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.
By
Maaike DE SCHEPPER
Supervisor: DVM. Cyrillus Ververs Co-Supervisor: Prof. dr. Peter E.J. Bols.
Research thesis as part of the Master's Dissertation
© 2016 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.
By
Maaike DE SCHEPPER
Supervisor: DVM. Cyrillus Ververs Co-Supervisor: Prof. dr. Peter E.J. Bols.
Research thesis as part of the Master's Dissertation
© 2016 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
1
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
2
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.
3
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)
4
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.
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
Fig 2: Caracal (Caracal caracal) (from http://les-felins.com)
5
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
6
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.
7
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.
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.
Fig 3: Anatomy of the male reproductive organs of the Lion (from: http://vetmed.wsu.edu).
8
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.
9
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).
10
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
11
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).
12
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
13
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.
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.
Fig 7: The cytoskeletal components of the central piece of a spermatozoa (from: Physiology of Reproduction, 2015).
14
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
15
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.
16
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
17
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.
18
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.
19
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.
Fig 8: Conducting a urethral catheterization on a Lion (Panthera leo) (© Maaike de Schepper.)
20
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.
21
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).
22
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.
Graph 1: The distribution of live and death spermatozoa from all seven samples.
87
89
68
46
40
88
83
13
11
32
54
60
12
17
1
2
3
4
5
6
7
Distribution of live and death spermatozoa
% Live % Dead
23
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%.
18
19
14
15
9
19
5
82
81
86
85
91
81
95
1
2
3
4
5
6
7
DISTRIBUTION OF NORMAL AND PLEIOMORPHIC SPERMATOZOA
% Normal % Abnormal
Graph 2: Distribution of normal and pleiomorphic spermatozoa of al seven samples.
24
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).
Diadem Knobbed 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.
25
Graph 3: Boxplot of the most prevelant abnormalites seen in Lion (Panthera leo) and Caracal (Caracal caracal).
26
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.
27
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
28
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.
29
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31
9 Appendix I
© Ilse Luther, personal communication.
