Semen characteristics of free-ranging African elephants (Loxodonta africana)

301 Pages·2017·7.14 MB·English

by  Ilse Luther

Semen characteristics of free-ranging African elephants (Loxodonta
africana) and Southern white rhinoceros (Ceratotherium simum simum)
using Computer-aided sperm analysis, Electron microscopy and
Genomics as diagnostic tools

by
Ilse Luther

A thesis submitted in partial fulfilment of the requirements
For the degree of Doctor of Philosophy in the Department of Medical Bioscience,
University of the Western Cape

July 2016
Supervisor: Prof Gerhard van der Horst
Co-supervisors: Dr Liana Maree and Prof Antoinette Kotze
Semen characteristics of free-ranging African elephants (Loxodonta africana) and Southern
white rhinoceros (Ceratotherium simum simum) using Computer-aided sperm analysis,
Electron microscopy and Genomics as diagnostic tools

Ilse Luther

KEYWORDS
Sperm characteristics
Sperm motility
Sperm kinematics
Sperm morphology
Sperm morphometric
African elephant (Loxodonta africana)
Southern white rhinoceros (Ceratotherium simum simum)
Computer-aided sperm/semen analysis (CASA)
Electron microscopy (EM)
Genomics
CatSper1 gene (Loxodonta africana)

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ABSTRACT
Semen characteristics of free-ranging African elephants (Loxodonta africana) and
Southern white rhinoceros (Ceratotherium simum simum) using Computer-aided
sperm analysis, Electron microscopy and Genomics as diagnostic tools
Ilse Luther
PhD, Department of Medical Bioscience, University of the Western Cape

The survival of free-ranging (in situ) African elephant and Southern white rhinoceros populations are
currently being challenged on a daily basis in Africa. Reproductive health is considered a vital
component of species conservation. Conservation of the last mega land mammals may ultimately
require intervention by breeding management or combined with assisted reproductive technologies
(ART). There is a strong case for gathering baseline information, both physiological and biological, of
any species, as opportunities arise.
During this study a total number of 21 ejaculates collected over two seasons from 12 free-ranging
African elephant bulls were characterised, as well as 10 ejaculates collected from 10 free-ranging
Southern white rhinoceros bulls from two populations. Ejaculates were collected from adult bulls by
means of electroejaculation under anaesthesia. Routine semen analysis was combined with
Computer-aided sperm analysis (CASA), Computer-aided sperm morphology analysis (CASMA),
Transmission electron microscopy (TEM) and Genomics as diagnostic tools. Additionally, sperm
functionality within different media was investigated and sperm subpopulation classification according
to the motion pattern displayed. The results presented is based on the evaluation and classification
of ≈ 45 000 individual African elephant spermatozoa and ≈ 18 000 individual Southern white
rhinoceros spermatozoa.
The average elephant ejaculate contained a total number of 47 x 109 spermatozoa (volume of 56 ±
38mL x concentration of 818 ± 750 x 106/mL) that recorded a total motility of 81 ± 29% of which 62 ±
26% were progressively motile. CASA recorded velocities for curvilinear velocity (VCL 241 ± 58µm/s),
straight-line velocity (VSL 173 ± 181µm/s) and average path velocity (VAP 201 ± 54µm/s), and
kinematics at straightness of track (STR 86 ± 85%), linearity of track (LIN 67 ± 16%), amplitude of
lateral head displacement (ALH 4 ± 0.75µm) and beat cross frequency (BCF 21 ± 3Hz). Structural
analysis revealed 68 ± 11% of the spermatozoa were viable (intact plasma membrane) and 77 ± 11%
maintained acrosome integrity. Ejaculates contained 55 ± 14% morphologically normal spermatozoa,
CASMA measured sperm head lengths at 6.83 ± 0.26µm and width 3.32 ± 0.18µm (total head area
of 20.17 ± 1.96µm2) of which 38.95 ± 0.92% is covered by an acrosomal cap.
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The average rhinoceros ejaculate contained a total number of 1.1 x 109 spermatozoa (volume of 24 ±
24mL x concentration of 83 ± 96 x 106/mL) that recorded a total motility at 82 ± 8% of which 28 ± 23%
were progressively motile. CASA recorded velocities for VCL (85 ± 29µm/s), VSL (44 ± 25µm/s) and
VAP (69 ± 30µm/s, and kinematics at STR (63 ± 14%), LIN (51 ± 16%), ALH (2 ± 0.16µm) and BCF
(16 ± 6Hz). Structural analysis revealed 73 ± 10% of the spermatozoa were viable (intact plasma
membrane) and 76 ± 4% maintained acrosome integrity. Ejaculates contained 62 ± 14%
morphologically normal spermatozoa, CASMA measured sperm head lengths at 5.5 ± 0.17µm and
width 2.9 ± 0.19µm (total head area of 14.8 ± 1.43µm2) of which 36.3 ± 0.59% is covered by an
acrosomal cap.
Based on a Boolean argument and CASA data exploration it was possible to derive elephant and
rhinoceros CASA cut-off criteria to sort between activated and hyperactivated motile spermatozoa.
For the genomic component of this study, the CatSper1 (Loxodonta africana) gene was identified,
sequenced and verified in a free-ranging (natural) African elephant population. Multivariate analysis
(MVA) was applied to examine the associations between the semen and sperm parameters and the
traits they accounted for in this study. Our understanding of wildlife reproductive sciences can
substantially progress as the analytical techniques applied and the combination thereof is expanded.
This investigation presents a new set of comprehensive semen and sperm threshold values for future
investigations.

July 2016

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DECLARATION

Hereby I, the undersigned, declare that the thesis “Semen characteristics of free-ranging African
elephants (Loxodonta africana) and Southern white rhinoceros (Ceratotherium simum simum) using
Computer-aided sperm analysis, Electron microscopy and Genomics as diagnostic tools.” is my own
work, that it has not been submitted previously for any degree or examination at any other university,
and that all the sources I have used or quoted have been indicated and acknowledged as complete
references.

Ilse Luther

31 July 2016
Signed: ………………………... Date: …………………….

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“If I have seen further than others, it was by standing on the shoulders of Giants”
Isaac Newton

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ACKNOWLEDGEMENTS

To all the giants. With the highest regard and utmost respect thank you very much.
Prof Gerhard van der Horst (University of the Western Cape), Dr Liana Maree (University of the
Western Cape) for your guidance, assistance, patience and ongoing enthusiasm during this study.
Mr Christiaan Labuschagne (Inqaba Laboratories), Dr Sahar Abdul-Rasool (University of the Western
Cape), Ms Majda Valjavec-Gratian (NIH/NLM/NCBI), Me Nolan Muller (Electron microscopy Unit,
Tygerberg Hospital), Prof Martin Kidd (Centre for Statistical Consultation, Stellenbosch University),
Dr Helene Steenkamp (NZG), Dr Imke Lueders (Geolifes), Prof Johan Terblanche (University of
Pretoria), Dr Carla Langley (Equis Veterinary Practise).
Thank you for all those involved in making the sampling opportunity possible from free-ranging
elephant and rhinoceros populations; Dr Douw Grobler (Catchco), Dr Romain Potier (Zooparc du
Beauval, Paris), Barbara Baker (Pittsburgh Zoo and Aquarium, USA.), Dr Thomas Hildebrandt
(Institute for Zoo and Wildlife Research, Berlin), Dr Frank Goeritz (Institute for Zoo and Wildlife
Research, Berlin), Dr Robert Hermes (Institute for Zoo and Wildlife Research, Berlin), Jitte Dietrich
(Institute for Zoo and Wildlife Research, Berlin).
Special thank you to the National Research Foundation of South Africa (NRF) for funding a three-
year scholarship under the DST/NRF Professional Development Programme and providing the
National Zoological Garden of South Africa (NZG) as a platform that allowed me to pursue this study.
Thank you to all the giants of the Section Reproduction, Veterinary Faculty, University of Pretoria,
Onderstepoort (2001 – 2008) for teaching me the basic principles of Veterinary Science.
To the foundation on which all past, present and future giants stand;
My parents and family, whom without this accomplishment would not be possible. Thank you for your
constant never-ending support, motivation and presence during this journey.

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LIST OF ABBREVIATIONS

°C Degree Celsius
µL Microliter
µm Micrometre
AC Acrosome
ALH Amplitude of lateral head displacement
AN Annulus
APV Apoptotic vesicle
ART Assisted Reproductive Techniques
AV Artificial Vagina
AX Axoneme
BC Berlin Cryoextender
BCF Beat Cross Frequency
BSA Bovine Serum Albumin
CAP Capitulum
CASA Computer-Aided Sperm Analysis
CASMA Computer-Aided Sperm Morphology Analysis
CD Cytoplasmic Droplet
CPP Coiled Principal Piece
DCT Distal Centriole
DD Dag Defect
EE Electroejaculation
EY Egg Yolk
EY_H Egg yolk and Ham’s F10
EY_I Egg yolk and INRA96®
FB Fibrous Sheath
H Ham’s F10
HA Hyperactive Motility
HA C Hyperactive Circular Motility
HA S Hyperactive Starspin Motility
I INRA96®
IF Implantation Fossa
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IU Unit International
IUCN International Union for Conservation of Nature
IZW Institute for Zoo and Wildlife Research, Berlin, Germany
kV Kilovolt
LIN Linearity
LIN Linearity of Track
M Midpiece
mA Milli Ampere
mg Milligram
mL Millilitre
MS Manual Stimulation
N Nucleus
Non-HA Non-Hyperactive Motility
NRF National Research Foundation of South Africa
NT Neat
NT_BO Neat semen and BO media
NT_H Neat and Ham’s F10
NT_I Neat and INRA96®
NZG National Zoological Gardens of South Africa
ODF Outer Dense Fibres
PAS Post Acrosomal Sheath
PCT Proximal Centriole
pH- Negative phase contrast objective
PM Progressive Motility
PM Sperm Plasma Membrane
PP Principal Piece
RNE Redundant Nuclear Envelope
S.A. South Africa
SCA® Sperm Class Analyser®
STR Straightness of Track
TEM Transmission Electron Microscopy
TM Total Motility
US University of Stellenbosch
UWC University of the Western Cape
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V Volt
V Nuclear Vacuoles
v/v Volume per Volume
VAP Average Path Velocity
VCL Curvilinear Velocity
VSL Straight-Line Velocity
W/V Weight per Volume
WOB Wobble
α Alpha

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