A Comparative Study in Morphological Defects of Semen from African Lions

GHENT UNIVERSITY FACULTY OF VETERINARY MEDICINE

Academic year 2015 – 2016

A comparative study in morphological defects of semen from African Lions (Panthera leo) and Caracal (Caracal caracal): collected by urethral catheterization and electro- ejaculation.

Maaike DE SCHEPPER

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 2015 – 2016

A comparative study in morphological defects of semen from African Lions (Panthera leo) and Caracal (Caracal caracal): collected by urethral catheterization and electro- ejaculation.

Maaike DE SCHEPPER

Preface

First of all I want to thank DVM. Cyrillus Ververs for being my supervisor. Thank you for helping brainstorming about all the interesting different topics and fully believing in the one it became! Thank you for reading everything until the last moment and always staying positive about my deadlines!

Second of all I want to thank Ilse Luther, for taking me under your wings, without even knowing me! Teaching me everything about semen and semen collection, but also for the nice visits I could bring to your place and the lovely talks and wine! Hope to see you soon again!

I want to thank Willie and Jill Jacobs from UKUTULA research centre for the help with my research, without you I wouldn’t have anything to research about and I hope this is useful for you in the (near) future.

DVM. Gerardus Scheepers, thank you for being the vet during my research and teaching me about ultrasound and anaesthetics.

Thank you Professor Dr.Peter Bols for reviewing my research and giving comments and tips if necessary.

DVM Kristof Hermans, for helping me with my statistics, which all sounds like Chinese to me!

Dr. Johan Marais, thank you for taking me with you on a Saving the Survivors mission. And be such an inspiration about wildlife conservation and medicine. Hope to see you and work with you again any time soon!

And of course a big thank you for my parents, who are always supporting me and made it possible to go Pretoria and Onderstepoort for my Erasmus and my research.

And last but not least, my friends and classmates, either in Ghent or in Pretoria. Specially Tom, Carlien, Dennis and Ezit for being always there for me to talk, chill, laugh and having my back!

Table of content

Preface ......

Table of content ......

Abstract ...... 1

Samenvatting ...... 2

Introduction ...... 3

1 Anatomy of the male reproductive organs ...... 7 1.1 Testes ...... 7 1.2 Epididymis ...... 8 1.3 Accessory sex glands ...... 8 1.3.1 Prostate ...... 8 1.3.2 Bulbourethral glands ...... 8 1.4 Urethra and penis ...... 8

2 Spermatogenesis ...... 10 2.1 Spermatogonia ...... 10 2.2 Spermatocytes ...... 10 2.3 Spermiogenesis ...... 10

3 Classify origin of defects ...... 11 3.1 Morphology ...... 11 3.1.1 The head ...... 12 3.1.2 The tail ...... 12 3.2 Defects ...... 14 3.2.1 Head defects ...... 15 3.2.2. Midpiece defects ...... 15 3.2.3 Tail defects ...... 16

4 Collecting methods ...... 16 4.1 Urethral catheterization ...... 16 4.2 Electro-ejaculation ...... 17

5 Materials and Methods ...... 17 5.1 Animal species and management ...... 17 5.2 Immobilisation ...... 18 5.3 Semen collection ...... 18 5.4 Semen processing ...... 19 5.4.1 Macroscopic evaluation ...... 20

5.4.2 Microscopic evaluation ...... 20 5.5 Data analysis ...... 21

6 Results ...... 21

7 Discussion...... 27

8 References ...... 29

9 Appendix I ...... 31

Abstract

In this research semen of four African lions and two caracals was collected examined. The semen was collected through urethral catheterization and electro-ejaculation. Urethral catheterization is a more recent and friendly way to collect semen. This due to low cost, field friendly and simple ways to apply.

An α2agonist is used as a general anaesthetic and is necessary for the semen release into the prostate gland. Form this it can be collected by an urethral catheter.

Lions showed 66% live spermatozoa on average from which an average of 15% normal morphology. The caracal showed 88% living spermatozoa per ejaculate 12% normal morphology. The most dominant abnormalities seen by lions and caracals were: dag defects, diadem defects, distal droplets and knobbed acrosomes.

Key words: Lion (Panthera leo) – Caracal (Caracal caracal) – Urethral catheterization – Electro-ejaculation – Semen morphology

1 Samenvatting

In dit onderzoek is sperma van vier leeuwen en twee caracals onderzocht. Het sperma werd verzameld door middel van urethrale katheterisatie of elektro-ejaculatie. Urethrale katheterisatie is een meer recente en vriendelijke manier van sperma verzamelen dan elektro-ejaculatie. Dit doordat het een lage prijs heeft, veld vriendelijk is en gemakkelijk toe te passen is. Een α2agonist wordt gebruikt voor de algemene anesthesie, maar zorgt ook voor het loslaten van spermatozoa in de urethra en vervolgens het op stapelen van het sperma in de prostaat. Daarna kan het sperma via een urethrale katheter gecollecteerd worden vanuit de prostaat, doormiddel van capillaire zuigkracht in de katheter. De meest geziene morfologische afwijkingen bij de leeuw en de caracal waren: dag defecten, diadeem defecten, distale druppels en knobbed acrosomen.

De leeuwen hadden gemiddeld 66% levende spermatozoa waarvan er gemiddeld 15% normale spermatozoa waren. De caracals hadden gemiddeld 86% levende spermatozoa waarvan er gemiddeld 12% normale spermatozoa zijn.

Het niveau van teratospermie verergert bij een toename van het verliezen van de genetische diversiteit, dit valt duidelijk te zien bij inteelt populaties. Alle studie objecten zijn in gevangenschap geboren en grootgebracht in een gesloten populatie, dus er moet rekening gehouden worden met de inteelt factor. Pukazhenthi, et al. (2006) hebben in hun onderzoek gezegd dat een ejaculaat van katachtige geclassificeerd kan worden als teratospermisch als er meer dan 60% pleiomorfische spermatozoa aanwezig zijn. In dit onderzoek is dit voor beide diersoorten het geval, dus het gemiddelde dier in dit onderzoek heeft een teratospermisch ejaculaat.

2 Introduction

Nowadays different animal species are becoming extinct at a rate 100 times the natural background rates (Silva, et al. 2004). Impact and influence from mankind in different ways has resulted in a decline of biodiversity all over the world, mainly due to the loss of different ecosystems and the loss of genetic diversity. As the natural habitats of many species disappear or become smaller, the remaining animals stay in segmented areas leading to inbred populations with a loss of genetic variability. By the reason of this loss, animals are limited in their adaptation capacity and are more vulnerable to diseases and hazardous challenges. Therefore conservation plays an important role in the surviving of different species.

In this research semen from the African Lion (Panthera leo) and the Caracal (Caracal caracal) is collected by two different methods and evaluated.

The African Lion (Panthera leo) is part of the genus Panthera, family Felidae. The closest living relatives nowadays are the tiger, jaguar, snow leopard and leopard. They are all part of the genus Panthera, but classified into a different families. Eight different subspecies in the Panthera leo species are classified as: Panthera leo leo, Panthera leo persica, Panthera leo senegalensis, Panthera leo nubica, Panthera leo azandica, Panthera leo bleyenberghi, Panthera leo krugeri, and the Panthera leo roosevelti.

Fig 1: Cecil the African lion (Panthera leo). (from: http://bbc.co.uk)

3 The African Lion, is stated Vulnerable on the IUCN Red List of Threatened Species (International Union for Conservation of Nature), which means that the species is facing a high risk of extinction in the wild. The total population size in the wild is estimated to be fewer than 10.000 mature animals.

There is a continuing decline in numbers of mature individuals, and no subpopulation is estimated to contain more than 1000 mature individuals (IUCN, 2003). The lion population (mature animals) in whole of Africa is estimated between 23.000-39.000, with a decline of 42% over the last 21 years (approximately three lion generations 1993-2014) (Bauer, et al. 2015). According to the most recent IUCN Red List Assessment the African lion population meets the criterion to be listed as endangered for the majority of its population. However, there are a few stable, and even increasing populations, in Botswana, Namibia, South Africa and Zimbabwe, giving the species a vulnerable listing.

The Caracal (Caracal caracal) is part of the genus Caracal and the family Felidae. Seven different subspecies are recognised: Caracal caracal algira (in North Africa), Caracal caracal caracal (in Sudan, East-, Central and South Africa), Caracal caracal damarensis (Damaraland in Namibie), Caracal caracal limpopoensis (Northern part of South Africa and Botswana), Caracal caracal lucani (Gabon), Caracal caracal nubica, (Sudan and Ethiopia) Caracal caracal poecilotis (Niger, Nigeria and West- Africa), and the Caracal caracal schmitzi.

Fig 2: Caracal (Caracal caracal) (from http://les-felins.com)

However the African Caracal (Caracal caracal) is stated as least concern on the IUCN Red List of Threatened Species (IUCN, 2008). This does not mean that there is no need to preserve the species. There are flourishing populations in Western and Southern Africa, but in Northern Africa the habitats

4 are declining. Their main threats are humans, mainly farmers due to the hunting of the caracal on small livestock. For those animals it is the right time to start with researching their semen and reproduction manners, to counter future problems. It might also serve as a model animal for other wild Feline species that are more threatened.

If carnivores are conserved, a large number of other species will be protected as well, due to the fact that carnivores are ‘umbrella species’. These carnivores are also classified as indicator species (those that reflect critical environmental damage), keystone species (those that play a pivotal role in ecosystems). But also as flagship species (popular species that attract much attention), and vulnerable species (species most likely to become extinct) (Silva, et al. 2004).

The killing of Cecil the Lion in Zimbabwe by an American trophy hunter made the world aware of the big business of trophy hunting on lions (from http://www.bbc.co.uk). One of the main threats the lion population is facing these days is this trophy hunting business, as well as habitat loss and the human- wildlife conflict in rural areas.

Those topics mentioned above show the importance of conservation research. By all means, there are two different ways in conservation: in situ and ex situ. In situ conservation means visible conservation where a population of animals is saved and kept in national parks. Those animals are still visible and not to be forgotten, although very susceptible to infectious and other lethal diseases (Cseh and Solti, 2000). Ex situ conservation is forming a stock of different kinds of genetic materials, oocytes, semen and embryos that have been frozen into liquid nitrogen. This method is called cryopreservation. This is quite save and inexpensive, but the downside of this method is that people tend to forget about those animals.

Due to the fragmentation of the habitat of the lion in Southern Africa, isolated populations are created. Isolated populations seem to lead to inbreeding among the present animals. Inbreeding causes several problems, like, morphological abnormal spermatozoa, increasing homozygotes and thus correspondingly decreasing the heterozygotes. The homozygote alleles can lead to recessive lethal mutations. As well as being more susceptible to environmental inflicted mortality. And also a reduction in fertility and embryogenesis. Inbreeding also reduces the overall fitness of the inbred animals.

The best way to compare the influence of inbreeding on spermatozoa is to look at the proportion of motile spermatozoa in an ejaculate. But also look at the morphological abnormalities of the spermatozoa. Fitzpatrick and Evans (2009) found in their research that extensive inbreeding leads to depressed sperm quality. Those factors show us how important it is to collect and cryopreserve semen from different animals among the continent. Due to cryopreserving of semen, female oocytes and ovarian tissue, artificial reproductive techniques might help to prevent future bottleneck populations.

Recently a new way to collect lion semen has been described by Lueders et al (2012). In their study they used a commercial dog urinary catheter in lions sedated with an α2adrenergic agonist. They collect semen due to catheterization of the urethra up till the prostate, called the Zambelli method (Zambelli, et al. 2008). It is known that α-adrenergic drugs influence erections as well as ejaculations in stallions. The adrenergic agents act on the α-adrenoreceptors and regulate the contraction of the

5 vas deferens and the participation of the contraction of the trigone and the sphincter of the urinary bladder (Zambelli, et al. 2007).

The goal of this research thesis is to collect semen and evaluate sperm morphology of the African lion and caracal and compare it with domestic cats. We want to set a baseline for morphological abnormalities in different populations and species. So that this is all well known for the future if both population decline further more.

6 1 Anatomy of the male reproductive organs

Starting from the testis, the spermatozoa are transported through the epididymis, the ductus deferens and the urethra during ejaculation. Meanwhile the accessory sex glands add their secretions into the urethra.

Fig 3: Anatomy of the male reproductive organs of the Lion (from: http://vetmed.wsu.edu).

1.1 Testes

The function of the testes is to produce spermatozoa and to synthesize and secrete hormones, mainly testosterone. Spermatogenesis starts in the tightly coiled seminiferous tubules, supported by the Sertoli cells. Sertoli cells are connected to each other by tight junctions and are attached to the basal lamina of the seminiferous tubules. In the interstitial spaces of the seminiferous tubules the Leydig cells are present. Leydig cells are responsible for the androgen production of the testes. Spermatozoa are transported from the testis via the rete testis into the efferent ducts (ductuli efferentes), into the epididymal ducts, where the spermatozoa mature and become motile.

7 1.2 Epididymis

The epididymis can be divided into the caput epididymis, corpus epididymis and cauda epididymis. The ductus efferentes and the caput epididymis resorb fluids, the corpus epididymis is secretory and the cauda epididymis is relatively inactive (Asa, 2010). Spermatozoa move from the cauda epididymis through the ductus deferens into the urethra.

1.3 Accessory sex glands

Accessory sex glands vary among the different mammal species, including their location, size, morphology and function. In the order of the Carnivores, family Felidae, genus Panthera the prostate and the bulbourethral glands are the only accessory sex glands present.

Secretions produced by the accessory sex glands contain fructose, which is used as an energy source for the spermatozoa. The secretions also facilitate the movement of the spermatozoa and are a physiological buffer against the acidic pH of the female reproductive tract.

1.3.1 Prostate

Embryonically speaking the prostate originates from the epithelium of the urogenital sinus. And thus serves as an accessory sex gland as well as an urethral gland.

The prostate is small and located at the cranial aspect of the rim of the pelvis in the abdominal cavity of the lion. It is bi-lobular in the transverse plane and oval shaped in the longitudinal plane. The general function of the prostate secretion is related to semen gelation, coagulation and liquefaction. Proteins consisted in the secretion are part of the coating and un-coating of the spermatozoa and the interaction with the female cervical muscles. Prostatic secretions are not absolutely required for fertility; however, fertility is impaired in the absence of a prostate (Hayward and Cunha, 2000).

1.3.2 Bulbourethral glands

The bulbourethral gland, also called the Cowper’s gland, embryonically originates from the distal urogenital sinus and is present in the female as well as in the male.

The bulbourethral gland consists of two lobules and is located lateral of the membranous part of the urethra at the base of the penis. This is just before the urethra leaves the pelvic cavity. Secretion of the bulbourethral glands is responsible for the seminal fluid clotting.

1.4 Urethra and penis

The urethra leaves from the neck of the bladder and leads through the pelvic cavity. After a short distance the urethra is surrounded by the prostate and is called the pre-prostatic urethra.

The penis of a lion and a caracal points caudally and the external opening is ventral of the anus, in the perianal area (see Fig. 4 and 5). In the penis the os penis is present, which is a tunnel-like bone and where the urethra runs through. Due to the os penis there is an increase of rigidity of the penis, which simplify the entry into the female during the early stage of the copulation process.

8 On the glans penis small-keratinized penile spines can be found, they are pointing backwards. The penile spines are hormonal dependent and mostly testosterone dependent. This is shown by tomcats, rats and hamsters. When males are castrated the spines disappear. When treated with testosterone after castration the penile spines growth is restored, even until a complete restoration (Arteaga-Silva, et al., 2008).

Fig 4: Backwards pointin penis of a Lion (Panthera leo) (© Maaike de Schepper).

Fig 5: Backwards pointing penis of a Caracal (Caracal caracal) (© Cyrillus Ververs).

9 2 Spermatogenesis

Spermatogenesis is the process of the cellular transformation from a stem cell to a spermatozoon. It occurs in the seminiferous epithelium of the testis and can be divided into three different phases. During the first phase the spermatogonia proliferate into spermatocytes and maintain their number by renewal. The second phase consists of the formation of haploid cells due to meiotic division of the primary and secondary spermatocytes, resulting in spermatids. Throughout the third phase the spermatids undergo a complex series of changes into spermatozoon.

2.1 Spermatogonia

Spermatogonia are diploid cells (2n), in contact with the epithelial basal lamina of the seminiferous tubule of the testis. There are two main types of spermatogonia, type A and type B. Type A are the dust like spermatogonia, those consists of a nucleus with a fine and palely stained chromatin granulation (Clermont, 1972). Type B are the crust like spermatogonia. Consisting of coarse granules or flakes associated with the nuclear membrane and nucleolus (Clermont, 1972).

The spermatogonia are divided by mitosis and keep in contact with the epithelial basal lamina during this process, resulting in the primary spermatocytes (2x2n).

2.2 Spermatocytes

The primary spermatocytes enter meiosis and leads to the production of two successive divisions. Leading to the production of haploid cells, the spermatids (n) (Clermont, 1972). The first step is meiosis I, forming two secondary spermatocytes (nx2) out of every primary spermatocyte. Thereafter the secondary spermatocytes undergo meiosis II, a common cell division, into the haploid spermatids (n).

2.3 Spermiogenesis

Newly formed spermatids do have a typical small spherical nucleus. They also have a normal cluster of cytoplasmic organelles, such as, the Golgi zone, mitochondria and centrioles. The Golgi zone forms different small granules that collide into one larger granule, the acrosomic granule. Around the acrosomic granule the head cap forms, growing over the surface of the nucleus. This is called the acrosomic system, covering two-thirds of the nucleus. During the growth and formation of the acrosomic system the Golgi-apparatus secrets glycoprotein. Glycoproteins contribute to the growth of the system and eventually separates from it. After separation the glycoproteins float around in the cytoplasm. The flagellum is formed on the opposite pole, due to the close binding of the centrioles, of the nucleus from where the head cap is formed. During the spermiogenesis process the nucleus rotates resulting in changing of the orientation of the acrosomic system. The system is lined in the direction of the limiting membrane of the seminiferous tubule. Together with the change in orientation the nucleus is replaced towards the periphery of the cytoplasm. After the displacement of the nucleus

10 the nuclear chromatins starts to condense and become more chromophilic (Clermont, 1972). During the displacement of the apex the nucleus stays closely to the acrosomic system. It also readjust its shape to the anterior portion of the nucleus. At the same time the caudal tube or machete, a fine fibrillar structure, is present in the cytoplasm and start to surround the flagellum. The chromatid body approaches the flagellum as well and start forming a delicate ring around the flagellum. The ring continuously moves down the flagellum and comes at rest at the caudal part of the midpiece. The midpiece of the spermatozoon is the part of the flagellum between the modified centrioles and the ring; the mitochondria concentrate around the flagellum at this point. The caudal tube disappears soon after the formation of the midpiece (Clermont, 1972). During the maturation phase the nucleus takes its final shape and completes its condensation. Residual body is formed during the second half of the spermiogenesis. When the cytoplasm that occurred around the flagellum flows towards the nucleus and separates from the cell.

3 Classify origin of defects

3.1 Morphology

Spermatozoa can be divided into two different functional parts, the head and the tail. The sperm head consists the paternal DNA and different materials to fertilize the female ovum. The sperm tail consist the apparatus for the energy production and motility of the spermatozoa.

Fig 6: Anatomy of a spermatozoa (from: Physiology of Reproduction, 2015).

11 3.1.1 The head

Spermatozoa have an oval shaped and flattened head and are divided into two different compartments. An anterior acrosome and the posterior post-acrosome region. The posterior region is also known as the nuclear ring or equatorial segment. Under the anterior acrosome and persisting onto the base of the head, lays the nucleus.

3.1.1.1 The nucleus

Condensed DNA is incorporated into the nucleus, which is the largest component of the head. The condensation of DNA is the result of the involvement of sperm-specific proteins, the protamines (Ward and Coffey, 1991). Due to the condensation of the DNA the spermatozoa is non-dividing and inactive. As well as the very small volume the DNA occupies compared to the DNA in the mitotic chromosome. The post-acrosomal sheet is a cytoskeletal complex of the perinuclear theca and surrounds the nucleus

3.1.1.2 The acrosome

The acrosome originates from the Golgi apparatus; it is formed during the early stage of the spermatogenesis, and therefore also called a specialized lysosome. It consists of an inner and an outer membrane and a matrix filled with protease. The outer acrosomal membrane lies underneath the plasma membrane and the inner acrosomal membrane lies directly over the nucleus. During the post- testicular maturation of the spermatozoa in the epididymis, the post-acrosomal region of the head goes through different changes. Those changes are important for the sperm-specific binding and fusion with the plasma membrane of the female oocyte, after penetration through the corona radiata and zona pellucida.

Different enzymes are present and formed in the acrosome, like: hyaluronidase, proacrosin, acrosin, neuraminidase and corona penetrating enzyme (CPE). Hyaluronidase is released from the acrosome and is responsible for the digestion of the cumulus oophorus of the female oocyte. Proacrosin is converted into acrosin at the inner acrosomal membrane. Acrosin itself is a trypsine-like enzyme, which is responsible for the penetration of the spermatozoa through the zona pellucida of the female oocyte. Neuraminidase is also a catalyst in the penetrating process of the spermatozoa through the zona pellucida. As well as CPE, who is associated with the outer acrosomal membrane and is responsible for the penetration through the zona pellucida.

3.1.2 The tail

The main function of the tail is motility, since there is no fertility without motility. The tail itself can be divided into four different sections, the neck, the midpiece, the principal piece and the endpiece. Every section is enclosed by a common cell membrane and the different sections have primary structural parts: the axoneme, the mitochondrial sheath, the outer dense fibres and the fibrous sheath. The axoneme is located centrally in the tail. It includes a central pair of single microtubules surrounded by nine even spaced double microtubules, called the A and B microtubules. The C-shaped B microtubules are attached to the A microtubules. Two tendon-like arms connect the A microtubules

12 with the previous pair. Those tendon-like arms are connecting in a clockwise direction. The helically arranged mitochondrial sheath is only found in the midpiece of the tail and surrounds the outer dense fibres. Those outer dense fibres consist of nine fibres that surround the axoneme and extend from the neck to the principal piece. The outer dense sheath surrounds the outer dense fibres only in the principal piece.

Fig 7: The cytoskeletal components of the central piece of a spermatozoa (from: Physiology of Reproduction, 2015).

3.1.2.1 The neck

The neck, also called the connecting piece, connects the neck and the tail of the spermatozoa. At the caudal end of the nucleus the basal plate, a concavity lining the implation fossa of the nucleus is formed. Which is connected by fine filaments to the capitulum, a convex articular region of the tail. The neck as well as the axoneme is formed by a pair of centrioles that are composed of nine circularly arranged microtubular triplets (Kaya et al., 2014). This pair of centrioles is formed in the spermatid during the sperm tail formation. The pair of centrioles can be divided into a proximal and distal centriole. The proximal centriole is related to the formation of the capitulum whereas the distal centriole is related to the formation of the axoneme. This means that the connecting piece originate from two different origins.

13 3.1.2.2 The midpiece

The midpiece is the part of the tail between the neck and the annulus, also called the Jensen’s ring. The annulus is the connective part between the midpiece and the principal piece. A helically wrapped mitochondrial sheath surrounds the outer dense fibre-axoneme present in the midpiece. Energy is generated in the inner mitochondrial membrane, by the mitochondria in the form of ATP. This energy is used for semen motility. The elongated mitochondrial helix surrounds approximately 80% of the midpiece (Kaya et al., 2014).

3.1.2.3 The principal piece

The principle piece is the part of the tail between the annulus and the endpiece. It is the longest part of the sperm tail. However, due to the ending of the present mitochondria, the diameter is getting smaller all the way to the endpiece. The fibrous sheath is present around the principle piece. It is a elastomechanic structure contributing into the flagellar waveform.

3.1.2.4 The endpiece

The endpiece, also called the terminal piece, is the last remaining part of the spermatozoa flagellum. This part of tail only consists the terminal segment of the axoneme and is only surrounded by a cell membrane.

3.2 Defects

Spermatozoa defects can be classified in three different ways. First, as head or nuclear defects and acrosome and tail or flagellar defects. Secondly as primary, secondary or tertiary defects and thirdly as compensable or un-compensable defects.

The first way is as nuclear defects originate in the testis during the spermatogenesis and have a noticeable effect on the fertility. Acrosome and flagellar defects originate in the epididymis or lower down the tract. Those abnormalities have often less effect on the fertility of the male lion and caracal.

Secondly as primary abnormalities, those are associated with the spermatozoa heads, midpieces and some type of tail abnormalities. Those abnormalities are mainly caused by an abnormal spermiogenesis before spermiation. Those are defects like: Dag defects, proximal cytoplasmatic droplets and primordial cells. Secondary abnormalities are those associated with tail abnormalities, caused during the transportation of the ejaculate from the testes. The main causes for those abnormalities are due to altered epididymal maturation, prolonged retention of the sperm cells in the male genital tract and abnormal content of the seminal plasma. Tertiary abnormalities are caused by improper handling of semen after semen collection, like cold-shock, contamination with urine or improper slide preparing (Chenoweth, 2005).

Thirdly as compensable or un-compensable defects, this is based on the fact if the spermatozoa can overcome the presence of the abnormality present (Saacke, 2008). And if the spermatozoa are still able to fertilize a female ovum. An un-compensable defect has mostly a genetic or molecular origin, like primary defects (Chenoweth, 2005). A way to increase the fertility chances by a compensable

14 defect is to increase the number of spermatozoa in an artificial semen sample. Spermatozoa with un- compensable defects are mostly able to reach the female ovum and are able to penetrate the zona pellucida how ever, they are not capable of zygotic and embryonic development.

3.2.1 Head defects

There are different head defects like decapitated sperm defects, diadem defects, rolled head, nuclear crest and abnormal DNA integrity and condensation.

3.2.1.1 Decapitated sperm defects

Loose heads, cacephalic, microcephalic and pinhead are all covered by the term of decapitated sperm defects. Various things like environmental factors affecting the spermiogenesis or sperm maturation can cause those defects. If the attachment between the head and tail is not formed during the spermiogenesis loose heads and tails can be seen. Another problem can be seen during the maturation of the spermatozoa, if the centriole-tail fails to attach normally to the nucleus. Both will develop separately from each other and will separate around spermiation. The Sertoli cells will phagocytose those loose heads (Chenowtha and McPherson, 2014).

3.2.1.2 Knobbed acrosome defect

A knobbed acrosome defect is expressed as a refractile, thickened acrosomal apex. Also a back bended of abrupt termination of the nuclear material is seen. A knobbed acrosome defect can either be caused by environmental or genetic factors. Environmental factors are transitory and mostly associated with other spermatogenic dysfunction signs. Genetic factors are seen when there are high proportions of spermatozoa with knobbed acrosome defects in the absence of frequent numbers of other spermatozoa abnormalities (Chenoweth, 2005). Due to the knobbed acrosome the spermatozoa lack the ability to attach to the female follicle.

3.2.1.3 Diadem defect

Diadem defects, also called crater defects, are believed to be caused by different responses to a wide range of spermatogenic insults. Also the reactive oxygen species (ROS) is seen as an element responsible for diadem defects during the spermiogenesis. There is believed that high temperature during the spermiogenesis is for some kind of influence on diadem defects (Chenoweth and McPherson, 2014).

3.2.2. Midpiece defects

3.2.2.1 Pseudo-droplet

A pseudo-droplet is a local thickening on the midpiece, with an irregular shape and visual dense. The ultra structural examination made clear that the pseudo-droplets mainly consist out of accumulated or displaced mitochondria.

15 3.2.2.2 Corkscrew defect

A corkscrew defect is an irregular disruption of the mitochondria in the tail. It is thought of that the defects have a common aetiology with the pseudo-droplet defect due to its ultra structural appearance (Chenoweth 2005).

3.2.2.3 Dag defect

The dag defects are described as a strong folding, coiling or fracturing of the distal part of the midpiece. It can be seen with or without a retained cytoplasmic droplet. This defect is associated with elevated zinc levels, disturbance in the testicle and/or epididymis and fever (Chenoweth and McPherson, 2014).

3.2.3 Tail defects

Tail defects have an influence on the motility of the spermatozoa. It is seen in a number of cases that the tail defects are shared with systematic sperm defects. Causing an overlap in classification of sperm defects (Chenoweth and McPherson, 2014).

3.2.3.1 Tail stump defects

Dysplasia of the fibrous sheath (DFS) needs to be differentiated from accessory tail defects. Those two defects share a common aetiology, but the accessory tail defect does not have much impact on the male fertility. Spermatozoa with DFS have a short, thick and irregular flagellae. Also a complete or a partial lack of motor proteins (dynein) and abnormal mitochondrial disposition is seen in those tails.

3.2.3.2. Coiled tails

A coiled tail, or also called a distal midpiece reflex is one of the most common sperm defects. An increased prevalence of those defects has its origin in non-genetic etiologies.

3.2.3.3 Proximal and Distal droplet

The proximal and distal droplets are the remains of the spermatid residual cytoplasm, which is still attached to the neck region of the spermatozoa. During the maturation process of the spermatozoa, along the transit through the head of the epididymis, the droplet moves from the proximal neck position of the neck to the distal part of the midpiece .

4 Collecting methods

During this study two different semen collection methods are used. The urethral catheterization method and the electro-ejaculation method.

4.1 Urethral catheterization

Urethral catheterization is a fairly new method, developed by Zambelli et al. (2008). This method is low in cost, non-invasive, practical and repeatable, which is very important in wildlife. Mainly due to the

16 lack of training possibilities, lack of close contact and the danger of handling animals. The adrenergic agents of α2-agonistic sedatives act on the α-adrenoreceptors and regulate the contraction of the vas deferens and the participation of the contraction of the trigone and the sphincter of the urinary bladder (Zambelli, et al. 2007). Resulting in the release of semen from the testis and pooling into the prostate. Which is collected by the urethral catheter due to capillary forces. Due to the Zambelli method higher semen concentrations, lower pH and a lower total volume is seen in an ejaculate collected by this method compared to an ejaculate collected by electro-ejaculation method (Zambelli, et al. 2008).

4.2 Electro-ejaculation

Electro-ejaculation (E.E.) is a diffrent method to collect semen of immobilized. A rectal probe is connected to an adjustable power source, which can be controlled manually. The amount of power given through the probe depends on the patient and his reaction to the current during the procedure. The rhythmic delivery of the electrical pulses depends on the manual adjustments of the handler of the power source. The voltage delivery is increased progressively over a few seconds. After a few stimulations the voltage is reduced to zero to give the animal some rest. Soon after, the voltage is increased until erection and/or ejaculation occurs. During the procedure the spastic contractions of the muscles of the hind limbs can occur, due to the electrical stimulation of the muscles. The semen samples collected have a high volume and a higher alkalinity. Concentration of the ejaculate is lower compared to urethral catheterization and there is an increased chance of urinary contamination due to stimulation of the urinary bladder. Semen collection is furthermore described in literature as slightly uncomfortable for the paitient. However sometimes it is the only possible way to get an ejaculate.

5 Materials and Methods

5.1 Animal species and management

Four adult male African lions (Panthera leo), captive bred at a breeding and research facility in South Africa, were used for this research. All males were between four and seven years old and were held either in a bachelor group, together with different males, or housed in a breeding group, together with four to six females. All four males are proven breeders. The housing consist of an outdoor enclosure with shelter, trees, water provided and different rocks. The bachelor groups, as well as, the breeding groups can have olfactory and visual contact with other lions, male and/or female, in their surroundings.

The two adult male caracals (Caracal caracal) used for this study are captive bred in Belgium and are three and seven years old. Both males are proven breeders and kept in separate outside enclosures, but together with one female each. The enclosures are outdoors, with a shed to seek shelter into. Also trees and foliage covering is present. In the neighbouring enclosures Servals (Leptailurus serval) and a variety of birds are present. Visual and olfactory contact between the different animals is present.

17 5.2 Immobilisation

All lions were sedated because of regular health checks and/or translocation. As a surplus, semen was taken from all four lions. Just before the procedure the animals were separated into a small enclosure, which was part of the big enclosure. The animals were sedated with an average of 21,4 mg Ketamine Hydrochloride (Ketalar®,Par Sterile Products, LLC) and 1,72 mg Medetomidine (Domitor®, Orion Pharma). A remote dart gun, with 3,0 ml darts, was used to administer the sedative cocktail intramuscular. 15 minutes after darting the lions were fully sedated and transported to a temporary clinical lab. There the semen collection could start. The procedures of each of four animals took between 60-90 minutes and the sedative was reversed with a total of 7 ml Yohimbine (Yohimbine®, Twins Pharmaceuticals) per lion injected Intramuscular in the Muscles Gluteus. All four lions were awake within 2-4 minutes.

The Caracals where starved for 12 hours before the procedure started. The animals where sedated on 26th of April with Medetomidine (Domitor®, Orion Pharma) first, intramuscular and after approximately 13 minutes Ketamine was administered IM. 10 minutes after the Ketamine (Nimatek®, EUROVET Animal Health B.V.) injection the animals where taken inside to start the procedure. The sedative was reversed with Atipamezole (Antisedan®,Zoetis), one hour after the Ketamine administration.

5.3 Semen collection

On all four lions an enema was conducted, to clean out the rectum to be able to do a rectal ultrasound. While doing the ultrasound the prostate was examined and the distance to the prostate was determined for the urethral catheterization. The ultrasound was performed by using a portable ultrasound (Logic e, General Electrics Healthcare, GmbH, Solingen, Germany) assembled with a linear probe on a PVC extension stick. The extension stick was approximately 30 cm long and had a slightly bended handle. After the ultrasound the preputial area was cleaned with fresh water and the penis was extruded and cleaned with ProntoSan spray (polyhexanid-betain-complex, Prontomed, GmbH, Hiddenhausen, Germany) while holding the shaft of the penis with a surgical swab. After 30 minutes into sedation the urethral catheterization started, to give the medetomidine a chance to release the semen into the prostate. A commercial dog urinary catheter (Buster, sterile dog catheter, WDT, Garbsen, Germany) (2.6 mm x 500 mm) was lubricated at the tip with non-spermicidal sterile lube (Priority Care, First Priority, Inc, IL., USA) and inserted into the urethral opening of the penis (see Fig. 8). The catheter was moved forward until the tip was visible on the ultrasound in the prostate. Due to capillary filling the semen moved into the catheter and the catheter was pulled back. The semen was removed from the catheter into a pre-warmed 1,5 ml Eppendorpf tube.

The caracals did not receive an enema nor did their penises get cleaned before the procedure. Both caracals where catheterised with a dog urinary 6FG Catheter (1.0x300 mm) into the ureteral opening approximately 30 minutes after the Domitor administration. The catheter was moved forward under the guidance of a measuring tape. This to make sure that the bladder was not reached during catheterization and setting a baseline for each individual caracal for future semen collection., since reference values could not be found in literature. Caracal one was catheterised four times and the catheter was inserted a maximum of 17.5 centimeters into the urethra without having urinary contamination. Samples were collected into pre-warmed 1.5 ml Eppendorf tubes for further analysis. Caracal two was catheterised four times as well with a maximum of 20 centimetres without having urinary contamination.

Electro-ejaculation was conducted on Caracal two after the failure of getting sufficient semen trough urethral catheterization. An electro ejaculator (e320, Mini Tüb, Germany) was used during the procedure. A dog probe was used and covered in lube and entered into the rectum. Cycles of 5 pulses each time were administered at 1.0 V, 1.5 V and 2.0 V. This was repeated 3 times with adequate rest in between. From 1.5 V onwards nice erection was seen and from 2.0 V the ejaculate was released and recovered into a sterile funnel.

5.4 Semen processing

The semen samples were marked with the name of each lion and/or caracal and kept warm on a heated plate with a temperature of 37°C.

19 5.4.1 Macroscopic evaluation

The total volume of the semen sample was measured in the Eppendorpf tube and the colour was evaluated, differentiating from milky, yellow, clear to even grey. Also the contamination was evaluated, differentiating from mucus, pus, debris, urine and/or faecal. The presence of a marbled texture on close examination of the semen sample is the macroscopic manifestation of the mass motility, and therefore a good indication of the concentration and motility of the semen sample. A pH strip was used to determine the pH value of the samples in a range from 6,0-8,0.

5.4.2 Microscopic evaluation

A Nikon E501 microscope with a warmed stage 37°C was used to evaluate the motility and an Olympus BX51 microscope was used to evaluate the morphology of the semen samples. Pictures of the samples where taken with an Ikegami colour camera (ICD-879P) connected to the micorscope.

4.4.2.1 Concentration

The concentration of the semen sample is determent by using a Buerker counting chamber. The chamber is placed under the microscope and looked at with a 40x objective.

4.4.2.2 Motility

Individual motility is assessed by putting a drop of semen on a pre-warmed microscopic slide and covered by a pre-warmed coverslip. Lion semen does not need to be diluted, contrary to bull semen. The motility was assessed by estimating the ratio of motile to non-motile spermatozoa, taking the average of different assessed fields. Meaning, for every non-motile sperm how many motile sperm are present on the slide.

Individual motility is scored as aberrant motility, progressive motility and immotile spermatozoa. Aberrant motility is abnormal motile sperm, the spermatozoa move in oscillatory, tight circular and reverse directions. Progressive motile sperm moves in a relatively straight and linear direction. Immotile sperm has no movement at all.

Individual sperm motility is easily influenced and reduced by cold-shock and/or osmotic shock. This could bias the outcome and needs to be accounted for. Cross-checking the outcome with mass motility, morphology and percentage live spermatozoa can be used to detect errors.

5.4.2.3 Morphology

Sperm morphology is examined under a light microscope, Eosin-Nigrosin smears are made on microscopic slides with frosted ends, the details of the lion and caracals and the date are recorded on the frosted end of the slide. A total of 100 randomly chosen spermatozoa have been counted, under a high power (100 x and oil immersion) microscope. From these 100 spermatozoa the percentage dead and alive sperm was determined. Here after 100 live spermatozoa are counted to determine the morphological abnormalities present. A datasheet was used to keep record of the different morphological abnormalities (see appendix I) seen on the slide. The datasheet classifies the abnormalities into two different categories, nuclear defects and acrosome and tail defects.

20 5.4.2.4 Evaluation of % live sperm

The evaluation of percentage live sperm on the Eosin-Nigrosin smear is used as a crosscheck for the results of the motility assessment. If the individual motility assessment has been done correctly the live/dead count will be favourable with the percentage motile sperm.

Spermatozoa are divided into dead sperm cells and live sperm cells. The dead spermatozoa have pink stained heads, the membrane isn’t intact anymore and the heads can take up the Eosin-Nigrosin stain. Live sperm cells have unstained heads. The results are divided in percentage live and percentage dead.

5.5 Data analysis

Microsoft excel was used to analyse the data retrieved during this research, descriptive statistics where used to construct various tables, charts and a boxplot. A table was constructed to identify all different morphological abnormalities. From this table a graphic was constructed. To construct the boxplot, R studio was used, in this program the GG plot was used.

6 Results

Semen was successfully collected from all seven animals during the procedure. In six animals semen was collected due to urethral catheterization and in one animal (Caracal no. 2, sample no.7) due to electro-ejaculation. The colour of the ejaculates varies between milky white and cream yellow and with an average volume of 340 μl in lions and an average of 240 μl in caracals. The concentration of the ejaculates ranged from 8x10^6/ml to 236x10^6/ml in caracals and between 0.125x10^9/ml and 2.53x10^9/ml in lions.

Lion Volume % Dead % Live % Normal 1 450 μl 13% 87% 18% 2 450 μl 11% 89% 19% 3 100 μl 32% 68% 14% 4 500 μl 54% 46% 15% 5 200 μl 60% 40% 9% Mean 340 μl 34% 66% 15% Table 1: Ejaculate characteristics of lions (Panthera leo).

Caracal Volume % Dead % Live % Normal 1 200 μl 12% 88% 19% 2 280 μl 17% 83% 5% Mean 240 μl 15% 86% 12% Table 2: Ejaculate characteristics of caracals (Caracal caracal).

21 In graph 1 the results of the sperm plasma membrane integrity was evaluated. It shows that sample 4 and sample 5 have the largest percentage of dead spermatozoa in their ejaculate of all animals assessed. This varies between 54% and 60% of dead spermatozoa in the ejaculate of those animals. Semen from sample 1 and sample 2 are from the same animal and have a percentage of 87% and 89% of live spermatozoa. Sample 3 has a percentage of 68% live spermatozoa. Sample 6 as well as sample 7 has a higher percentage of spermatozoa with an intact sperm plasma membrane, specifically a percentage of 83% and 88%, both samples are from the caracals. A difference between the two animal species can be seen in the percentage of live and dead spermatozoa.

Distribution of live and death spermatozoa

% Live % Dead

7 83 17

6 88 12

5 40 60

4 46 54

3 68 32

2 89 11

1 87 13

Graph 1: The distribution of live and death spermatozoa from all seven samples.

22 From the percentage live spermatozoa the percentage of normal and pleiomorphic spermatozoa has been evaluated. In graph 2 an overview of all animals and their percentage normal and percentage pleiomorphic spermatozoa can be seen. All animals have a very high percentage of pleiomorphic living spermatozoa, which is common in feline species. Differences between the two feline species are present and differences within the two species are present as well. The variation of percentage live and dead and percentage normal and pleiomorphic within one animal are small, as can be seen in sample 1 and 2. Sample number 5 has the lowest percentage living spermatozoa, 40%, and the lowest percentage normal spermatozoa, 9%.

DISTRIBUTION OF NORMAL AND PLEIOMORPHIC SPERMATOZOA

% Normal % Abnormal

7 5 95

6 19 81

5 9 91

4 15 85

3 14 86

2 19 81

1 18 82

Graph 2: Distribution of normal and pleiomorphic spermatozoa of al seven samples.

23 The dominant abnormality seen in this research can be seen in graph 3, in table 3 values are stated. The Dag-like defect is the main defect in lions, with the median around the average pleiomorphism for all samples. The pre-dominant abnormality is the diadem defect and this can be seen in both species, with the median just above the average. Third is the knobbed acrosome defect, mainly seen in lions. The knobbed acrosome has a median of just below average. The distal droplet has a median below average and a big outlier, which is sample 3, a lion. The main abnormalities are part of the acrosome and tail defects. However, the diadem defect is the pre-dominant defect and is classified as a nuclear defect (see Fig.9).

Knobbed Diadem Acorsomes Dag-defect Distal droplet Min 16.0 2.0 22.0 0.0 Lower quartile 24.0 5.5 34.5 1.5 Median 31.0 11.0 48.0 3.0 Upper quartile 36.5 21.0 65.5 9.5 Max 39.0 29.0 79.0 10.0 Table 3: Values of the boxplot.

24 Graph 3: Boxplot of the most prevelant abnormalites seen in Lion (Panthera leo) and Caracal (Caracal caracal).

25 Fig 9: Most seen pleiomorphic spermatozoa. A-D=Lion spermatozoa. E-H=Caracal spermatozoa. A+E=Dag defect. B+F=Diadem defect. C+G=Knobbed acrosome. D+H=Distal droplet.

26 7 Discussion

In six out of seven animals the urethral catheterization was successful. The one caracal (sample 7) that did not respond to the α2-agonistic sedatives as a semen collection method underwent electro- ejaculation. As seen in previous studies (Zambelli et al. 2008; Lueders, et al. 2012) and in this study, the semen concentration of the ejaculate collected through urethral catheterization (236x10^6/ml) was much higher than the semen concentration collected through electro-ejaculation (8x10^6/ml). A possible explanation is due to a lower volume in the ejaculate because the seminal fluid is not added to the ejaculate during a urethral catheterization.

The main morphological difference between the two species, Panthera leo and Caracal caracal, is the head shape. The lion spermatozoa have an elongated oval shape, like tomcats (Schmehl and Graham, 1989). Where as the caracal spermatozoa have a longer and thinner oval shape than lions. The tomcat spermatozoa show a midpiece that tapers inwards, this is not seen in lion or caracal spermatozoa.

Axnér, et al. (1999) found in their research that most of the pleiomorphic spermatozoa originates in the testis of the domestic cat. It is seen that the head abnormalities and the midpiece abnormalities decrease when they are transported from the ductus efferentes to the cauda epididymis. However, the acrosomal defects and the tail defects increase when the spermatozoa are transported from the ductus efferentes to the cauda epididymis.

It is believed that in domestic cats the appearance of distal droplets is a sign of not fully matured spermatozoa, because in a normal ejaculate the distal droplets disappear. In this study all the lions had more distal droplet abnormalities than has been seen in the caracal. Normally the distal droplets at the tail of the spermatozoa are either shed at the tail of the epididymis or after the spermatozoa got mixed with the secretions from the accessory sex glands. But due to the urethralthere is no mixing of spermatozoa and the secretion of the de accessory sex glands. This can be cause for those abnormalities seen in this researach.

Wildt, et al., (1983) found that cheetah semen has a higher abnormal spermatozoa percentage (65% abnormal living spermatozoa) than domestic cat. Semen of the domestic cat is characterised by the few primary and secondary defects of spermatozoa. However, cheetah semen contains mostly both primary and secondary spermatozoa defects. Lion and caracal semen on the other hand contain mostly primary defects, like dag defects, caused by abnormal spermiogenesis. A possible cause for those abnormalities in captive cheetah is the chronic stress associated with captivity (Wildt, et al., 1983). As the study animals are in captivity as well, this is something to keep in mind. Extra research needs to be done, to compare stress levels in captive animals correlated with semen abnormalities.

Due to the differences in the pleiomorphic spermatozoa it can be said that the members of the Felidae family have evolved all uniquely qua ejaculate and morphology. If we compare the lion, caracal and the cheetah with the domestic cat, the cheetah has 65% abnormal living spermatozoa in their

27 ejaculate (Wildt, et al, 1987). Where we found an average percentage of 85% pleiomorphic spermatozoa in the lion and an average of 88% of pleiomorphic spermatozoa in the caracal. The domestic cat shows an average of 47,4% ± 19.0% abnormal spermatozoa in their ejaculate (Prochowska, et al., 2015). Wildt et al. (1987) found that the most prevalent abnormalities in tiger, cheetah, leopard and puma semen where coiled or bent flagellum, bent midpiece of a residual cytoplasmatic droplet. Prochowska, et al. (2015) found a high occurrence of distal droplets, bent tails and dag-like defects in ejaculates from domestic cats collected trough urethral catheterization. The dag-like defects and the distal droplets are also found in this study for the two wild feline species. Prochowska, et al. (2015) also describe the high occurrence of the dag defect in semen collected from domestic cats. They suggest that this defect is caused by changes of osmotic pressure instead of a hereditary base as seen in bulls.

In the lion and caracal the Dag defect, diadem defect and the knobbed acrosome where most prevelent.

The level of teratospermia increases with increased loss of gene diversity, which can be seen by inbred populations. As all our study subjects are born and raised in captivity in a closed population, inbreeding is a factor that should be encountered for. Pukazhenthi, et al. (2006) stated that in ejaculates of teratospermic felids a percentage over 60% of pleiomorphic spermatozoa could be seen. In this study the lions had an average of 85% of pleiomorphic spermatozoa and the caracals had an average of 88% of pleiomorphic spermatozoa. So the average animal studied had a teratospermic ejaculate.

Two consistent observations can be made regarding sperm morphology. First, there are species- or population-specific sperm concentration and motility characteristics. Second, the taxon as a whole exhibits a higher incidence of teratospermia than most other mammals.

The assimilation of spermatozoa, follicles and genetic material will ensure a continuance of genetic variation in the near future for captive and/ or wild animals. Due to preservation of genetic material, a bottleneck population in the future can be prevented. As the wild and captive animals are not closely related, it is important to preserve the underrepresented captive individuals for the future generations.

28 8 References

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29 17. Pukazhenthi, B.P., Neubauer, K., Jewgenow, K., Howard, J. G., Wildt, D.E. (2006). The impact of potential etiology of teratospermiain the domestic cat and its wild relatives. Theriogenology 66, 112-121. 18. Prochowska, S., Niżański, W., Ochota, M., Partyka, A. (2015). Characteristics of urethral and epididymal semen collected from domestic cats-A retrospective study of 214 cases. Theriogenology 84, 1565-1571. 19. Saacke, R.G. (2008). Sperm morphology: Its relevance to compensable and uncompensable traits in semen. Theriogenology 70, 473-478. 20. Schmehl, M.L., Graham, F. (1989). Ultrastructure of the domestic tom cat (Felis domestica) and tiger (Panthera tigris altaica) spermatozoa. Theriogenology 31, 861-874. 21. Silva, A.R., Marato, R.G., Silva, L.D.M. (2004). The potential for gamete recovery from non- domestic canids and felids. Animal Reproduction Science 81, 159-175. 22. Washington State University (2014). Internet reference: http://www.vetmed.wsu.edu, consulted at 19/04/2016 23. Ward, W.S., Coffey, D.S. (1991). DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biology of reproduction 44, 569-574. 24. Wildt, D.E., Bush, M., Howard, J.G., O’Brien, S.J., Meltzer, D., van Dyk, A., Ebedes, H., Brand, D.J. (1983). Unique seminal quality in the Southe African cheetah and a comparative evaluation in the domestic cat. Biology of reproduction 29, 1019-1025. 25. Wildt, D.E., Philips, L.G., Simmons, L.G., Chakraborty, P.K., Brown, J.L., Howard, J.G., Teare, A., Bush, M. (1987). A comparative analysis of ejaculate and hormonal characteristics of the captive male cheetah, tiger, leopard and puma. Biology of reproduction 38, 245-255. 26. Zambelli, D., Cunto, M., Prati, F., Merlo, B. (2007). Effects of ketamine or medetomidine administration on quality of electroejaculated sperm and on sperm flow in the domestic cat. Theriogenology 68, 796-803. 27. Zambelli, D., Prati, F., Cunto, M., Iacono, E., Merlo, B. (2008). Quality and in vitro fertilizing ability of cryopreserved cat spermatozoa obtained by urethral catheterization after medetomidine administration. Theriogenology 69, 485-490.

30 9 Appendix I