, if the sulphur content is not within the limited tolerance level, the interaction at bio-interfaces does not have optimal settings, which could influence the compounds effectiveness as cryoprotective agent.

A previous study made by Jagodzinski et al. [27], demonstrated that high molecular weight dextran sulphates are involved in the activation of immune cells, as the compounds increased the concentration of interleukin-8 in the medium of monocyte cell culture. Thus, it appears that excessive sulphur content negatively impacts human cells. However, as the results of this study were uncertain, possibly due to the applied freezing protocol, it is difficult to determine whether the excessive sulphur content of DSSS 80 and DSSS 140 affect their potential cryoprotective properties.

4.1.2. Molecular weight

Like the sulphur content, the molecular weight of the two compounds was not in good agreement with their respective tolerance level (Table 2 and 3). As mentioned in section 4.1.1., the variation of molecular weight could be because of changes in temperature during the synthesis of the compounds, since temperature is one of those factors that could influence the polymer composition. From Table 3, it is noticeable that the molecular weight of DSSS 140 is approximately 6000Da lower than Dextran 70 (70.000Da). As DSSS 140 was synthesised from Dextran 70, and therefore should have increased its molecular mass (due to the two sulfonating species for each glucose monomer) , it appears that Dextran 70 has cleaved. In the manufacturing process of the Dextrans, the desired molecular weight is obtained by hydrolysis in acid. Hence, a too high reaction temperature in the synthesis of DSSS 140, could have cleaved the Dextran 70, and as a consequence the molecular mass decreases.

Whether the deviation of molecular weight has impact on DSSS 80 and DSSS 140’s abilities as cryoprotective agents, is also difficult to conclude, as more experiments using the correct molecular weights of the two compounds, should be performed. Since no similar studies have been performed previously, it is challenging to evaluate, whether the molecular weight of dextran sulphate has influence on their possible cryoprotective properties.

According to a study made by Kundu et. al [5], there is a relation between recovery of sperm motility and the molecular mass of dextran, as the authors state that the cryorecovery decreases with increasing molecular mass. The study found that Dextran of 10 kDa offered the most effective cryoprotection, where the lowest was shown by the 2000 kDa Dextran. Moreover, the researchers state that the optimum concentration of the sugar polymer for cryoprotection is inversely related to its molecular mass. Therefore, it is possible that dextran sulphates of high molecular masses do not display superior cryoprotective properties, when compared to conventional types of cryoprotectants.

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During freezing in cryopreservation, ice crystals are formed, but it is the formation of ice crystals in the cytoplasm that are thought to cause damage on the cell membrane, and thereby leading to cell death [28]. The formation of ice crystals is thought to occur in several steps. First, single ice crystals form, from five water molecules that interact via hydrogen-bonds to formation of tetrahedral arrangements [29]. Next, the unit ice crystals unite to form larger crystals, also through hydrogen-bonds.

The exact mechanism for cryoprotection of cells is not well founded. However, it is hypothesized that cryoprotectants function through hydrogen-bonds formed between their –OH groups and the oxygen atoms of the phospholipid head groups in the cell membrane [30,31]. Moreover, it is believed to be a possibility that dextrans, due to their high molecular mass, cause mechanical obstruction during the formation of large ice crystals. Thus, it can be speculated that a very high molecular mass might cause too much obstruction on the cell environment, cause changes in osmotic pressure etc. Therefore, dextrans of lower molecular weights might be more beneficial for the purpose of cryopreservation.

As Dextrans have many -OH groups, they might form a layer on the sperm surface. But a previous study made by Bamba and Miyagawa [32] showed that hydrophobic aromatic compounds function as cryoprotectants for boar spermatozoa. The fact that these compounds have cryoprotective properties, despite that they do not have –OH groups, and thus are incapable of forming hydrogen bonds with the phospholipids of the cell membrane, suggest that the proposed cryoprotective mechanism of cryoprotectants through hydrogen bonding might be incorrect. These observations imply that more cryopreservation studies should be made, so that the exact mechanism can be comprehended, and thereby aid the design of future cryoprotective agents.

4.2. Effect of dextrans on cryopreservation of spermatozoa

The aim of this study was to investigate the effect of two novel cryoprotectants on cryopreservation of sperm cells. Furthermore, the aim was to find an optimum concentration for the two compounds in relation to cryorecovery. Moreover, this study meant to compare the cryoprotective efficiency of the two new polymers to Dextran 40 and Dextran 70. Other studies have demonstrated the cryoprotective potential of dextran of various molecular masses on mammalian cells, but this study was the first of its kind to investigate if dextran sulphates can act as cryoprotective agents.

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Based on the results in this study (Table 4, which shows 0% viable cells regardless compound or concentration) the effect of DSSS 80 and DSSS 140 on cryopreservation cannot be validated nor compared to other cryoprotective agents. Since previous studies (1,5) have proven Dextran 40 and Dextran 70 to be able to cryoprotect sperm cells, the results indicate that the lost post-thaw motility was not due excessive time for the cells in the freezer. And while the sperm cells were provided from different male participants, the loss of viability could not be due to the property of the testing material. Thus, it indicated that the lost post-thaw motility was due to the used cryopreservation procedure. This indication was further enforced, as the results (section 3.2.3.) show that cells in the freezer die within the first hour.

Figure 8: The formation of intracellular ice, osmotic balance and level of

hydration is dependent on the cooling rate [33].

Freezing cells include intracellular and extracellular multifaceted actions that are not completely understood [34]. During the freezing procedure, ice forms on both sides of the cell membrane. When ice form on the extracellular side of the cell, water diffuses out of the cell. This leads to dehydration, shrinkage and finally cell death. In case too much water remains inside the cells during the freezing procedure, intracellular ice crystals form. This event damages the organelles and

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As it can be seen from figure 8, rapid cooling minimizes the solute imbalance between the internal and external side of the cell, but the disadvantage is that more intracellular ice is formed. In slow cooling, water from the intracellular side of the cell diffuses out of the cell, leading to dehydration and shrinkage. Thus, if cells are to be successfully frozen, the cooling rate should be slow enough to prevent intracellular ice formation, but fast enough to prevent dehydration and shrinkage. In this study, the accessible equipment for cryopreservation was limited. So, the cryopreservation protocol, developed for this study, was a combination of previous studies and available equipment. In the successful cryopreservation study on goat spermatozoa, reported by Kundu et al. [5], the cooling rate was controlled by a bio-freezer, so that the temperature decreased slowly over time. In another recent study published by Rajan et. al [26], mouse fibroblast cells were successfully cryopreserved, as the cells were suspended in the cryopreservation solution in a vial and stored at -80°C without controlling the cooling rate. Thus, it appears that for cryopreservation to be successful, several factors including specific cooling rates for specific type of cells, type and optimum concentration of cryoprotectant and the sufficient equipment must be coordinated.

The motility of human spermatozoa is based on the specialized structure of the sperm flagellum, which consist of the axoneme (skeleton), the mitochondrias surrounding the axoneme and a thin cell membrane [35]. The sperm movements are activated by changes in the intracellular ion concentration. Ex. a rise in the pH activates ATPase that leads to a decrease in intracellular potassium, and thereby induces membrane hyperpolarization. But sperm movements can also be activated by an increase in calcium ion or cAMP [35].

The high molecular masses of dextrans make it impossible for it to penetrate the sperm cells [1,3,5]. Consequently, dextran only cryoprotects cells from the damaging formation of ice crystals on the extracellular side. This is why dextrans are commonly used for cryopreservation in combination with glycerol, as glycerol’s low molecular weight allows it to penetrate the cell membrane, and thus also protects the intracellular side of the cell.

Since the spermatozoa, in this study, were rapidly cooled without controlling the rate of cooling, it is most likely that the ice crystals on the intracellular side have damaged the cell membrane and/or organelles, to such an extent that the sperm cells eventually lost their motility and viability. And while the high molecular weight of dextrans do not allow for the compound to penetrate the cell membrane, this event could probably not have been avoided when using the cryopreservation procedure, which was applied during this study. Consequently, since the cells were not cryopreserved under optimum conditions, it is not possible to justly validate the potential cryoprotective properties of DSSS 80 and DSSS 140.

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5. CONCLUSIONS AND PERSPECTIVES

This study aimed to investigate the cryoprotective properties of two novel synthesized biopolymers, DSSS 80 and DSSS 140 on human spermatozoa. The main objective, for the synthesis of the compounds, was to be in accordance with the tolerance level of sulphur content (%) and molecular weight, which was specified in internal specifications at PKC. Furthermore, the aim was to validate the cryoprotective efficiencies of DSSS 80 and 140 and compare them to Dextran 40 and Dextran 70. In case DSSS 80 and DSSS 140 provided cryoprotection, the objective was moreover to investigate if there was a correlation between concentration of the compounds and motility of the cells.

The results showed that all motility was completely lost, regardless cryoprotectant, concentration, in the absence of dextrans or donor of cells. Further experiments were conducted, which indicated that the loss of motility was due to the applied freezing protocol. Thus, since the method used in study was not optimal for cryopreservation of sperm cells, it is not possible to determine whether DSSS 80 and DSSS 140 display cryoprotective properties or not, nor if they are superior to conventional types of cryoprotectants.

In order to properly be able to conclude on the subject, several factors are essential; firstly, the specifications of the compounds should be in agreement with the tolerance level in order to avoid bias due to the content. Secondly, since the formation of intracellular ice, osmotic balance and level of hydration is dependent on the cooling rate, the right freezing protocol must be used. This could be done by e.g. using a computer-controlled bio-freezer, which other successful studies have proven effective for the use in cryopreservation of spermatozoa of different species. Lastly, more experiments should be conducted, using several different types of dextrans and dextran sulphates at additional various concentrations. Also, the dextrans should be used in combination with penetrating cryoprotective agents, such as DMSO and glycerol.

As previous mentioned, cryopreservation is of great importance for treating e.g. infertility. Thus, not only spermatozoa but also oocytes and embryos could be subjected to cryopreservation using dextrans as cryoprotective agents. Furthermore, other types like stem cells, tissue etc. could be included in the experiments. By this, the cryoprotective efficiency and potential of DSSS 80 and DSSS 140 could rightly be analysed and validated, and prospectively aid treating infertility.

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6. Acknowledgements

I would like to address an acknowledgement to my supervisor Carsten Johnsen, who provided great help and support when conducting the project. Moreover, I would like to thank Christian Sørensen, Rie Meyer Scheuermann and Mona Nielsen for performing the analysis of DSSS 80 and DSSS 140. Also, I would like to say my appreciation to the employees at PKC, for their incurring and warm sense of humour.

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REFERENCES

1. Pharmacosmos. Cryoprotecting agent, cryoprotecting and cryopreserved compositions, uses thereof, and methods of cryopreservation. Patent number: CA2891198A1.

2. Pegg D. (2007). Principles of Cryopreservation. Methods Molecular Biology. 2007;368:39-57.

3. Shukla A and Tiwari R. (2011). Biomedical Engineering and Information systems: Technologies, Tools and Applications. Medical Information Science Reference. ISBN 978-1-61692-004-3.

4. Curry M. (2007). Cryopreservation of Mammalian Semen. Methods Molecular Biology. 2007;368:303-11.

5. Kundu C.N., Chakrabarty J, Dutta P, Bhattacharyya D, Ghosh A and Majumder GC (2002). Effect of dextrans on cryopreservation of goat cauda epididymal spermatozoa using a chemically defined medium. Reproduction 2002 Jun;123(6):907-13.

6. The scientist (2013). Freezing cells

>https://www.the-scientist.com/infographics/freezing-cells-39834< HTML (2018-01-25)

7. Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature. 1949; 164:666.

8. Lovelock J.E., Bishop M.W. (1959). Prevention of Freezing Damage to Living Cells by Dimethyl Sulphoxide. Nature 183, 1394-1395.

9. Matsumura K, Bae J, Hyon S. (2010). Polyampholytes as cryoprotective agents for mammalian cell cryopreservation. Cell Transplant. 2010;19(6):691-9.

10. Sand O, Sjaastad Ö, Haug E and Bjaalie J (2008) Menneskets anatomi og fysiologi. Gads Forlag 2nd ed. ISBN 978-87-12-04298-3.

11. Belder A.N. (2003). Dextran. Amersham Biosciences Edition AA.18-1166-12.

12. Socratic (2018). Biology. >https://socratic.org/questions/what-is-the-main-component-of-the-cell-membrane-why-is-it-the-main-component< HTML (2018-06-02)

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13. Kundu C.N., Das K and Majumder G.C (2001). Effect of Amino Acids on Goat Epididymal Sperm Cryopreservation Using a Chemically Defined Model System. Cryobiology 41, 21-27.

14. Matsumura K, Hyon S. H. Polyampholytes as low toxic efficient cryoprotective agents with antifreeze protein properties. Biomaterials 2009, 30, 4842-4849. 15. Figgs G (2013). Dextran: Chemical structure, Applications and Potential Side Effects. Nova Science Pub Inc. ISBN 978-1629489605.

16. Breed R.S. (1957). Manual of Determinative Bacteriology. Williams and Wilkins Co. 7nd ed. ISBN 978-0683006032.

17. Internal document, PKC.

18. World Health Organisation (WHO) (2015). 19th WHO Model List of Essential Medicines (April 2015). WHO Essential Medicines and Health Products Annual Report 2016.

19. Brimacombe J.S. (1973). Carbohydrate Chemistry, Specialist Periodical Reports. The Chemical Society Volume 6.

20. Caki M, Glisic S, Nicolic G, Nicolic G.M., Caki K and Cvetinov M. (2016).

Synthesis, characterization and antimicrobial activity of dextran sulphate stabilized

silver nanoparticles. Journal of Molecular Structure 1110:156-161

21. Konc J, Kanyó K, Kriston R, Somoskői B and Cseh S (2014). Cryopreservation of Embryos and Oocytes in Human Assisted Reproduction. Biomed Res Int. 2014; 2014: 307268.

22. Steirteghem A. (2009). The International Committee for Monitoring Assisted Reproductive Technology (ICMART) and the World Health Organization (WHO) Revised Glossary on ART Terminology, 2009. Human reproduction, Volume 24, Issue 11, 1.

23. Internal document, PKC.

24. Mandal M, Banerjee S and Majumder GC (1989). Stimulation of forward motility of goat cauda epididymal spermatozoa by a sperm glycoprotein factor. Biology of reproduction 41 983-989.

25. Roy N and Majumder GC (1989). Purification and characterization of an antisticking factor from goat epididymal plasma that inhibits sperm-glass and sperm-sperm adhesion. Biochimica et Biophysica Acta 991 114-122.

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26. Rajan R, Hayashi F, Nagashima T and Matsumura K (2016). Toward a Molecular Understanding of the Mechanism of Cryopreservation by Polyampholytes: Cell Membrane Interactions and Hydrophobicity. Biomacromolecules 2016, 17, 1882-1893.

27. Jagodziński P.P., Lewandowska M, Januchowski R, Franciszkiewicz K, Trzeciak WH. The effect of high molecular weight dextran sulfate on the production of interleukin-8 in monocyte cell culture. Biomed Pharmacother. 2002 Jul;56(5):254-257.

28. Mazur P (1984). Freezing of living cells. Mechanism and implication. American Journal of Physics 243, 125-142.

29. Luyet B and Rapatz G (1958). Patterns of ice formation in some aqueous solutions. Biodynamics 8, 1-68.

30. Crowe JH, Whittam MA, Chapman D and Crowe LM (1984). Interaction of phospholipid monolayers with carbohydrates. Biochemia et Biophysica Acta 769, 151-159.

31. Woelders H (1997). Fundamentals and recent development in cryopreservation of bull and boar semen. Veterinary Quarterly 19, 135-138.

32. Bamba K and Miyagawa N (1992). Protective action of aromatic compounds against cold-shock injuries in boar spermatozoa. Cryobiology 29, 533-635.

33. Biological Industries (2018). Cryopreservation Guide. >https://www.bioind.com/worldwide/cryopreservation-guide< HTML (2018-05-23)

34. Radojcic L, Vukotic-Maletic V and Balint B (1998). Current knowledge on cryopreservation of spermatozoa, ovum cells and zygotes. Medical Pregl 51, 29-36.

35. Luconi M and Baldi E (2003). How do sperm swim? Molecular mechanisms underlying sperm motility. Cellular and Molecular Biology (Noisy-le-grand). 2003 May; 49 (3): 357-69.

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Appendix 1

Information letter

Project title: Effect of Dextrans on Cryopreservation of Human Spermatozoa Study manager: Fie Barbara Nordskov Harder

E-mail: fieharder@gmail.com Studying at Malmö University, Faculty of Health and Society, S-205 06 Malmö,

Phone +46 40 665 70 00 Education:

Biomedical surface science Level: Master

Involuntary childlessness is a disabling problem. Worldwide is it estimated that 12-28% of all heterosexual couples experience involuntary childlessness for at least one year. 20-30% of infertility cases are due to male infertility, which is mostly due to deficiencies in the semen. Treatment of infertility can include a technique, where a single healthy sperm directly is injected into the mature egg. In case of numerous tries, it can sometimes be necessary to freeze the semen. But the disadvantage of freezing semen is that it in some cases can decrease the viability and motility.

The aim of this study is to see if a new type of sugar compound, named dextran, can avoid that the semen’s motility and viability is reduced after freezing. Therefore, we need healthy male volunteers, who will help giving the specimens, and thereby hopefully in the future we can help unwillingly childless couples.

The study will be carried out during spring 2018, where we will need one sample from healthy male volunteers, aged 18-50. The participants will be selected by age, number of children, healthiness etc. The timescale for respondents is two weeks, and there are no risks involved in taking part. The sperm sample can be made at your own home. Glass containers for the sperm will be provided for you, but the sperm sample will be needed latest 1 hour after ejaculation. No money, or any other forms of goods will be provided though. Nor will the results of your semen analysis be specified.

Your participation in the study is completely voluntary, and you can stop participating at any time and no explanation is necessary. We strive to guarantee confidentiality in the study in that no unauthorised person may have access to the material. The material is stored so that it is only accessible for the individual leading the study, and once the analysis is completed the sperm sample will be destroyed. In the reporting of results in the form of a master degree project paper at Malmö University/or in another form of publication, the respondents will be unidentifiable and it will not be possible to link the results to individuals.

If you have any further questions regarding the study, you are most welcome to ask or send an email.

Hereby, are you willing to participate in this study?

_________________________________________ Signature and date

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Appendix 2

Cryopreservation protocol

Samples must be collected latest 1 hour after ejaculation. Pre-freeze

Spermatozoa

Assumption: approximately 10 x106 cells pr. ml. The plasma is added in order to avoid sperm adhesion to glass.

Plasma

1. Centrifuge the sample until the pellet and plasma separates

2. Determine motility by incubating 50µL cells with 50µL plasma and 50µL medium at room temperature for 1 min.

3. Inject 10µL of sperm suspension into a haemocytometer and determine total motility in percentage.

Cryopreservation procedure

Freezing

1. Dissolve the specific concentration of respective dextran in medium. 2. Slowly add 50µL of the semen pellet together with 50µL plasma to 1ml

cryopreservative solution, mix properly. 3. Seal the open end of the tubes.

Cooling process/Freezing protocol

Freeze the samples (-80°C) in triplicates for 7 days.

Thawing

1. Plunge the tubes into a 37°C water-bath for 2-3min, then disperse the spermatozoa in medium.

2. Centrifuge the samples until the pellet and plasma separates. 3. Resuspend the pellet, plasma and medium in a 1:1:1 ratio.

4. Mix the sperm suspension with trypan blue in a 1:1 ratio, mix properly. 5. Inject 10µL of sperm suspension into a haemocytometer and determine

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Appendix 3

Table 6. Cryopreservation of human spermatozoa for 24h. Recovery of sperm motility at various concentrations for different molecular masses of dextran and DSSS used as cryoprotectant. Cryoprotective agent Pre-freeze forward motility (%) Post-thaw motility 24h (mean %) 1.25 mmol/L Post-thaw motility 24h (mean %) 2.5 mmol/L Post-thaw motility 24h (mean %) 5.00 mmol/L Dextran 40 43 0 ±0 0 ±0 0 ±0 Dextran 70 43 0 ±0 0 ±0 0 ±0 DSSS 80 43 0 ±0 0 ±0 0 ±0 DSSS 140 43 0 ±0 0 ±0 0 ±0

Table 7. Cryopreservation of human spermatozoa for 24h. Recovery of sperm motility in the absence of dextrans.

Pre-freeze motility (%) Post-thaw motility 24h

(mean %) Controls (absence of

Figure

Figure 3: Structure of the cell membrane bilayer, consisting of two inverted   phospholipid layers, in which the non-penetrating cryoprotectants can form  hydrogen bonds with the extracellular side  [12]
Figure 4: Chemical structure of Dextran  [17] .
Figure 5: Molecular structure of Dextran Sulphate Sodium Salt  [20] .
Table  1.  The  various  concentrations  of  cryoprotective  agents  used  for  cryopreservation of spermatozoa
+5

References

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