GHENT UNIVERSITY

FACULTY OF VETERINARY MEDICINE

Academic year 2016 – 2017

SEMEN PREPARATION FOR EQUINE ICSI – A COMPARISON OF COMMONLY

USED PROCESSING METHODS

by

Sander DAMEN

Promotors: Prof. dr. Ann van Soom Research Report

Dr. Katrien Smits as part of the Master’s Dissertation

© 2017 Sander Damen

DISCLAIMER

Universiteit Gent, its employees and/or students, give no warranty that the

information provided in this thesis is accurate or exhaustive, nor that the content

of this thesis will not constitute or result in any infringement of third-party rights.

Universiteit Gent, its employees and/or students do not accept any liability or

responsibility for any use which may be made of the content or information given

in the thesis, nor for any reliance which may be placed on any advice or

information provided in this thesis.

GHENT UNIVERSITY

FACULTY OF VETERINARY MEDICINE

Academic year 2016 – 2017

SEMEN PREPARATION FOR EQUINE ICSI – A COMPARISON OF COMMONLY

USED PROCESSING METHODS

by

Sander DAMEN

Promotors: Prof. dr. Ann van Soom Research Report

Dr. Katrien Smits as part of the Master’s Dissertation

© 2017 Sander Damen

PREFACE

This research report represents the second and final part of the author’s Master’s Dissertation at Ghent

University’s faculty of Veterinary Medicine. Acquiring new skills and competences during the synthesis

of a solid literature review were main objectives of the first part of the dissertation. This second piece of

work has been an interesting opportunity for the author to advance his knowledge on the subject and to

learn more about the elementary principles of research. Naturally, this has been a challenging, but at

the same time very enjoyable experience. Enjoyable, in particular, because of the pleasant collaboration

with the members of the Department of Reproduction, Obstetrics and Herd Health.

In special, the author would like to express his gratitude and appreciation towards the promotors of this

dissertation, prof. dr. Ann van Soom and dr. Katrien Smits, for their dedicated assistance during the

process of writing this work. The author would also like to thank dr. Bart Leemans, for assisting the

statistical analysis of the results of the experiments, and ms. Petra van Damme, for her technical support

and genuine advice.

Sander Damen

Ghent, 12th of February, 2017

CONTENT PREFACE

CONTENT

ABBREVIATIONS

ABSTRACT ............................................................................................................................................ 1

SAMENVATTING .................................................................................................................................. 1

INTRODUCTION ................................................................................................................................... 4

1. Artificial reproductive techniques in the horse ................................................................................... 5

1.1 Embryo transfer ........................................................................................................................ 5

1.2 In vitro fertilization (IVF) ........................................................................................................... 6

1.3 Intracytoplasmic sperm injection (ICSI) ................................................................................... 7

1.3.1 History of ICSI in equines ................................................................................................ 8

1.3.2 Oocyte collection for ICSI ................................................................................................ 9

1.3.3 Spermatozoa and ICSI .................................................................................................. 10

2. Semen preparation for ICSI in horses ............................................................................................. 11

2.1 Types of spermatozoa ........................................................................................................... 11

2.2 Preparation methods ............................................................................................................. 11

2.2.1 Washing method ............................................................................................................ 11

2.2.2 Swim-up method ............................................................................................................ 12

2.2.3 Density gradient centrifugation method ......................................................................... 12

2.2.4 Other preparation methods ............................................................................................ 13

2.2.3 Comparison of techniques ............................................................................................. 13

MATERIALS & METHODS .................................................................................................................. 15

1. General methods ............................................................................................................................. 15

1.1 Semen collection .................................................................................................................... 15

1.2 Use of media ........................................................................................................................... 15

1.2.1 Sp-CZB medium ............................................................................................................ 16

1.2.1 Calcium-free TALP medium .......................................................................................... 16

2. Experimental set-up ......................................................................................................................... 17

2.1 Washing .................................................................................................................................. 17

2.2 Swim-up .................................................................................................................................. 17

2.3 Percoll density gradient centrifugation .................................................................................... 18

3. Sample analysis .............................................................................................................................. 18

3.1 Computer assisted sperm analysis ......................................................................................... 18

3.2 Statistical analysis .................................................................................................................. 19

RESULTS ............................................................................................................................................. 20

DISCUSSION ...................................................................................................................................... 23

REFERENCES .................................................................................................................................... 26

ABBREVIATIONS

ART Artificial reproductive techniques

AI Artificial insemination

IVP In vitro production

ET Embryo transfer

OPU Ovum pick up

IVF In vitro fertilization

ICSI Intracytoplasmic sperm injection

ZP Zona pellucida

eFSH Equine follicle stimulating hormone

DMEM/F-12 Dulbecco’s modified eagle medium/Ham’s nutrient mixture F-12

hCG Human chorionic gonadotrophin

MACS Magnetic activated cell sorting

BSA Bovine serum albumin

TALP Tyrode’s albumin lactate pyruvate

Ca- TALP Calcium-free TALP

CASA Computer assisted sperm analysis

1

ABSTRACT & KEYWORDS

Artificial reproductive techniques in horses have become more important in recent years, and although

progressions are continuously made, some difficulties still remain. Conventional in vitro fertilization, like

performed in human and cattle, is yet not commercially applicable in horses. Intracytoplasmic sperm

injection (ICSI) is currently the best alternative. As ICSI requires only a singular viable sperm cell, semen

samples can be frozen, thawed and diluted, and then re-frozen to be used in ICSI later on. The protocol

to process these diluted semen samples prior to performing ICSI are yet to be determined. In this study,

the author compared three common processing methods to assess the best way to treat diluted sperm

cells prior to ICSI: basic washing, swim-up, and Percoll density gradient centrifugation. All methods were

executed twice, comparing two different media: Sp-CZB and calcium-free TALP (Tyrode’s albumin

lactate pyruvate). Results show the highest percentage of progressively motile sperm cells were

obtained by the combination of swim-up and Sp-CZB (19.7 ± 9.2%, P = 0.048 when compared to

washing, and P = 0.026 in comparison with the Percoll method), indicating this to be the combination of

choice. In absolute numbers, swim-up (in both media) recovers the lowest amount of total spermatozoa,

suggesting an adequate selection to occur. Swim-up based upon Sp-CZB as a medium is recommended

to be the processing method of choice in the protocol performed to treat diluted, frozen-thawed and re-

frozen sperm cells prior to the ICSI procedure.

Keywords: Horse – ICSI – Swim-up – Processing method – Artificial reproduction

SAMENVATTING

Artificiële reproductie is sedert tientallen jaren een belangrijk onderwerp in de landouwindustrie. In de

veehouderij tracht men immers al lange tijd om de hygiënische omstandigheden te verbeteren en

overdracht van ziekten, zoals venerische, te verminderen. Het gebruik van artificiële

reproductietechnieken (ART), zoals artificiële inseminatie (AI) bij runderen en varkens, is daar een

voorbeeld van. Toepassing van artificiële reproductietechnieken in de paardensector heeft eerder een

individueel karakter. Merries met medische problemen aan de genitalia of met een sportcarrière die nog

niet stilgelegd mag worden, zijn situaties waarin artificiële reproductie uitkomst kan bieden.

Hedendaags zijn er bij paarden hiertoe velerlei mogelijkheden: naast het wijdverspreide AI, dat in

veldomstandigheden wordt toegepast, kan in kliniekomstandigheden embryo transfer en ovum pick up

plaatsvinden, evenals intracytoplasmatische sperma-injectie (ICSI) als voorlopig enige praktisch

toepasbare vorm van in vitro fertilisatie (IVF) bij het paard. De conventionele IVF, zoals dit bijvoorbeeld

bij runderen veelvuldig wordt gedaan, stuit bij paarden nog op een aantal problemen en kan nog niet

commercieel worden aangeboden. In conventionele IVF worden spermatozoa van de hengst

gecoïncubeerd met een gematureerde eicel in geschikt medium, waardoor een potente spermacel in

vitro deze eicel kan gaan bevruchten. Hiertoe moet de spermacel een activatieproces zijn ondergaan,

‘capacitatie’, waardoor het mogelijk wordt uiteindelijk de zona pellucida en de plasmamembraan van de

eicel te penetreren. Dit proces kan in vitro nog niet naar voldoening herhaald worden.

2

ICSI is bij paarden hierdoor voorlopig het alternatief voor de in vitro productie van embryo’s. Bij deze

techniek wordt één spermacel rechtstreeks in het cytoplasma van de mature eicel geïnjecteerd, door

middel van een pipet onder laboratoriumomstandigheden. Het criterium van slechts één leefbare

spermacel zorgt ervoor dat in ICSI gewerkt kan worden met sperma dat na invriezen en ontdooien,

verdund mag worden en opnieuw ingevroren, waarna het alsnog geschikt blijft voor de ICSI procedure.

Dit biedt vele voordelen, zoals het gebruik van hengsten met suboptimale spermakwaliteit, zowel

kwalitatief (slechte motiliteit of morfologie) als kwantitatief (lage concentraties).

De optimale bewerking van verdunde en ontdooide spermacellen voorafgaand aan het uitvoeren van

de ICSI procedure is nog niet goed gekend. Optimalisatie van het protocol voor de behandeling van dit

verdunde sperma is dus nodig. Het onderzoek dat in deze thesis werd uitgevoerd hoopt hieraan bij te

dragen.

In de studie die werd uitgevoerd, werden drie veel gebruikte bewerkingsmethodes van sperma met

elkaar vergeleken, in de hoop inzicht te verwerven welke bewerking de best leefbare spermacellen

(hiertoe worden beschouwd, deze met de hoogste progressieve motiliteit) overhoudt na bewerking van

ontdooid en verdund sperma. De onderzochte methodes zijn enerzijds de basisbehandeling ‘wassen’,

waarbij verdunning met medium en centrifugeren volgt. Dit is eerder een opzuivering van het sperma

dan dat er selectie van hoog-motiele spermacellen plaatsvindt. Ten tweede werd de ‘swim-up’ methode

vergeleken, een techniek waarbij het spermastaal op de bodem van een buis wordt gedeponeerd, en

de meest motiele cellen het verst naar boven doorheen het medium zullen migreren (de bovenste laag

medium wordt geanalyseerd). De laatste techniek omvat de ‘density gradient centrifugation’ methode,

waarbij het spermastaal bovenop een medium (hier Percoll®) wordt gedeponeerd, en waarbij het

medium in een concentratie aanwezig is die toeneemt naar de bodem van de tube (dit kan gradueel

zijn, of door middel van twee lagen met een scherpe overgangslijn). In deze studie werd gekozen voor

twee lagen, met een 90% Percoll®-laag op de bodem van de tube, en een 45%-laag daarbovenop. De

meest motiele spermacellen zullen door hun hogere dichtheid via centrifugatie het meest naar de bodem

van de tube verplaatsen.

Elk van de drie methodes werd daarnaast uitgevoerd met twee verschillende media, in de hoop het

medium te kunnen vinden dat in combinatie met een bewerkingsmethode de beste spermacellen zou

overhouden. Het betrof Sp-CZB medium en calcium-vrij TALP (Tyrode’s albumine lactaat pyruvaat). Op

die manier ontstonden zes verschillende testsituaties .

De onderzoeksresultaten tonen een significant hoger percentage progressief motiele spermacellen voor

de swim-up methode op basis van Sp-CZB (19.7 ± 9.2% met een P-waarde = 0.048 wanneer vergeleken

met de wasmethode, en P = 0.026 vergeleken met de Percoll® methode). Op basis van calcium-vrij

TALP is er geen significant verschil tussen de drie technieken. De wasmethode levert absoluut gezien

de meeste spermacellen (142.0 en 67.1 miljoen·mL-1 in Sp-CZB of calcium-vrij TALP, respectievelijk).

De swim-up methode levert absoluut gezien de minste hoeveelheid cellen (0.6 en 2.4 miljoen·mL-1). Dit

is verwacht, aangezien enkel de meest motiele cellen naar boven zullen migreren, terwijl de

wasmethode de minst selectieve functie heeft en aldus verwacht wordt veel cellen, ook de minder

leefbare, over te houden.

3

Het onderzoek dat in het kader van deze thesis werd uitgevoerd, toont duidelijk de significant betere

resultaten van swim-up op basis van Sp-CZB aan, in vergelijking met de wasmethode en de Percoll®-

methode op basis van hetzelfde medium. De combinatie van deze methode met dit medium lijkt aldus

de beste om te komen tot een populatie spermacellen met zo hoog mogelijke progressieve motiliteit.

Het feit dat bij swim-up het absolute aantal cellen laag is, is geen onoverkoombaar punt, gezien de

vergevingsgezindheid van de ICSI procedure, waar immers slechts 1 potente spermacel vereist is.

Uiteraard is verder, meer uitgebreid en grootschaliger onderzoek aangewezen, eventueel bij meerdere

hengsten om de invloed van individuele factoren te minimaliseren. Desondanks hoopt de auteur van

deze thesis dat deze studie een bijdrage heeft kunnen leveren aan de optimalisatie van de

bewerkingsprotocollen van verdund sperma voor toepassing van ICSI bij paarden.

4

INTRODUCTION The development of artificial reproductive techniques (ART) has been an important research subject for

decades. Advances that were made in livestock species, for example developing artificial insemination

(AI) in cattle as a way to improve hygienic standards and to minimalize disease transmission1, can partly

be attributed to the motivation and efforts of breeders, who are continuously trying to increase

productivity. Nowadays, ART in cattle, including the in vitro production (IVP) of embryos, is even more

important, as genotype determination and genetic improvement of the herd have become priorities1.

Livestock species thus play an essential role in the advancement of our knowledge with regard to ART,

enabling animal scientists to perform empiric research that ultimately leads to better understanding and

implementation in humans2.

In contrast with livestock, reasons for using ART in humans and horses are more related to individual

problems with the genitalia causing sub- or infertility in both males and females3. Although ART costs a

fair amount of time and money, the use of these techniques in horses can be justified by the greater

economic value of the individuals, compared to livestock.

The first successful pregnancy in mares as a result of artificial insemination was already described in

the late nineteenth century3. Since then, AI has become a widely used ART in the equine breeding

industry and assisted reproduction in the horse has evolved greatly3,4. Apart from AI and in contrast to

other species like cattle, sheep and pigs, however, development of other ART has evolved only in recent

years and has taken a rather slow pace3. This has been postulated to be, in part, the result of the horse

industry having a lack of interest in the development of ART5. On the other hand, one should not

undervalue the differences between species with regard to their reproductive characteristics. The

physiology of the mare and the nature of her reproductive cycle can thus be considered when

investigating reasons for the slow evolution of ART in horses6. Given the fact that a mare has a

seasonally polyoestrous cycle, for example, there are several months each year in which her ovaries

are inactive (winter anoestrus) and AI and embryo transfer (ET) are impossible. In addition to this, oocyte

collection per ovary, either post mortem or in living horses using ovum pick up (OPU), is typically

relatively little compared to other species, like cattle, due to the strong attachment of the oocyte to the

follicular wall3,6.

Another concern in regard to assisted reproduction, is the fact that in vitro fertilization (IVF, see below)

has proven quite unsuccessful in horses; this in contrast with the livestock industry, where IVF is being

commercially exploited for years7. This all indicates the horse to be quite a challenging species for

researchers. Currently, with IVF not being an option in horses, the method of choice for the in vitro

production of equine embryos is intracytoplasmic sperm injection (ICSI)8. This technique has progressed

swiftly in the last decade until, having its advantages and disadvantages, it has grown to a commercially

applicable method for IVP of equine embryos. One of these advantages, that will be discussed more

elaborated later on, is the fact that ICSI requires less sperm cells to be successful – theoretically, only

one viable spermatozoon that is capable of doing the job is needed7. Therefore, with criteria less strict

than in other ART, clinical practitioners performing ICSI have been successfully using diluted semen

5

samples to produce embryos in the last years. The semen is thawed, diluted to a grade thought suitable

and then frozen again (at -80° Celsius) until needed for ICSI. Semen having low sperm numbers or

inferior quality, that in conventional ART like AI would not meet the qualitative requirements and would

be lost for artificial reproduction, can now be used with ICSI7.

Despite the technical improvements and the ongoing advancement of our knowledge with concern to

ICSI, the technique still is in need of more research to be able to optimize the procedure. A consensus

on how to adequately treat diluted semen samples prior to ICSI, thereby optimizing the sperm samples

in ways of selecting the most viable spermatozoa, seems non-existent.

In this dissertation, the author will try to contribute to this subject by investigating the influence of several

processing methods on the viability of diluted semen. It is hoped that the results of these experiments

will give more insight in the best way to treat diluted sperm cells prior to ICSI, and that this will ultimately

lead to a protocol most ideally for equine sperm, to guarantee that the most vital sperm cells are

preserved and selected to be used in the procedure.

First, this study will provide in an overview of the literature on the ART currently available in horses, with

special attention given to ICSI, as it is the current method of choice for the IVP of equine embryos and

the subject of the experimental set-up later on in this dissertation. Indeed, after discussing the literature

with regard to ART, the knowledge gap in concern to the several processing methods for the equine

semen will be situated. Finally, the study that was set-up to try elucidating this lack of information on

how best to treat diluted sperm prior to ICSI, will be described and discussed.

1. Artificial reproductive techniques in the horse

ART that are nowadays used in equine clinical practice include, apart from AI, ET, oocyte transfer and

ICSI, nuclear transfer, embryo biopsy (for genetic diagnosis) and vitrification of early embryos or

expanded blastocysts7. Those techniques most relevant for clinical practice will be overviewed in this

chapter.

1.1 Embryo transfer (ET)

Transferring embryos from a donor mare to an acceptor mare, by flushing the uterus and recovering the

embryo that was fertilized in vivo, has evolved into a common clinical procedure for modern equine

practitioners. Reasons for performing these transfers could be the individual value of mares and the

sportive aspirations of their owners, who perhaps would welcome a foal but do not want to postpone

any successes in sport, or do not want to expose their expensive horse to the (natural) risks of gestation

and birth. When performing ET in valuable mares, owners will be capable of increasing the total offspring

of those animals and will create a larger progeny than possible through insemination and normal

gestation of each mare.

6

Next to this, ET can be used when individual problems to the mare’s genital system impede a normal

gestation. For instance, when ovaria and oviducts are normal (and fertilization of the oocyte succeeds),

but implementation and carrying of the embryo proves impossible, due to local anatomical or

physiological abnormalities in the uterus, ET could be the technique of choice.

The mare has an oestrous period of 5-7 days and generally ovulates only one follicle at a time.

Superovulation, like in cattle, can be tried but is not as effective as in cows. It can be achieved by

treatment with equine pituitary extract, purified equine follicle stimulating hormone (eFSH) or

recombinant equine FSH7. The typical follicle ovulation rate will raise to three, although the embryo

recovery rate does not increase linearly9. Insemination is then supposed to occur as close to the moment

of ovulation as possible. In practical conditions, this means insemination preferably between 24 hours

before and 12 hours after ovulation. When fertilization succeeds, the embryo arrives in the mare’s uterus

approximately 6 days after ovulation (day 0) and will stay in a ‘migration phase’ until day 16. Embryo

recovery will be performed by repeatedly flushing the uterus, a technique that has been adequately

described by others7.

1.2 In vitro fertilization (IVF)

Although not yet commercially applicable in horses, and its relevance thus rather limited for the equine

practitioner until this moment, IVF will shortly be discussed, for its widespread application in humans

and cattle for the production of in vitro embryos indicates the major role of this technique in assisted

reproduction.

As the name already indicates, in this technique it is tried to fertilize the oocyte in an experimental,

controlled environment. A mature oocyte is co-incubated together with capacitated spermatozoa in a

medium that should facilitate the capacitation of the sperm cell. After this, binding of the spermatozoon

to the oocyte can occur, and finally, the penetration and conception. ‘Capacitation’ is the process of

activation of ejaculated spermatozoa, in normal circumstances occurring in the female genital tract, in

order to be able to bind the zona pellucida (ZP) of the oocyte10,11. This binding of the spermatozoon to

the oocyte induces, on the other hand, changes in the sperm that generally are referred to as the

‘acrosomal reaction’11. When this happens, the outer membranes of both sperm cell and oocyte can

ultimately fuse, which leads to incorporation of the sperm cell in the oocyte and conception. It is this

process that researchers want to induce in vitro when incubating an oocyte together with capacitated

sperm in an IVF set-up.

Until now, however, IVF has proven inconsistently repeatable in horses12. Only two case reports have

described foals from IVF, using OPU-collected oocytes that were matured in vivo and using

spermatozoa that were treated with calcium ionophores for capacitation12,13,14. In cattle, on the other

hand, IVF already has developed into a commonly applied ART, resulting in 13,780 transferrable bovine

embryos in 2015 on the European continent that were produced in vivo15,16. The first living IVF calve

even dates from 198117. Since then, major advances have been made, not only in cattle, but in other

farm animals as well on the subject of in vitro fertilization. IVF nowadays is a routinely applied artificial

7

reproductive technique in the modern breeding industry and improves the animal’s prolificacy per

cycle12.

Reasons for IVF to be problematic in equines seem multiple. Some authors postulate the reliance on

the ZP for hardening of the oocyte and reduced in vitro capacitation of equine spermatozoa to be main

limiting factors in this regard13. Others assume the attempt to fit equine IVF procedures in other species’

requirements (especially bovine) to be of major importance12. It was indeed proven that equine sperm

capacitation conditions can differ from the conditions that have been found suitable for other species;

this was observed when equine semen was subjected to other species’ requirements, like bovine’s or

porcine’s19,20. Researches report a low incidence of the acrosomal reaction (see above) following binding

of the ZP. Other local biological interactions are suggested to be contributing to the induction of this

acrosomal reaction, like local activity of hormones (progesterone) and other molecules (leptin) in the

follicular fluid or plasm membrane13. When breaching the ZP, high rates of fertilization and even

polyspermy were observed, indicating the zona to be of major importance in the failure of the IVF

protocol7. The capacitation of stallion semen in vitro, which facilitates the spermatozoa to penetrate the

ZP, is thus still not yet an efficient procedure and is subject of ongoing investigation. Until then, IVF will

not be able to fulfil the role of the commercial exploitable in vitro embryo production method that it does

in cattle nowadays. In the meantime, for horses, ICSI will continue to be the alternative, as will be

discussed later on.

1.3 Intracytoplasmic sperm injection (ICSI)

Given the disappointing repeatability of IVF in equines, in which the inability of spermatozoa to

adequately penetrate the zona pellucida (ZP) is recognized to be a major reason for failure of the

technique, researchers have put efforts in developing procedures that circumvent this interaction with

the zona. ICSI is an artificial reproductive technique that meets this criterion, by direct inoculation of a

single stallion spermatozoon into a mature (meaning, in metaphase II21) oocyte’s cytoplasm. This is

done by a micromanipulation procedure using little pipettes to fixate the sperm cell (pipette diameter 6-

8 µm) and oocyte (diameter 15-20 µm)14. The sperm cell is selected from a drop of ejaculate that is kept

in a medium of polyvinylpyrrolidone (having a higher viscosity, this product slows down the movement

of the spermatozoa). Selection is based on visual parameters like morphology and progressive motility.

Nonetheless, it should be mentioned that nonmotile sperm cells have been found capable of producing

blastocysts22, indicating the ‘motility’ parameter not to be absolute for successful ICSI.

After selecting a sperm cell, it is immobilized mechanically or with Piezo pulses that disintegrate the

cell’s plasm membrane. This ensures the sperm cell’s immotility and facilitates the release of cytosolic

factors that are important in oocyte activation21. The sperm cell then is ready to be injected into the

oocyte’s cytoplasm, for which it will need to penetrate both the ZP and the oolemma. This can be done

through several methods (see below)23.

8

ICSI can be a method of choice in case a mare suffers from subfertility and therefore is incapable of

delivering a vital oocyte into the oviduct to be fertilized. Local problems in the oviduct could be a reason

for this subfertility, for instance. Uterine abnormalities, like chronic persistent endometritis, and

dysfunctionalities at the level of the cervix, are also postulated as situations in which ICSI might be of

help14. Next to this, a major advantage of ICSI is the requirement of only one single competent sperm

cell to perform the procedure, which means that the technique can be used for stallions with low quality

semen or, quantitatively, with low numbers of spermatozoa in their semen7. This is in contrast with

conventional IVF that, assumed it would be applicable in horses, would require a population of sperm

cells competent of migrating to the oocyte and penetrating its ZP and oolemma.

Disadvantages with regard to ICSI are the requirement of great expertise of the operator performing the

procedure and the need of rather expensive and specific equipment, which makes the total procedure

quite costly, time-consuming and complex.

1.3.1 History of ICSI in equines

The first pregnancy after ICSI in horses was announced by Squires et al. in 199621,23,24. Due to difficulties

in the beginning to develop blastocysts in vitro after the ICSI procedure, the first studies used surgical

transfer of the sperm-injected oocytes into the oviducts of recipient mares7. Later on, as knowledge in

this regard extended, researchers were found able to attain repeatable in vitro embryo development

rates of 25-35% when working with a culturing medium, in mixed-gas environment, that normally was

used to support oviductal cells (Dulbecco’s modified Eagle’s medium/Ham’s F-12, or ‘DMEM/F-12’)7,22.

These rates are in line with the typical embryo development rates in cattle and significantly higher than

the disappointing blastocyst development rates of equine embryos that were obtained until then, which

were typically <10%21.

Techniques for performing the ICSI procedure have evolved over time. Originally, the conventional ICSI

procedure was characterized by the use of a bevelled injection pipette to mechanically penetrate the

zona pellucida and plasm membrane of the oocyte, subsequently followed by injection of an immobilized

sperm cell23. In this conventional technique, the tail of the spermatozoon is pressed against the bottom

of the dish to immobilize it26. This treatment damages the sperm plasm membrane and facilitates the

release of sperm-borne oocyte-activating factors27. Conventional ICSI has also been associated with

damage of the oocyte, through mechanical deformation of the ZP, thereby exposing the oocyte’s plasm

membrane to negative pressure during the injection phase23.

An alternative technique was introduced in 2002 when researchers used a Piezo drill (Prime Tech Ltd.,

Japan) to penetrate the ZP. This Piezo drill-assisted ICSI was associated with improved cleavage rates

and was found to be less traumatic than the conventional technique26,28. With this method, Piezo pulses

are used both for the ZP and the oolemma to penetrate these structures. It was found that when using

mercury in the injection pipette, the lateral oscillations of the pipette are bigger, which significantly

contributes to the penetration of the ZP29. The exact mechanism on how mercury influences the lateral

oscillations of the pipette is still unknown. Despite this positive interaction of mercury, however, the

popularity of injection pipettes with the metal has decreased over time, for mercury has been associated

with toxicity in humans29.

9

After penetrating the oolemma, fusion of the nuclei of the sperm cell and the oocyte can occur, ultimately

leading to the zygote. After conceptus, it is the sperm cell that induces the cleavage of the oocyte. There

has been some controversy over the necessity for chemical activation to induce pronucleus formation

after ICSI, but studies with frozen-thawed sperm have shown that good pronucleus formation rates can

be obtained without activation treatment in the horse, thus putting this treatment in a better

perspective26,30.

1.3.2 Oocyte collection for ICSI

Sources of oocytes in horses can be excised ovaries (from either deceased or live mares) or

preovulatory and immature follicles in live mares. The optimal oocyte for embryo production would be

the natural ovulated, with the oocyte from preovulatory follicles of live mares being the best alternative

in comparison6.

When collecting preovulatory oocytes from follicles in live mares, maturation advances in vivo. In clinical

practice, this can be accelerated by administration of hormone derivatives. A substance regularly applied

in this regard is human chorionic gonadotropin, also referred to as ‘hCG’ (Chorulon®, MSD Animal

Health BVBA, Brussels, Belgium31), which is extracted from women’s urine and administered to induce

ovulation in mares. Ovulation often occurs approximately 36 hours after treatment of the mare with hCG.

An alternative for hCG can be found in derivatives of gonadotropin releasing hormone (GnRH).

Deslorelin, for example, is a GnRH-analogue commonly used in veterinary practice. It can be

administered to the mare as a subcutaneous implant. Alternatively, the mare can be treated with both

medicaments simultaneously, as Carnevale et al. found that after administration of hCG alone, follicles

sometimes fail to react, whereas treatment with both hCG and deslorelin adequately induced maturation

of the follicles32,33.

These substances induce a swift maturation that ultimately leads to ovulation, so the oocytes need to

be collected from the follicle before this happens (at least 24 hours after stimulation, but no more than

35 hours afterwards7). In general, a follicle is recognized to be preovulatory when its diameter meets 35

mm or more, but this depends on factors like breed and individual variation. Most of the time, only one

preovulatory follicle can be obtained per aspiration cycle, sometimes two, but the recovered oocyte has

matured in vivo and the developmental competence is high34.

Apart from collecting oocytes that were matured in vivo after medical stimulation, it is also possible to

collect immature oocytes from the follicles of live mares followed by in vitro maturation. Immature equine

oocytes have a firm and dense layer of cumulus cells surrounding the oocyte. These cells provide in a

firm attachment of the oocyte to the follicular wall35. As a result, oocyte recovery rates are known to be

lower than those after collecting mature oocytes (often less than 50%, compared to 65-80% in

preovulatory follicles7,35). When performing ovum pick up (OPU) to collect immature oocytes, all the

follicles with a diameter of at least 5 mm are aspirated14. In this technique, the follicles are aspirated

under assistance of ultrasound technology, in general under transvaginal approximation (the

transabdominal approach is mostly applied post mortem in slaughterhouses) of the ovary14. The 5 mm

diameter seems a minimum for efficient oocyte collection, in agreement with results of a study conducted

10

by Galli et al. in 20073. One of the advantages of collecting immature oocytes, is that the mare not

necessarily has to be in preovulatory stage of oestrus (which would be the case for collecting mature

cells). Hence, the time interval in which immature oocytes can be collected is much wider. Research

has even postulated that it is possible to collect oocytes from mares in winter anoestrus, as results

showed no evidence of difference in maturation competency that would be related to seasonality or the

mare’s cycle stage36. Another advantage of collecting immature oocytes, is the fact that more immature

follicles can be aspirated per cycle, although the oocyte recovery rate is relatively low. Jacobson et al.

show, for example, an oocyte recovery rate of 54% after aspiration of immature follicles34.

A third way to obtain oocytes is by collecting immature follicles from excised ovaries. Extraction from

ovaries obtained in slaughterhouses is mostly done for research purposes in experimental set-ups.

Ovaries can also surgically be removed in case a mare has to be euthanized for other reasons and the

owner wants to collect oocytes for potential offspring5,35.

Excised ovaries need to be handled and transported with care and require optimal transportation media

to facilitate the best conditions for sufficient oocyte recovery. Details on the procedure of collecting

oocytes from excised ovaries and on media best suitable transportation have earlier been provided by

others and will not further be discussed in this study35.

1.3.3 Spermatozoa and ICSI

Given the vital role of the stallion’s sperm cell in performing the intracytoplasmic sperm injection,

research has been aiming at advancing our knowledge with regard to spermatozoa for quite a while. As

mentioned before, with ICSI only a single sperm cell is theoretically needed and used to complete the

procedure and, ultimately, obtain an embryo. This is undoubtedly a great advantage for stallions with

low quality semen or insufficient amounts of spermatozoa in their ejaculate. Those horses would have

low potential of producing offspring when using ART like AI, but this would not automatically apply to

them when using ICSI. It was already put forward that nonmotile sperm cells have been found capable

of producing blastocysts, which puts motility as an absolute benchmark in to perspective22. Indeed, it

was shown that development rates of oocytes are not correlated to the stallion’s fertility status in the

field37.

Next to this, a study conducted by Choi et al. in 2002 revealed that embryo development rates after

performing ICSI with frozen-thawed stallion spermatozoa were comparable to those obtained with fresh

equine sperm cells26. This means that developing embryos is possible with the use from stallions that

cannot provide in fresh spermatozoa. Moreover, it was shown that thawing and refreezing of semen

samples can be performed without negatively affecting the ability to initiate embryo development38,39.

Next to this, when recovered within 24 hours post mortem, it is even possible to collect spermatozoa

from the epididymis of the deceased or euthanized stallion40.

Given this essential role of the sperm cell in the process of ICSI (and ART in general), the importance

of extending our knowledge on this subject is indisputable. More elaborate research is required to live

up to this intention and advance our understanding and knowledge with regard to the best conditions to

maintain, manipulate and use spermatozoa in the ICSI procedure.

11

2. Semen preparation for ICSI in horses

As mentioned before, there is a necessity to adequately manipulate and prepare semen samples that

are supposed to be used for ICSI. Indeed, by processing the sperm cells prior to performing ICSI, one

could try to select the sperm cells with the highest viability based upon certain selection criteria (in this

study, motility parameters are considered to reflect the vitality of the spermatozoon). Hence, in this

chapter, before continuing to the experimental set-up and the results of the study that was performed,

possible preparations and processing steps after collection of the ejaculate will first be discussed.

2.1 Types of spermatozoa

Three types of semen can theoretically be used in equine ICSI: fresh, cooled or frozen-thawed41.

Research by Choi et al. suggested comparable embryo development rates after ICSI when using fresh

or frozen-thawed spermatozoa26. By all means, a first important step after collecting the semen is to

separate the seminal plasm from the vital sperm cells, in order to limit potential damage and reduction

of viability of the spermatozoa as much as possible. It is recommended to perform this separation as

soon as possible after ejaculation, preferably within 30 minutes42.

After separation of viable sperm cells from the plasm, one could continue to further process and prepare

the cells for freezing, or directly use the fresh spermatozoa and continue to isolate the preferred sperm

cells prior to performing ICSI. A third option that has been showed successfully applicable in ICSI, as

previously described, is the double freeze-thaw cycle, in which frozen semen samples are thawed, then

further diluted using an extender until the optimal dilution is reached, after which refreezing can be

done38,39. In the study that was performed in this dissertation, it was chosen to use this diluted sperm

that had undergone an thawing and refreezing cycle (see below).

2.2 Preparation methods

Several sperm separation techniques have been developed to adequately prepare and select the sperm

cells most suitable for the ICSI procedure. These techniques rely on different working mechanisms and

use different sperm cell parameters.

2.2.1 Washing method

In this simplest of methods, a culture medium is added to the semen sample, and centrifugation is

performed twice to remove the extender (or seminal plasm, when using the washing method to wash an

fresh ejaculate). This technique thus acts more like a separation technique, rather than one that can be

used to select sperm cells. Hence, it is mostly used when the semen has optimal parameters and

selection of sperm cells (for example, based on motility or morphology) is of minor importance. As a

result of this low grade selection, a high yield of sperm cells is obtained43. A washing step can often be

12

implemented in the protocol of other preparation methods, like the swim-up technique, in order to purify

the semen sample prior to further processing. In literature, there seems consensus to address to this

method with the term sperm washing when speaking of it, whereas the term sperm preparation should

be used for the techniques that will be discussed hereafter44.

2.2.2 Swim-up method

Conventionally, this popular technique is executed by placing a semen pellet or liquefied semen sample

in a conical tube, overlaid with a culture medium of choice. This swim-up method is a migration-based

technique and relies on the intrinsic motility of spermatozoa.43 To enable the use of this capacity, the

tube with the semen sample and culture medium needs to be incubated in a 45° angle in the incubator

(37-38° Celsius), for a variable amount of time (often 30 to 45 minutes45). In principle, the swim-up

method is used to collect highly motile sperm cells and separate them from the less mobile46. By using

round-bottomed tubes, the interface area between semen and medium is maximized42.

The swim-up method is a relatively simple technique and requires no great costs, but the amount of

motile spermatozoa retrieved is considered to be quite low; in general, only 5-10% of the spermatozoa

initially subjected to the swim-up, are retrieved43.

2.2.3 Density gradient centrifugation method

A second migration-based technique is the density gradient centrifugation method. This technique

separates sperm cells based on their densities. In human, for instance, it is known that mature and

morphologically normal spermatozoa have a density of at least 1.10 g·mL-1, whereas immature and

morphologically abnormal sperm cells have a density between 1.06 and 1.09 grams per millilitre47.

Subsequently, after centrifugation, each spermatozoon will be located at the gradient level that is

associated with its density43. The highly mobile and morphologically normal sperm cells form a pellet on

the tube’s bottom (highest density). It has been showed that density gradient centrifugation yields higher

amounts of motile sperm cells than the swim-up technique. This technique is therefore appropriate for

sperm with suboptimal parameters (oligozoospermic or teratozoospermic samples).

Density gradients can be either continuous or discontinuous. In case of the former, a density gradient is

composed that increases from top to the bottom of the tube. In case of a discontinuous gradient, there

is a clear demarcation line between the two density layers. When using the latter, it is important to keep

in mind that the separated layers might blend into each other over time. It is therefore recommended to

use the density gradient within one hour43. This demarcation line that represents the border of the two

layers is known to be the location of the greatest amount of abnormal spermatozoa48.

Studies in the early 1960’s showed that colloidal silica was capable of separating sperm cells, and this

because of several intrinsic properties: due to being a mineral substance, interference through osmotic

effects when adding to the medium did not occur; secondly, it allowed high density media to be prepared,

which is useful, as sperm cells themselves are quite dense cells in their mature phase; and lastly, as a

colloid more than a solution, and therefore having a low viscosity, it did not heavily interfere with sperm

cell sedimentation44.

13

In 1978, Pertoft et al. commercialized modified colloidal silica, and called it ‘Percoll’ (Pertoft’s Colloid).

It is composed, in its purest form, of solid silica spheres that are coated with polyvinylpyrrolidone49. In

this original study by Pertoft, chemical analysis revealed a polyvinylpyrrolidone content between 11.4%

and 12.4%49. From the early 1980’s, Percoll evolved into the colloid predominantly used in human sperm

preparation, both in clinical circumstances and in concern to researching purposes. However, in 1996,

Percoll was withdrawn from the market for clinical use in humans by the manufacturer (Pharmacia

Biotech, Uppsala, Sweden)44. From then on, the manufacturer would only recognise the use of it for

research purposes. One of the reasons for this withdrawal was perhaps the suspicion that some batches

of Percoll contained high concentrations of endotoxin contamination, far above the levels permitted for

use in non-research situations44.

Nowadays, several alternatives for Percoll have emerged on the market, among which are PureSperm™

(Nidacon International AB, Göteborg, Sweden), Isolate™ (Irvine Scientific, Santa Ana, California, United

States) and SupraSperm™ (Origio, MediCult, Copenhagen, Denmark)44. In contrast to Percoll, which is

typically used in conjunction with a 10% buffer to create a 90% Percoll solution, these new preparations

are available as ready-to-use, isotonic media, based on colloidal silica covalently bound to silane

molecules (silanized silica). Many other products have lately become available, based upon the original

composition of these silanized silica44.

2.2.4 Other preparation methods

Apart from the preparation methods described above, some less common techniques are also applied,

for instance magnetic activated cell sorting (MACS) or glass wool filtration. MACS is a method capable

of separating apoptotic sperm cells from non-apoptotic. This capacity is based on recognizing changes

in the outer membrane of the apoptotic cell (highly specific antibodies bind to annexin V, indicating a

loss of integrity of the cell’s outer membrane)43.It has been suggested to use MACS as a preparation

technique in combination with, and following after, density gradient centrifugation, as results showed

higher motility percentages and a lower fraction of cells expressing apoptotic markers (annexin V-

negative spermatozoa) when performing density gradient centrifugation followed by MACS50.

Glass wool filtration, on the other hand, separates motile spermatozoa from unwanted fractions like

immotile sperm cells, leukocytes and debris43. This technique is also effective for processing semen with

suboptimal parameters. Research has showed that the glass wool filtration technique filtrates

approximately 87.5% of the leukocytes in the semen sample51. By doing so, sperm cell damage can be

decreased, for the leukocytes are known the be the largest producers of radical oxygen species in

semen.

2.2.5 Comparison of techniques

Despite specific advantages of some of the formerly mentioned preparation methods, and despite the

variety of processing techniques that nowadays exist, the density gradient centrifugation technique is

generally considered the method of choice by the majority of ART laboratories43,52.

With regard to ICSI, however, a general consensus on what preparation method should be preferred, is

non-existent. Indeed, the current situation seems to be that different laboratories use different

14

preparation methods prior to their ICSI procedure, based on their own considerations, habits, resources

or additional argumentations26,53. An evaluation of the influence of different preparation methods prior

to ICSI on the sperm cell parameters that are recognized to represent the spermatozoon’s fitness for

ART, would be valuable. As mentioned before, these parameters are mainly recognized to be the sperm

cell’s motility and morphology. By comparing the methods, and obtaining insight into their efficiency and

capability to obtain the most viable sperm cells (with adequate morphology and sufficient motility), this

knowledge might contribute to creating more of a consensus between laboratories and researchers in

the future.

The main of this study was to compare three of the basic and commonly used preparation methods for

ICSI in horses, by measuring sperm concentration and motility parameters (total percentage of motile

sperm cells, percentage of progressively motile sperm cells) of the sperm cells after processing the

semen samples, prior to the ICSI procedure. This was performed on diluted sperm (see below), for the

quantitative amount of sperm cells needed for ICSI is very low (theoretically, one), which gives ART

laboratories the opportunity to work with less concentrated semen samples. It would therefore be of

most value to investigate which processing method is best suitable based upon diluted sperm.

The methods that were chosen to be compared, are the basic washing method, the swim-up method

and the density gradient centrifugation method using Percoll. In addition, all three experiments will be

executed by using two different media, in order to elucidate the optimal combination of medium and

method in this regard: the first medium being a CZB medium for sperm culture (Sp-CZB), the second

being calcium-free TALP (Ca- TALP)54,55.

15

MATERIALS & METHODS

1. General methods

1.1 Semen collection

Fresh semen was recovered from a stallion using the facilities at the Department of Reproduction,

Obstetrics and Herd Health at Ghent University (Belgium). The stallion was owned by the university and

is commonly used to collect semen for research purposes.

The ejaculate was separated from the seminal plasm and prepared for freezing (in a -196° Celsius liquid

nitrogen storage tank), and then stocked at the faculty’s depot until needed for the experiment. This non-

diluted, unprocessed semen sample was used as a control. Furthermore, samples from the same

ejaculate were frozen, thawed and then diluted 50 times compared to the original sample. Then they

were re-frozen as described above. The experimental set-up was performed using this diluted,

unprocessed and re-frozen sperm. To facilitate a thorough comparison, motility parameters and sperm

concentration numbers of the unprocessed frozen-thawed semen samples of this stallion, both diluted

and non-diluted, were analysed (Table 1).

Table 1. Concentration rates and motility parameters of unprocessed frozen-thawed semen.

The straws that were used to freeze the semen, had a volume of 450 µL, containing typically 50-100

million sperm cells56.

1.2 Use of media

Each processing method was performed twice, using two different media in order to obtain more insight

into the influence of the medium used when processing the semen samples on the motility of the sperm

cells.

Total spermatozoa Motile sperm cells Progressively motile

sperm cells

Non-diluteda

Concentration (million·mL-1) 211.2 140.9 40.5

Percentage 100.0 66.7 19.2

Dilutedb

Concentration (million·mL-1) 61.1 45.3 7.6

Percentage 100.0 70.0 11.7

a The non-diluted sample was only separated from the seminal plasm and frozen in a nitrogen tank. b The diluted samples were obtained by diluting the former 50 times after thawing, after which they were re-frozen and used for further analysis later on.

16

1.2.1 Sp-CZB medium

The common CZB medium for sperm culture, shortly Sp-CZB, is modified from the original CZB medium

by eliminating NaHCO3 and glutamine and by changing the concentrations of NaCl and D-glucose

(Table 2)54. Bovine serum albumin (BSA) was added only short before executing the processing

methods, thereby resulting in the modified Sp-CZB composition as presented in the table. All CZB

components were purchased from Sigma Chemicals (St. Louis, Missouri, United States).

1.2.2 Calcium-free TALP medium

The second medium used in this experiment was Tyrode’s albumin lactate pyruvate (TALP). As for CZB,

some alterations to the TALP composition are made to optimise the medium for use together with

spermatozoa (Sp-TALP, Table 2)55. In addition to the composition of Sp-TALP, as displayed in the table,

it was chosen to use calcium-negative TALP (Ca- TALP), this because calcium has been proven to

facilitate the capacitation of the sperm cell in vitro57. This would lead to unwanted early modifications in

Table 2. Qualitative analysis of the modified Sp-CZB and Sp-TALP media (adapted from Reference 54, 55).

Components Sp-CZB

NaCl (mM) 116.7

KCl (mM) 4.83

KH2PO4 (mM) 1.18

MgSO4·7H20 (mM) 1.18

NaHCO3 (mM) –

CaCl2·2H20 (mM) 1.70

D-glucose (mM) 0.55

Sodium lactate (mM) 31.30

Sodium pyruvate (mM) 0.27

EDTA (disodium salt) 0.11

Glutamine (mM) –

BSA (mg·mL-1) 5.00

Sodium penicillin G (U·mL-1) –

Streptomycin (mg·mL-1) –

Phenol red (mg·mL-1) 0.001

Gentamicin (mg·mL-1) 0.025

MEM non-essential amino acids

solution (x100) –

Fetal bovine serum –

Osmolarity (mOsmol) 298 ± 3

Components Sp-TALP

NaCl (mM) 100.0

KCl (mM) 3.1

NaHCO3 (mM) 25.0

NaH2PO4 (mM) 0.3

Lactate (sodium salt) (mM) 21.6

CaCl2 (mM) 2.0

MgCl2 (mM) 0.4

HEPES (mM) 10.0

Pyruvate (mM) 1.00

Glucose (mM) –

Bovine serum albumin

(BSA) (mg·mL-1) 6.0

Penicillamine (µM) –

Hypotaurine (µM ) –

Epinephrine (µM) –

Gentamycin (µg·mL-1) 50.0

17

the membrane of the sperm cell. The mechanism of this capacitation is the interference of the calcium

ionophores with the plasma membrane, thereby stimulating a massive Ca2+ influx into the cytoplasm58.

The calcium ionophores bypass the local calcium-depending regulatory mechanisms and can cause

damage to the spermatozoon.

The Ca- TALP medium was stocked at -80° Celsius until needed for the experiment. Then it was allowed

to thaw at room temperature. All components of the TALP medium were purchased from Sigma

Chemicals (St. Louis, Missouri, United States).

2. Experimental set-up

Given the use of two different media for all three processing methods that need to be evaluated, a total

of six different protocols was performed: washing the semen sample with either Sp-CZB (1) or Ca- TALP

(2); executing the swim-up method using Sp-CZB (3) or Ca- TALP (4) as a medium; and lastly,

performing the Percoll density gradient centrifugation method with use of Sp-CZB (5) or Ca- TALP (6).

2.1 Washing

After thawing the semen sample in water (32° Celsius for approximately 30 seconds), washing was

performed by diluting the sample with 4 mL of medium (Sp-CZB in (1), Ca- TALP in (2)), then centrifuging

at x405g59 for ten minutes.

The supernatant was removed and the sperm pellet was washed again, as described above. After

another centrifugation at x405g for ten minutes, the

supernatant was removed and the sperm pellet was

ultimately resuspended in 30 µL of medium, ready for

analysis (see below).

2.2 Swim-up

The protocol that was followed to execute the swim-

up method was in accordance with Choi et al. who

adequately described the protocol in 200354. The

semen sample (one straw, approximately 450 µL)

was thawed (see 2.1), then placed at the bottom of a

pre-warmed (38.2° Celsius) conical tube containing 2 mL of medium, Sp-CZB in (3) and Ca- TALP in

(4). The deposition of the semen sample on the bottom of the tube was done by holding the tube in a

45 degrees angle, after which the semen was placed into the tube by using a 1 mL syringe with needle.

Subsequently, the tube was incubated in air at 38.2° Celsius for 20 minutes while maintaining the angle

Figure 1. In the swim-up method, the semen sample

is deposited at the bottom of the tube, which is held

under a 45 degrees angle.

18

of the tube to allow the swim-up of the spermatozoa to proceed (Figure 1). After this, the supernatant

with the migrated motile sperm cells (0.6 mL) was collected and placed in a new, smaller tube containing

1 mL of medium (Sp-CZB or Ca- TALP). This smaller tube was centrifuged at x327g for three minutes,

then the content was removed and the sperm pellet at the bottom of the tube was resuspended with 1

mL of medium. Re-centrifugation was performed, and the sample was washed and resuspended in 30

µL medium once again. The semen sample then was ready to be analysed.

2.3 Percoll density gradient centrifugation

In the density gradient centrifugation method, a 15 mL Falcon tube was

filled with 2 mL of a 90% Percoll solution. In a second tube, a 45%

Percoll solution was synthesized by adding 1.5 mL of medium (either

Sp-CZB or Ca- TALP in experiment (5) and (6), respectively) to 1.5 mL

of 90% Percoll in the tube. Henceforth, 2 mL of this solution was

aspirated in a glass pipette and carefully layered on top of the 2 mL 90%

Percoll solution in the first tube(Figure 2, left). To be able to maintain

the sharp demarcation between the two layers and to avoid mixing

them, the layering has to be performed very gently. After this, the semen

sample (thawed as described in 2.1) was placed on top of the Percoll

layers by using a 1 mL syringe with needle.

The tube was then centrifuged for 40 minutes at 38.2° Celsius at x720g.

Then, the supernatant was removed. The sperm pellet containing the

healthy and progressively motile sperm cells that migrated through the

Percoll gradient (Figure 2, right) was resuspended in 4 mL of medium, followed by centrifugation for ten

minutes at x405g. Finally, the supernatant was removed and the pellet was resuspended in 30 µL of

medium.

3. Sample analysis

3.1 Computer assisted sperm analysis

After performing all three methods with both media and thereby obtaining 6 samples, analysis was done

electronically by means of the so-called ‘computer assisted sperm analysis’ (CASA) system (Figure 3).

The system automatically counts the static and motile sperm cells and processes the counting to ready-

to-use data (concentration of spermatozoa, total numbers of sperm cells, absolute and relative numbers

of motile cells). The CASA system used in this experiment was owned by and present at the Department

of Reproduction, Obstetrics and Herd Health at Ghent University (Merelbeke, Belgium). The table of the

Figure 2. Representation of the

Percoll gradient before (left) and

after (right) centrifugation

(adapted from Reference 43).

19

microscope connected to the digital CASA system was

pre-heated to body temperature (38° Celsius) prior to

analysis of the samples. This was done to avoid cold

shock. Simultaneously, a separate heated table with the

same temperature (this was checked by placing an

analogue thermometer on the surface of the plate) was

used to keep the material (pipette tips, microscope slides)

warm when they were not immediately used.

3.2 Statistical analysis

The differences between methods were analysed using analysis of variance. Statistical analysis and

graph plotting was performed using SPSS version 20 for Windows (SPSS IBM, Brussels, Belgium).

Values of P < 0.05 were considered significant.

Figure 3. A pre-heated microscope, connected

to the CASA system, was used to assess the

motility of the sperm samples.

20

RESULTS

In experiment 1 and 2, performing the washing method with both media, a large amount of sperm cells

was recovered (142.0 versus 67.1 million·mL-1 spermatozoa for experiment 1 and 2, respectively, see

also Table 4). In contrast to this are the results obtained with the swim-up method, which show a total

amount of sperm cells of 0.6 and 2.4 million·mL-1 using Sp-CZB and Ca- TALP, respectively. The

absolute sperm recovery rates when using the Percoll density gradient technique seem intermediate in

this regard (Table 6).

When comparing the percentages of total progressively motile sperm cells of the replicates, one could

find that by using the washing method (experiment 1 and 2), the littlest percentage of progressive motility

in this study was obtained: 3.8 ± 1.3% for Sp-CZB, and 4.8 ± 4.7% progressivity when using the Ca-

TALP medium (Table 3). Although a little higher, there was no significant difference in the results

recovered with the Percoll density gradient method (5.6 ± 4.4% in experiment 5 and 6.8 ± 6.2% in

experiment 6) when compared to the other techniques for both media.

The total percentage of progressive motile sperm cells in Sp-CZB using the swim-up method was found

to be 19.7 ± 9.2%, whereas Ca- TALP and swim-up showed only 2.9 ± 1.8%. When comparing this

swim-up in Sp-CZB (experiment 4) with the other methods in the same medium, there was a significant

difference (P = 0.048 when compared to washing, and P = 0.026 when compared to the Percoll density

gradient method). There was no significant difference between the washing technique and the Percoll

method in Sp-CZB. When using the Ca- TALP medium, there was no significant difference between the

three processing methods at all (Figure 4).

Table 3. Results (in percentages) of experiment 1-6.

Total spermatozoa

(in %)

Motile sperm cells

(in %)

Progressive motile

sperm cells (in %)

Washing

(1) Sp-CZB 100.0 47.0 ± 16.9 3.8 ± 1.3

(2) Ca- TALP 100.0 25.0 ± 11.3 4.8 ± 4.7

Swim-up

(3) Sp-CZB 100.0 28.8 ± 6.6 19.7 ± 9.2

(4) Ca- TALP 100.0 16.2 ± 2.6 2.9 ± 1.8

Percoll density gradient

(5) Sp-CZB 100.0 25.6 ± 8.7 5.6 ± 4.4

(6) Ca- TALP 100.0 21.9 ± 10.2 6.8 ± 6.2

21

Figure 4. Visualisation of the percentages total progressively motile sperm cells (left) and total motile sperm cells

(right). When comparing the three media in Sp-CZB for progressive motility (dark grey, left), a significant difference

can be found between swim-up (B) and both the other media (A), washing (P = 0.048) and Percoll (P = 0.026).

Table 4. Results (in million·mL-1) of the washing method using Sp-CZB (1) or Ca- TALP (2).

No. of washing replicate Total spermatozoa

Motile sperm cells

Progressive motile

sperm cells

Experiment 1: Sp-CZB

1 143.9 50.5 4.2

2 140.0 41.9 3.4

Mean 142.0 46.2 3.8

Experiment 2: Ca- TALP

1 21.3 8.0 2.2

2 110.9 24.6 2.9

3 69.0 10.7 1.1

Mean 67.1 14.4 2.1

B

A

A

22

Table 5. Results (in million·mL-1) of the swim-up method using Sp-CZB (3) or Ca- TALP (4).

Table 6. Results (in million·mL-1) of the Percoll density gradient method using Sp-CZB (5) or Ca- TALP (6).

No. of swim-up replicate Total spermatozoa

Motile sperm cells

Progressive motile

sperm cells

Experiment 3: Sp-CZB

1 0.7 0.3 0.1

2 0.6 0.2 0.2

3 0.6 0.2 0.2

Mean 0.6 0.2 0.2

Experiment 4: Ca- TALP

1 1.3 0.2 0.1

2 1.1 0.2 0.0

3 4.8 0.9 0.2

Mean 2.4 0.4 0.1

No. of Percoll replicate Total spermatozoa

Motile sperm cells

Progressive motile

sperm cells

Experiment 5: Sp-CZB

1 48.9 12.1 1.8

2 25.0 3.7 0.7

3 50.8 20.8 4.2

4 51.4 13.7 6.9

5 64.0 13.5 2.1

6 47.1 12.2 1.0

Mean 47.9 12.7 2.8

Experiment 6: Ca- TALP

1 8.8 1.2 0.3

2 4.5 0.6 0.3

3 11.1 3.4 1.8

4 18.7 5.9 0.4

Mean 10.8 2.8 0.7

23

DISCUSSION

The aim of the study described in this dissertation, was to investigate the role of three different

processing methods for semen on viability parameters of diluted semen that is meant to be used in ICSI.

These methods were compared in two media to try elucidating which medium is best suitable to select

and recover the most viable sperm cells, in combination with the best processing method. The ultimate

goal would be to discover a combination of medium and processing method that would facilitate the

selection of diluted spermatozoa with high progressive motility numbers – assuming this to be a

parameter representing the viability of the sperm cell (see above). In accordance with the current

knowledge considered the ICSI technique, it is more important to collect qualitatively good sperm cells,

than to quantitatively recover large amounts of cells in absolute numbers, for only one viable

spermatozoon is theoretically needed to be able to fertilize an oocyte when using ICSI, and this is why

one has begun to use diluted semen and wants to optimize processing and selection protocols for these

diluted samples.

A major outcome of the results of this study, is the superiority of the combination of swim-up in Sp-CZB

medium, that was revealed by a progressive motility of nearly twenty percent. The large difference with

the other methods is intriguing. Indeed, it can be suspected that when performing a swim-up, only sperm

cells with an adequate progressive motility are capable to reach the upper region of the medium in the

tube. The same can be said for the Percoll density gradient method, however. The results indicate

indeed the washing method to be least effective in selecting progressively motile sperm cells, but the

difference with the Percoll method is not significantly large. Thus, when suspecting both swim-up and

Percoll to be methods that are more efficient in selecting progressively motile sperm cells in comparison

with the basic washing method, then swim-up in Sp-CZB has proven to be superior to the Percoll density

gradient method in the same medium.

Furthermore, it is interesting to find that it apparently is the combination of method and medium that

does the job – the Sp-CZB medium was showed to provide lower progressive motility rates in both the

washing technique and the Percoll method compared to Ca- TALP, although differences were not

significant.

Next to this, it is important to mention the difference in total amount of spermatozoa collected by the

three methods. It is no surprise to observe the basic washing technique having the highest total recovery

of spermatozoa in absolute numbers, as the selection of sperm cells is minimal and the least of the

three. More interesting is the finding that the swim-up method, in both Sp-CZB and in Ca- TALP, was

capable of collecting only low total amounts of sperm cells (0.6 and 2.4 million·mL-1, respectively). The

low recovery of total spermatozoa in combination of the high progressive motility rates in Sp-CZB,

suggest an adequate selection during the process when using this medium. In Ca- TALP, with

progressive motility numbers not significantly differing from the other two methods, this selection seems

less distinct.

The capacity of the swim-up method to provide in a low recovery of highly motile spermatozoa has been

described by others. Loomis et al. mention a yield of approximately 10-20% and discuss the usefulness

24

of the swim-up method when obtaining these low recovery rates: the density gradient method then is

preferred over swim-up when having semen samples with suboptimal quality60. In human artificial

reproduction, hyaluronic acid, a glycosaminoglycan that can be found in the female reproductive tract,

is added to the swim-up, to significantly increase the recovery of viable and motile male

spermatozoa60,61,62. This is also done in bovine artificial reproduction60,62.

The Percoll method seems to hold an intermediate position in concern to absolute total recovery rates.

Its total recovery of spermatozoa is less than when using the washing method, but far more than when

using the swim-up method (mean amounts of 47.9 and 10.8 million·mL-1 in experiment 5 and 6). Indeed,

it was already described in earlier work by Beydola et al. that Percoll density gradients are preferred

over the swim-up technique when using suboptimal sperm samples (in terms of concentration or

motility), as the density gradient method is thought to lead to higher recovery of motile sperm cells43.

This study partly agrees with this, as the findings reveal the Percoll method to be capable of recovering

far higher absolute numbers of spermatozoa than the swim-up method (in both media). However, the

results of this study suggest a higher capability of the swim-up method (in combination with Sp-CZB) to

obtain high percentages of progressively motile sperm cells, indicating a more distinct selection than in

the density gradient method.

Another problem in regard to the Percoll density gradient, is the assumed endotoxin contamination that

led to the withdrawal of the solution from the human market (see above). Given the fact that alternative

colloidal silica are available nowadays, the question rises how the responsible use of Percoll still can be

justified in equine ICSI, regardless whether the assumed risks in concern to Percoll are undeniable or

not. Naturally, the alternatives would need to live up to the expectations, and this might be still a problem:

Söderlund et al., for example, investigated the potential of PureSperm® in comparison with Percoll63. A

four-layer variant of PureSperm® was found to result in comparable sperm motility rates, whereas the

two-layer PureSperm® variant had even lower motility numbers, compared to Percoll. Hence, Söderlund

et al. stick to the recommendation to use the swim-up technique when high progressive motility rates

are required.

In this study, it was chosen to compare three singular processing methods: washing, swim-up and

density gradient centrifugation. Lately, some works have been published investigating the influence on

viability and motility of spermatozoa when certain processing method are combined. Naturally, this

involves a prolonged protocol, which makes processing more time-consuming and more costly, but

nevertheless potentially useful when resulting in even higher viability of the sperm cells. Choi et al. found

a significant increase in blastocyst development rates after ICSI when using sperm cells that had

undergone density gradient centrifugation followed by swim-up, compared to density gradient

centrifugation alone38. Alternatively, Yamanaka et al. investigated the influence on ultrastructural

abnormalities in human spermatozoa after a combination of density gradient centrifugation and swim-

up64. A significant decrease in spermatozoa having DNA fragmentations was observed when using the

combination of the techniques. This shows the potential usefulness of combining multiple methods,

although more elaborate research obviously is required. Despite combining processing methods not

25

being the subject of this dissertation, it is worth mentioning to help placing the results of this study in a

greater perspective.

When evaluating the experimental set-up of this study and its outcome, one could find several

opportunities for improvement for future research. For example, it would be interesting to use semen of

more stallions to assess the processing methods, in order to decrease the role of stallion-specific

individual influences. Parallel to this, an experimental set-up collecting replicates on a larger scale would

be very informative in this regard. Given the limited amount of time, sources and experience of the

author of this dissertation, the number of replicates in this study was not very extended. The relatively

high variance of the findings might be a result of several factors, of which the author being unfamiliar

with the laboratory protocol and being new to laboratory work in general, can be one of them.

In conclusion, the findings of the study in this dissertation show that performing the swim-up method for

diluted sperm using Sp-CZB medium facilitates the highest percentage of progressively motile sperm

cells that can be used in ICSI. Hence, the practitioner wanting to use frozen-thawed, diluted and re-

frozen sperm cells to perform ICSI in horses, might find it the best choice to use the swim-up technique

in combination with Sp-CZB, in order to collect the most viable sperm cells of the sample.

26

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