β-carotene Supplementation Increases Progesterone Concentration and Glutathione Peroxidase Activity Following Alternative Progesterone Primed Oestrous Synchronization Protocol in Goats

Abstract

The present study evaluated the effect of β-carotene supplementation and oestrous synchronization protocol on ovarian activity and fertility of Saanen does during the breeding season. The supplemented group received 100 mg β-carotene during the breeding and all does were synchronised with Controlled Internal Drug Release dispenser (CIDR) and injected with cloprostenol at CIDR withdrawal. One group of does were injected with 300 IU of eCG, while in another group bucks wearing aprons were introduced at CIDR withdrawal. Does were artificially inseminated twice (48 and 60 h) with fresh undiluted semen. The onset and duration of oestrus, progesterone, oestrdiol-17β and glutathione peroxidase activity, oestrous response and conception rate were analysed. Synchronization protocol did not affect response to oestrus, onset and duration of oestrus and oestradiol-17β concentration. The male presence group had significantly higher conception rate (97%) than the eCG (72%) group. β-carotene supplemented group had higher progesterone concentration and glutathione peroxidase activity. Supplemental β-carotene during the breeding period therefore, could play an important role on establishment of pregnancy due to high progesterone concentration and glutathione peroxidase activity. Inclusion of male effect in progesterone based oestrous synchronization protocol improves conception rate. Therefore, male effect can be used as an alternative to equine chorionic gonadotropin in progesterone based oestrous synchronization protocols especially, where drugs for oestrous synchronization are not affordable.

Full text

© 2020 Dominic Lado Marino Gore and Khoboso Christina Lehloenya. This open access article is distributed under a
Creative Commons Attribution (CC-BY) 3.0 license.
American Journal of Animal and Veterinary Sciences
Original Research Paper
β-carotene Supplementation Increases Progesterone
Concentration and Glutathione Peroxidase Activity Following
Alternative Progesterone Primed Oestrous Synchronization
Protocol in Goats
1,2Dominic Lado Marino Gore and 3*Khoboso Christina Lehloenya
1Department of Animal and Wildlife Sciences, University of Pretoria, Hatfield, 0002, South Africa
2Department of Animal Production, University of Juba, P.O. Box 82 Juba, South Sudan
3Department of Agriculture, University of Zululand, Private Bag X1001, KwaDlangezwa 3886, South Africa
Article history
Received: 19-05-2020
Revised: 27-06-2020
Accepted: 07-08-2020
Corresponding Author:
Khoboso Christina Lehloenya,
Department of Agriculture,
University of Zululand, Private
Bag X1001, KwaDlangezwa
3886, South Africa
Email: LehloenyaK@unizulu.ac.za
khoboso.lehloenya@gmail.com
Abstract: The present study evaluated the effect of β-carotene
supplementation and oestrous synchronization protocol on ovarian activity
and fertility of Saanen does during the breeding season. The supplemented
group received 100 mg β-carotene during the breeding and all does were
synchronised with Controlled Internal Drug Release dispenser (CIDR) and
injected with cloprostenol at CIDR withdrawal. One group of does were
injected with 300 IU of eCG, while in another group bucks wearing aprons
were introduced at CIDR withdrawal. Does were artificially inseminated
twice (48 and 60 h) with fresh undiluted semen. The onset and duration of
oestrus, progesterone, oestrdiol-17β and glutathione peroxidase activity,
oestrous response and conception rate were analysed. Synchronization
protocol did not affect response to oestrus, onset and duration of oestrus
and oestradiol-17β concentration. The male presence group had
significantly higher conception rate (97%) than the eCG (72%) group. β-
carotene supplemented group had higher progesterone concentration and
glutathione peroxidase activity. Supplemental β-carotene during the
breeding period therefore, could play an important role on establishment of
pregnancy due to high progesterone concentration and glutathione
peroxidase activity. Inclusion of male effect in progesterone based oestrous
synchronization protocol improves conception rate. Therefore, male effect
can be used as an alternative to equine chorionic gonadotropin in
progesterone based oestrous synchronization protocols especially, where
drugs for oestrous synchronization are not affordable.
Keywords: Artificial Insemination, Buck Effect, eCG, Glutathione Peroxidase
Activity, Oestradiol-17β, Progesterone
Introduction
Nutrition affects all aspects of reproductive events
from gametogenesis to puberty in both males and
females (Scaramuzzi et al., 2006). In females, the most
prominent effect is around mating period influencing
the wave-like pattern of follicle development, ovulation
rate, embryo survival and twining rate (Viñoles Gil,
2003). Short-term supplementation with maize and
lupin before ovulation was reported to increase
ovulation rate (Nottle et al., 1990; Nogueira et al.,
2017). The period post ovulation is also very crucial for
survival of embryos in farm animals. Many reports have
associated the concentration of systemic progesterone
with early embryo loss and that progesterone
supplementation in cows, particularly those with low
progesterone concentration, can reduce this loss
(Morris and Diskin, 2008). Most embryonic mortality
occurs between day 8 and 16 in cattle (Diskin and
Sreenan, 1980) and during the first four weeks of
pregnancy in sheep (Petrović et al., 2012).
β-carotene is a primary precursor for vitamin A, but
also has been noted to play specific roles on some
aspects of ovarian activity such as being an antioxidant

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DOI: 10.3844/ajavsp.2020.211.219
212
for quenching singlet oxygen and scavenging peroxyl
radicals (Sies and Stahl, 1995) which are harmful during
steroidogenesis (Arellano-Rodriguez et al., 2009;
Meza-Herrera et al., 2013a; 2013b). As an antioxidant,
β-carotene supplementation has been reported to
decrease oxidative stress (Otomaru et al., 2018) and
increase total protein, glucose and cholesterol, while
decreases urea concentrations in the female goat
(Meza-Herrera et al., 2017). β-carotene has also been
implicated in the luteal cells function for the synthesis of
progesterone in goats (Arellano-Rodriguez et al., 2009).
Additionally, high concentration of β-carotene in corpora
lutea is reported to promote ovarian steroidogenesis and
increases plasma progesterone concentration in goats and
cats (Weng et al., 2000; Arellano-Rodriguez et al., 2009;
Meza-Herrera et al., 2013a). Studies have reported the
positive effect of β-carotene on the response to oestrus,
improved ovulation rate and conception rate in cattle and
goats (Aréchiga et al., 1998; de Ondarza et al., 2009;
Arellano-Rodriguez et al., 2007; 2009; Meza-Herrera et al.,
2013b; López-Flores et al., 2018). However, other
studies have refuted the positive effect of β-carotene on
ovarian activity and fertility. For example, plasma
progesterone concentration, incidence and duration of
oestrus and first conception rate were not influenced by
supplemental β-carotene in cattle (Wang et al., 1988b).
In addition, there were no effects of β-carotene
supplementation observed on progesterone production,
ovarian activity, cervix and uterine diameters in cows
(Kaewlamun, 2010). Due to the conflicting results and
limited studies in goats concerning β-carotene effect on
reproduction, more studies are necessary in order to
ascertain its effects on reproduction. Concerning few
studies in goats, β-carotene supplementation improved
follicular development, ovulation rate and progesterone
production (Arellano-Rodriguez et al., 2009;
Meza-Herrera et al., 2013b; López-Flores et al., 2018).
Progesterone or its analogues in combination with
eCG as a co-treatment has been widely used for oestrous
synchronization in goats. However, repeated eCG
treatments over a period of time are followed by
decreased fertility in inseminated goats (Baril et al.,
1996; Roy et al., 1999). The decreased fertility is
attributed to the presence of circulating anti-eCG
antibodies in the plasma of goats treated with eCG. As
a result, there is a need to find alternative co-
treatments. In this study we evaluated the possibility of
replacing eCG with the male effect in a synchronization
protocol, as the male effect induce similar attributes to
eCG such as the increase of both FSH and LH and
triggering ovulation (Ungerfeld, 2003).
Given the above positive attributes of β-carotene and
male effect, we hypothesized that supplemental β-
carotene and male effect would positively influence the
response to oestrus and fertility in goats. Therefore, the
aim of this study was to evaluate the effect of short-term
β-carotene supplementation around the breeding period
on ovarian activity and fertility of Saanen goats
following synchronised oestrus using male effect and
eCG progesterone based protocol.
Materials and methods
Ethical Approval
This study was approved by the animal ethics
committee of the University of Pretoria (Project
no.EC108-14).
Experimental Site
The study was undertaken at the experimental farm of
the University of Pretoria, South Africa. The farm lies
between latitude 25°44’30” S and longitude 28°15’30”
E, with an elevation of 1360 meters above sea level
(Van Niekerk et al., 2009).
Experimental Animals and Management System
Sixty (60) Saanen does of age between 1-6 years were
used for this study. The goats were managed under
intensive system and fed with a Total Mixed Ration (TMR),
the ingredients are presented in Table 1. Water access was
ad libitum throughout the duration of the study.
Experimental Design and Treatments
The does were allocated into two groups on their
weight and parity; Group 1: β-carotene supplemented (n
= 30) and Group 2: Non-supplemented (n = 30) group.
Further, the experimental groups were sub-divided into
two oestrous synchronization protocols; (1) Progesterone
(CIDR) + Prostaglandin (PGF2α) + equine chorionic
gonadotropin (eCG) and (2) Progesterone (CIDR) +
Prostaglandin (PGF2α) + Male effect (ME).
β-Carotene Supplementation
For the β-carotene groups, goats were supplemented
with β-carotene (100 mg/goat/day) (Pennville Pty Ltd,
Gauteng, South Africa) orally according to the company
instructions, for a period of 58 days starting 28 days
before oestrous synchronization and 17 days post artificial
insemination (AI). The control group received water as
placebo with similar quantities as in the treatment group.
Table 1: Feed ingredients for the total mixed ration
Ingredients Dry matter % Quantity in kg/animal
Lucerne hay 43.81 1.600
Eragrostis curvula hay 24.10 0.900
Maize meal 16.06 0.600
Molasses 9.05 0.350
Protein concentrate 6.98 0.250
Total 100.00 3.700

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DOI: 10.3844/ajavsp.2020.211.219
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Oestrus Synchronization Protocols
The bucks were kept away from females for a
duration of one month before the onset of the study.
Thereafter, all the does were intravaginally inserted with
Controlled Internal Drug Release dispenser (CIDR)
(Pfizer, New Zealand) containing 0.3 g progesterone and
the CIDRs were removed after a duration of 11 days. All
does were injected with 150 µg cloprostenol (Intervet
Schering-Plough Animal Health, South Africa) at CIDR
withdrawal. In addition, at CIDR withdrawal does were
divided into two groups: In the first one does were
injected with 300 IU (2.5 mL) of eCG (Intervet
Schering-Plough Animal Health, South Africa) and in
the second does were mixed with two bucks fitted with
aprons in another group and left with the does for 72 h.
Semen Collection and Artificial Insemination (AI)
Semen Collection
Semen from bucks was harvested using an electro
ejaculator (Ramsem, South Africa) following procedures
described by (Sundararaman et al., 2007). The rectal
probe was inserted in the rectum and following the
insertion of the rectal probe, buck was stimulated
through massaging and the machine button was pressed
to generate a voltage of 3-5 volts and paused for 4-5 sec
and again brought back to 0. It is replicated until a buck
discharged semen. The semen ejaculate volume was
recorded and semen mass motility was evaluated as
described by (Dogan et al., 2005). Ten (10) µL of semen
harvested from each buck was placed on a glass slide
and evaluated using a microscope (Olympus Cx21).
Only semen ejaculate scoring motility of 3 and beyond
were selected and used for insemination of the does.
Artificial Insemination
Does were artificially inseminated using the
method outlined by (Steyn, 2005). Briefly, each doe
was inseminated with 0.2 mL fresh undiluted semen
with a concentration between 300-800×106 sperm
cells/ml as outlined by (Lehloenya et al., 2005) with
modifications. All does were cervically inseminated,
at fixed times of 48 and 60 h following CIDR
withdrawal (Motlomelo et al., 2002).
Data Collection and Analysis
Blood Sampling for Hormonal and Enzyme Assay
Blood samples from 5 goats in each sub-group were
collected. The blood samples were collected at CIDR
insertion, 8 h and 48 h after CIDR removal as well as 12
days post AI. Immediately, following collection, the
blood samples were centrifuged at 3000× g for 20 min.
The blood plasma recovered was then stored at -20°C
until analysed for progesterone, oestradiol-17β and
glutathione peroxidase activity.
Analysis of Hormones and Enzyme Activity
The analyses were done using FC Microplate
Photometer Thermo Scientific Multiskan®. The plasma
progesterone and oestradiol-17β concentrations were
analysed using progesterone ELISA DE1561 kit
(Demeditec-Germany) with intra and inter assay
variability of 5.4-6.86 and 4.34-9.96 and oestradiol-17β
ELISA DE2693 kit (Demeditec-Germany) with 2.71-
6.81 and 6.72-9.39, respectively, while glutathione
peroxidase activity was analysed using glutathione
peroxidase assay kit (ab102530-Abcam) with minimum
detection sensitivity of 0.5 mU/mL.
Monitoring the Onset and Duration of Oestrus
The onset and duration of oestrous were monitored
following CIDR removal using two aproned buck for a
duration of 72 h at 8 h interval (06:00, 14:00 and 22:00 h
for 1 h period) according to (Lehloenya et al., 2005;
Ramukhithi et al., 2012). A doe considered to be on
oestrus when accepted mounting by the buck and out of
oestrus when it did not allow to be mounted.
Ultrasonographic Evaluation of Pregnancy
Pregnancy detection was done 35 days following
artificial insemination using real-time B-mode ultrasound
scanner (Aloka, 500 SSD, Japan) fitted with a transrectal
7.5-MHz linear array probe (UST-660-7.5 model).
Statistical Analysis
The onset and duration of oestrus, progesterone,
oestrdiol-17 β and glutathione peroxidase activity were
analysed using Analysis of Variance (ANOVA) and the
oestrous response and conception rate were analysed
using Chi-square test procedures of (SAS, 2014).
Significance level was set at P<0.05.
Results
One goat was removed from the experiment due to
reproductive tract abnormalities. The response to oestrus,
onset and duration of oestrus and conception rate were
not significantly different (P>0.05) between the
supplemented and control group (Table 2).
Additionally, most does expressed oestrus signs 32
h following CIDR removal in both groups (Fig. 4).
Supplemental β-carotene however, increased
significantly (P<0.05) the progesterone concentration at
day 12 following Artificial Insemination (AI) (Fig. 1).
Oestradiol-17 β concentrations were not affected by
supplemental β-carotene throughout the period of
measurement but result not presented. The
concentration of oestradiol -17β at CIDR withdrawal, 8
h after CIDR withdrawal, 48 h after CIDR withdrawal
and 12 days after CIDR withdrawal for β-carotene
supplemented group were 1.39±0.24, 3.90±0.13,

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9.12±0.42 and 0.69±0.16 and for control group were
1.47±0.24, 3.85±0.13, 8.70±0.42 and 0.75±0.16,
respectively. The glutathione peroxidase activity was
significantly (P<0.05) higher in β-carotene supplemented
goats at CIDR insertion, 8 h and 48 h following CIDR
withdrawal and at day 12 following AI (Fig. 2).
Table 2: Effect of β-carotene supplementation and synchronization protocol on estrous response and conception rate of Saanen does
(Mean ± SE)
No of does Oestrous Onset of Duration of Conception
responded response (%) oestrus (h) oestrus (h) rate (%)
Supplementation
β-carotene supplemented 28/29 96.550 28.41±0.91 30.06±0.81 86.670
Control 30/30 100.000 26.00±0.91 30.04±0.81 83.330
P-value 0.980 0.061 1.00 0.978
Synchronization
eCG 29/30 97.000 28.29±0.94 29.43±0.83 72.000
Me 30/30 100.000 26.13±0.90 30.67±0.80 97.000
P-value 0.980 0.104 0.287 0.028
Interaction
β-carotene supplemented × eCG 13/14 92.860 28.00±1.32 29.14±1.17 71.430
β-carotene supplemented × Me 15/15 100.000 24.00±1.32 30.93±1.13 93.330
Control × eCG 15/15 100.000 28.57±1.32 29.71±1.17 73.330
ControlxMe 15/15 100.000 28.27±1.28 30.40±1.13 100.000
P-value 0.980 0.161 0.633 0.759
Fig. 1: Effect of supplemental β-carotene on the progesterone (P4) concentration of Saanen goats. Bars with different letters are
significantly different (P<0.05; a,b significant within a time)
Fig. 2: Effect of supplemental β-carotene on Glutathione Peroxidase activity (GPx) of Saanen goats. Bars with different letters are
significantly different (P<0.05; a,b significant within a time)
a
P4 concentration (ng/ml)
10
9
8
7
6
5
4
3
2
1
0
At CIDR
insertion
a
a
a
a
a
a
b
8 h after CIDR
withdrawal
48 h after CIDR
withdrawal
12 d after artificial
insemination
Treatment group Control group
GPx activity (mU/ml)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
At CIDR
insertion
8 h after CIDR
withdrawal
48 h after CIDR
withdrawal
12 d after artificial
insemination
Treatment group Control group
a
a
a
a
b
b
b
b

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Fig. 3: Effect of synchronization protocols (Male effect (Me) and equine chorionic gonadotropin (eCG)) on response to oestrus in
Saanen goats. (P<0.05;a,a not significant within a time)
Fig. 4: Effect of β-carotene supplementation on response to oestrus in Saanen goats. (P<0.05; a,a not significant within a time)
On oestrous synchronization protocols, there was no
significant difference (P>0.05) (Table 2) between the eCG
and male effect group on the response to oestrus, onset
and duration of oestrus. However, in male effect group,
does expressed oestrus earlier than the eCG group and
majority of does expressed oestrus 32 h following CIDR
removal as shown in (Fig. 3). The oestrous
synchronization protocol had a significant effect (P<0.05)
on conception rate (Table 2). The male effect group had
significantly (P<0.05) higher conception rate than the eCG
group. The interactions were tested but all not significant.
Discussion
The current study has partly confirmed the hypothesis
that β-carotene supplementation and male effect based
oestrous synchronization protocol would positively
influence hormonal concentration, response to oestrus and
fertility in goats. The study found that supplemental β-
carotene increased progesterone concentration and
glutathione peroxidase activity, in goats. Additionally, the
study revealed that male effect based oestrous
synchronization significantly improved conception rate in
goats. Other parameters measured in the current study
however, were not affected by β-carotene supplementation
and male effect based oestrous synchronization protocol.
The high concentration of progesterone following
supplemental β-carotene in the current study agrees with
findings of other researchers (Weng et al., 2000;
Arellano-Rodriguez et al., 2009). This increase in
concentration of progesterone on day 12 following
artificial insemination could be attributed to implication
of β-carotene in progesterone synthesis through the
steroidogenic process. β-carotene concentration
increases in a dose dependent manner in Corpus Luteum
(CL) (Weng et al., 2000) and this could play role in
Percentage of does on oestrus
120
100
80
60
40
20
0
8 16 24 32 40 48 56 64 72
Synchronization Me Synchronization eCG
Time (hours following CIDR withdrawal)
a
a
a
a
a
a
a
a
a
a
Percentage of does on oestrus
120
100
80
60
40
20
0
8 16 24 32 40 48 56 64 72
Control Treatment
Time (hours following CIDR withdrawal)
a
a
a
a
a
a
a
a
a
a

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progesterone synthesis and prevention of lipid
peroxidation (Young et al., 1995; Arikan and Rodway,
2000; Weng et al., 2000). Lack of β-carotene effect on
progesterone concentration 8 and 48 h following CIDR
removal could be attributed to the fact that there was no
active CL and as such no synthesis of progesterone was
taking place.
The high GPx activity following supplemental β-
carotene in the current study agrees with other researchers
(Kamİloğlu et al., 2005) who observed improved
antioxidant activity of GPx in sheep before breeding and
during pregnancy period. This improvement in GPx
activity is attributed to increase in protection from oxygen
free radicals during oxidative stress (Festila et al., 2012).
However, supplemental β-carotene in the current study did
not improve concentration of oestradiol-17β and this
concurs with a study (Weng et al., 2000).
The current study did not influence conception rate
following β-carotene supplementation and this is in
agreement with some studies in cows (Bindas et al.,
1984; Wang et al., 1988a; 1988b). There are however,
other studies that disagreed with the current findings
who reported improved fertility rate following β-
carotene supplementation in cows (Aréchiga et al., 1998;
De Ondarza et al., 2009). These discrepancies could be
attributed to the species differences, goats are efficient
converters of β-carotene in their intestinal mucosa
compared to cattle (McDowell, 2000). Due to this reason,
cattle have more circulating β-carotene concentration in
their body tissues than in goats (Yang et al., 1992) and
thus the improved fertility in cattle could be attributed to
high β-carotene concentration which could influence the
ovarian activities positively.
Oestrous synchronization protocol did not
significantly influence oestrus response, onset and
duration of induced oestrus in the current study. This
could be attributed to the fact that both eCG and male
effect of possessed similar attributes of inducing
ovulation (Leboeuf et al., 1998). In line with the findings
of the current study, supplemental β-carotene did not
affect response to oestrus and onset of oestrus in cattle
(Wang et al., 1987; Aréchiga et al., 1998). In
disagreement with the current study, supplemental β-
carotene reduced the incidence of onset of oestrus
(Wang et al., 1982). The current study agrees with the
findings of previous studies who reported no effect of
supplemental β-carotene on the duration of oestrus
(Wang et al., 1982; 1988b). There was however, a
positive effect of oestrous synchronization protocol on
conception rate.
Interestingly, the male effect based protocol had
significantly high conception rate compared to the eCG
based protocol. The high conception rate in male effect
group could be attributed to the following: Firstly, the
onset of oestrus was slightly earlier in the male effect
group and consequently the time of artificial
insemination was more appropriate for male effect group
than eCG group. Secondly, the physical presence of male
is associated with lower stress in female animals. Male
effect is linked with the stable secretion pattern of
cortisol, whereas vomerolectomized goats experienced
increased concentration of cortisol due to failure in
sensing the male presence (Jansen van Vuuren, 2015).
The known link of stress (based on cortisol
concentration) is that cortisol releasing hormone is
directly affected by a similar pathway that stimulates the
release of gonadotropin releasing hormone and its
inhibition is a secondary effect to luteinizing hormone
and estradiol (Swenson and Reece, 1993). This positive
role of male existence on conception rate may possibly
encourage the use of the male effect when synchronising
oestrus in progesterone primed goats to avoid the
intensive application of eCG. The eCG has been associated
with reduced fertility in goats when used repeatedly
(Baril et al., 1996; Roy et al., 1999). Therefore, male
effect offers an alternative to eCG in progesterone based
synchronization protocols during the breeding season.
The option would not only reduce the quantity of
hormones used in a synchronization protocol but also
maintain fertility, since synchronization of oestrus using
eCG is implicated in reducing fertility over time,
attributed to the presence of circulating anti-eCG
antibodies in the plasma of goats treated with eCG
(Baril et al., 1996; Roy et al., 1999).
Conclusion
Supplemental β-carotene did not influence ovarian
activity and conception rate. β-carotene
supplementation however, improved plasma
progesterone concentration and GPx activity. The male
effect based oestrous synchronization protocol
improved conception rate compared to eCG based
protocol. Supplemental β-carotene during the breeding
period therefore, could play an important role on
establishment of pregnancy due to the high
progesterone concentration and glutathione peroxidase
activity. Inclusion of male effect in progesterone based
oestrous synchronization protocol improves conception
rate. Therefore, male effect can be used as an alternative
to equine chorionic gonadotropin in progesterone based
oestrous synchronization protocols especially, where
drugs for oestrous synchronization are not affordable.
Acknowledgment
The authors acknowledge the Office of International
Research, Education and Development (OIRED) through
Rebuilding Higher Education in Agriculture (RHEA)
project implemented by Virginia Tech and Borlaug
Higher Education for Agricultural Research and
Development (BHEARD) project implemented by

Dominic Lado Marino Gore and Khoboso Christina Lehloenya / American Journal of Animal and Veterinary Sciences 2020, 15 (3): 211.219
DOI: 10.3844/ajavsp.2020.211.219
217
Michigan State University under the umbrella of USAID
for providing financial assistance for this study as well
as the University of Pretoria for providing the
experimental animals.
Funding Information
This material is based upon work supported by the
United States Agency for International Development, as
part of the Feed the Future Initiative, under the CGIAR
Fund, award number BFS-G-11-00002 and the
predecessor fund the Food Security and Crisis Mitigation
II grant, award number EEM-G-00-04-00013.
Author’s Contributions
Dominic Lado Marino Gore: Contributed on the
original ideas of the manuscript, data collection, analysis
and interpretation and manuscript write up.
Khoboso Christina Lehloenya: Contributed on the
original ideas and preparation of the manuscript.
Ethics
The authors confirm that the manuscript has not been
published or not under consideration elsewhere for
publication in another journal. This work is part of the
master’s dissertation of the first author and the second
author was the supervisor.
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