The morphology and development of normal and abnormal spermatozoa in the emu, Dromaius ...

179 Pages·2015·12.92 MB·English

The morphology and development of normal
and abnormal spermatozoa in the emu,
Dromaius novaehollandiae

by

Lizette du Plessis

Submitted in partial fulfillment of the requirements for the degree
Doctor of Philosophy

Faculty of Veterinary Science
University of Pretoria
Pretoria

October 2012

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My journey the last few years was adventurous
At times it has been dark
the road steep and fraught with dangers for me
At times joyous
filled with sunshine, fun and laughter
even a bit of magic
Sometimes I have been foolish
sometimes wise
Often I have despaired
but continued in blind belief
I gave my everything, my heart, my soul
My journey continues

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CONTENTS

Declaration ii
Acknowledgements iii
Publications iv
Summary v

Chapter 1 General introduction and basic architecture of the
emu male reproductive tract 1

Chapter 2 Morphology of normal sperm 13

Chapter 3 Spermiogenesis 49

Chapter 4 A novel transient structure associated with
spermiogenesis 77

Chapter 5 Incidence and morphological classification of
abnormal sperm 98

Chapter 6 Morphology and origin of abnormal sperm I:
Head base bending and disjointed sperm 112

Chapter 7 Morphology and origin of abnormal sperm II:
Abaxial tail implantation 126

Chapter 8 Morphology and origin of abnormal sperm III:
Multiflagellate sperm 136

Chapter 9 Morphology and origin of abnormal sperm IV:
Miscellaneous defects 150

Chapter 10 General conclusions 169

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DECLARATION

I, Lizette du Plessis, declare that the thesis which I hereby submit for the
degree Doctor of Philosophy in Anatomy at the University of Pretoria, is
my own work, with the exception of the electron tomography which was
done at The University of Cape Town. I have not submitted the contents
of this thesis for a degree at this or any other tertiary institution.

October 2012

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ACKNOWLEDGEMENTS

I am grateful for the support that I received from family and friends, with special thanks to the
following people:

My supervisor, mentor and friend, Prof. John Soley, for introducing me to the vast and wonderful
world of sperm. Through our many talks and discussions, sharing your knowledge and guiding me,
you gave me confidence, motivated and inspired me to do the best I can;

My support team - my husband Dion du Plessis, my sister Anne-Marié Rogers and my friend Gerda
McDonald - without your constant encouragement this would never have happened. Dion, the last
two years were filled with hardship and heartache for us, thank you for your patience and loving
support;

Mrs. Erna van Wilpe, Manager of the Electron Microscope Unit at the Faculty of Veterinary Science,
Onderstepoort, always willing to help in whichever way possible, give advice (and a shoulder to cry
on) and allowing me freedom and time to work on my ‘spermpies’;

The Unit for Microscopy and Microanalysis, Main Campus, University of Pretoria for use of their
facilities;

Prof. Trevor Sewell, Electron Microscope Unit, University of Cape Town, for help with the electron
tomography;

The Eastern Cape Emu Breeders Association, in particular Mr Peter Bezuidenhout and The
Grahamstown Ostrich Abattoir, as well as Mrs Petra Rough from The Emu Ranch, Rustenburg for
making material and facilities available;

Prof. John Soley and Dr. David Phillips for supplying ostrich and rhea testicular blocks from their
personal collections;

The Jotello F. Soga library staff for friendly service and going the extra mile to help me find articles;

Ms. Estelle Mayhew, Department for Education Innovation,Onderstepoort, for the schematic
illustrations in Chapters 2 and 3;

The University of Pretoria for financial support.
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PUBLICATIONS

Du Plessis, L. & Soley, J.T. (2011) Incidence, structure and morphological classification of abnormal
spermatozoa in the emu (Dromaius novaehollandiae). Theriogenology 75, 589–601.

Du Plessis, L. & Soley, J.T. (2011) Head-base bending and disjointed spermatozoa in the emu
(Dromaius novaehollandiae): a morphological comparison of two closely related defects.
Theriogenology 76. 1275–1283.

Du Plessis, L. & Soley, J.T. (2012) Abaxial tail implantation in the emu, Dromaius novaehollandiae:
morphological character istics and origin of a rare avian sperm defect. Theriogenology 77, 1137–
1143.

Du Plessis, L. & Soley, J.T. (2012) Structural peculiarities associated with multiflagellate sperm in the
emu, Dromaius novaehollandiae. Theriogenology 78, 1094–1101.

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SUMMARY

The morphology and development of normal and abnormal spermatozoa
in the emu, Dromaius novaehollandiae

by

Lizette du Plessis

Supervisor: Prof. J.T. Soley

Department: Department of Anatomy & Physiology, Faculty of Veterinary Science,
University of Pretoria, Private Bag X04, Onderstepoort, 0110,
Republic of South Africa

Degree: Ph.D.

The morphological features and development of normal and abnormal sperm were studied in the
emu, Dromaius novaehollandiae, using both light and electron microscopy. Detailed descriptions of
normal as well as abnormal forms were documented. Where possible, the origin and development of
the defects were examined and interpreted. Based on these observations, a system for the
morphological classification of ratite sperm abnormalities was proposed.

Emu sperm typically revealed the basic filiform morphology which is common in other ratites and
non-passerine birds in general. At the ultrastructural level, emu sperm displayed a conical acrosome
covering the tip of the nucleus and a long distal centriole running the entire length of the midpiece.
Mitochondria of the pars spiralis were more numerous than in other ratites, but no intermitochondrial
cement was present in the mitochondrial sheath. Although sharing basic ultrastructural features, a
distinct difference between sperm of the emu and other ratites was the complete absence of a
perforatorium and endonuclear canal. Another distinctive feature revealed by all forms of microscopy
was the presence of a cytoplasmic appendage located at the base of the nucleus and which
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appeared to represent the point of cytoplasmic release during spermiation.
Based on the examination of a total of 15 semen samples collected during the middle of the breeding
season from the distal deferent ducts of commercially slaughtered adult emus, a total of 14%
morphologically abnormal cells were identified. Head defects were represented by bent,
microcephalic, macrocephalic or round heads. Acephalic sperm were also present in all samples.
Zones of incomplete chromatin condensation and retained cytoplasmic droplets appeared to be
implicated in head bending, while giant heads were often associated with multiple tails. In acephalic
sperm the tail was complete, but the head was absent, often being replaced by a small spherical
structure. Tail defects were subdivided into defects of the neck/midpiece and those of the principal
piece. In the neck/midpiece region two anomalies were noted, namely abaxial tail implantation and
disjointed sperm. Abaxial tail implantation involved the eccentric positioning of the centriolar complex
relative to the head base while disjointed sperm were characterized by the complete separation of
the head and midpiece in the neck region, but within the confines of the plasmalemma. Defects
observed in the principal piece were subdivided into short, coiled and multiple tails. Cytoplasmic
droplets were classified as a separate defect. A small percentage of sperm displayed multiple
abnormalities. Defective alignment and/or migration of the centriolar complex (diplosome) was
implicated in a number of the defects observed, including head-base bending, disjointed sperm,
acephalic sperm and abaxial sperm.

Spermiogenesis in the emu broadly paralleled the development of spermatids reported in other non-
passerine birds. However, a previously undescribed morphological feature of avian sperm
development was observed. During the circular manchette stage of spermatid development, a unique
transient structure appeared. It was associated with the outer nuclear membrane and formed regular
finger-like projections into the cytoplasm. This structure was present during both the circular and
longitudinal manchette stages of development and was particularly obvious towards the apical
aspect of the nucleus where it abutted the cone-shaped acrosome. There were no obvious
connections between the structure and the microtubules of the manchette. During the final stages of
spermatid development the structure abruptly disappeared. Despite employing immunogold labelling
techniques and electron tomography, the nature and function of this structure remained unresolved.
Descriptions of a similar structure in various lizard species and the Caiman crocodile, although
restricted in its location to the nuclear membrane immediately beneath the acrosome, would appear
to reinforce the phylogenetic link previously identified between birds and crocodiles.
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Chapter 1 General introduction and basic architecture of the
male reproductive tract

General background
Emus are the second largest living birds after the ostrich. Both belong to the flightless group of birds
called the ratites (order Struthioniformes, families Dromaiidae and Struthionidae, respectively). They
are native to Australia and are found predominantly in the state of Western Australia (Davies, 1963;
Serventy & Whittell, 1976). The first written description of the emu can be found under the common
name of ―New Holland cassowary‖ in Arthur Phillip‘s ―Voyage to Botany Bay” which was published in
1789 (Gould, 1865). The ornithologist, John Latham, who contributed towards Phillip‘s book, first
named and described the species Dromaius novaehollandiae, which means ‗fast footed New
Hollander‘. The origin of the common name, emu, dates back to the 17th century and originates from
an Arabic word for large bird, which the Portuguese explorers used to describe the related
cassowary in New Guinea (Anonymous, 2001). There were originally four different emu species,
three of which are now extinct. Apparently there was also one prehistoric species that is only known

from fossils. The extinction of the birds is ascribed to hunting, fires and change of habitat. Three

subspecies have been recognised within the remaining genus, D. novaehollandiae, namely, D.
novaehollandiae novaehollandiae, D. novaehollandiae woodwardi and D. novaehollandaie rothschildi
(Sales, 2007).

Commercial value
In Australia wild emus are considered a pest since the birds compete with other livestock for food
and water and trample and destroy crops when migrating (Minnaar & Minnaar, 1994; Thompson,
1997). During the early 1900‘s the Australian government set a bounty on emus and in 1932 even
attempted to exterminate the bird. Fences were put up to protect crops and livestock from migrating
emus and commercial farming of the birds was prohibited. Despite their status as pests, the
economic value of the birds has long been recognized. The meat was a source of food for the
Aboriginal people who also used the fat for medicinal purposes such as healing wounds (Whitehouse
et al., 1998; Sales, 2007) and to oil their wooden tools. The early European settlers used the fat as
fuel for their lamps. In 1987 the first permits were issued to a few farmers in Western Australia which
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made it possible to initiate the commercial farming of the birds, and the first farmed emus were
slaughtered three years later.

Today, the main commercial products are oil, meat and leather. Emu oil is harvested from the
subcutaneous and retroperitoneal fat of the bird (Sales, 2007) and has several pharmaceutical and
cosmetic applications (Zemtov et al., 1996; López et al., 1999; Yoganathan et al., 2003). It is
particularly valued for its anti-inflammatory properties (Thompson, 1997; Bennett et al., 2008;
Howarth et al., 2008). As in the ostrich, the meat is low in cholesterol and fat. The leather has a
distinctive pattern and is used for making shoes, wallets and other leather products, while the eggs
and feathers are used in the decorative arts and crafts.

In common with other niche industries world-wide such as ostrich and crocodile farming, the
commercial raising of emus is a relatively small enterprise and is severely hampered by high
production costs. One suggestion to overcome these inherent constraints is to educate the public
about emu products and to aim at exclusive, higher value markets (Sales, 2007). Despite these
drawbacks, the commercial farming of emus is now a world-wide enterprise. The first emus were
exported to the USA between 1930 and 1950 as exotic zoo animals. It has since become a viable
industry in many countries, including North America, China, Peru and South Africa (Wikipedia, 2012;
personal communication with farmers). Emu farmers, particularly the fledgling South African

operation, appear to be suffering the same teething problems as those experienced by the now well-

established ostrich industry regarding reproductive performance.

Breeding of emus
The ratite industry currently relies on natural reproduction which is not always cost effective, partly
due to the monogamous nature of ratites (Malecki et al., 1998a; 2000). This means that a large
number of males must be kept for the sole purpose of breeding, which adds an unnecessary financial
burden to farming operations. The emu is a seasonal breeder and breeding, in the southern
hemisphere, is restricted to autumn and winter (Malecki, et al., 1998a; personal communication with
farmers). Mating usually starts towards the end of summer, with eggs being laid from autumn through
to the spring months. The peak season is in winter (O‘Malley, 1994). Males often become quite
aggressive during the breeding season and can cause serious injuries to each other which leads to
additional veterinary expenses. In order to address these problems, it has been suggested that the
ratite industry should employ advanced reproductive technologies such as artificial insemination (AI),
which has been successfully employed in commercial poultry enterprises (King et al., 2000; Foote,
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