AABArchives Animal BreedingAABArch. Anim. Breed.2363-9822Copernicus PublicationsGöttingen, Germany10.5194/aab-59-139-2016Testosterone and cortisol patterns and the effects of electro-ejaculation
and copulation in Awassi ramsAlomarMazenscientific@aec.org.sySoukoutiAhmadAlzoabiMohee AldenZarkawiMoutazDivision of Animal Production, Department of Agriculture, Atomic Energy
Commission, P.O. Box 6091, Damascus, SyriaMazen Alomar (scientific@aec.org.sy)14March20165911391449November20158February201619February2016This work is licensed under a Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/This article is available from https://aab.copernicus.org/articles/59/139/2016/aab-59-139-2016.htmlThe full text article is available as a PDF file from https://aab.copernicus.org/articles/59/139/2016/aab-59-139-2016.pdf
The effects of month, electro-ejaculation (EE) and copulation
process on testosterone and cortisol levels were investigated in Syrian
Awassi rams. Jugular blood samples were collected from 10 rams at weekly
intervals for 1 year. During the breeding and non-breeding season,
samples were collected 60 min before EE and copulation as well as 0 (at the time of
ejaculation), 20 and 60 min after EE and copulation. Low testosterone levels
were detected from October to February (4.58–5.06 nmol L-1), while high
levels were noted from May to September (8.01–11.40 nmol L-1) with
significant differences among months (P<0.001). In contrast,
cortisol levels were low from March to October (0.63–2.27 nmol L-1) and the
highest level was recorded in December (11.30 nmol L-1) with a significant
month effect (P<0.001). Cortisol reached its maximum concentration
in the electrically stimulated rams 20 min post electro-ejaculation with no
significant difference between the two seasons at this end time point. Means
of testosterone levels differed between the breeding and the non-breeding
season for electro-ejaculated rams. An increase in testosterone level was
observed after 60 min of copulation process in the non-breeding, while no
differences were noted for this hormone over the four time periods during
the breeding season. Cortisol levels rose significantly in non-breeding
season at 0 and 20 min after copulation (P<0.001). In conclusion,
Syrian Awassi rams displayed a clear seasonality of testosterone and
cortisol. Cortisol levels indicate an acute stress response to EE treatment.
Females have an effect on testosterone and cortisol levels in Syrian
Awassi rams only during the non-breeding season.
Introduction
It is well known that sheep species have marked seasonality of their
breeding activity (Rosa and Bryant, 2003). Reproductive seasonality in the
ewe was characterized by changes at behavioural, endocrine and ovulatory
levels (Avdi et al., 1993). However, in the ram, testicular size and
efficiency of spermatogenesis have differed between breeding and
non-breeding season (Kafi et al., 2004). Moreover, seasonal changes in
testosterone concentrations during a 1-year period have been used to
determine that breeds such as Chios and Daglic are seasonal breeders
(Gundogan, 2007). Testosterone secretion in small ruminants was driven by
natural day length changes or artificial photoperiodic cycles (Langford et
al., 1999). In addition to photoperiod effect, copulation process altered
the levels of testosterone in males in a number of species such as humans,
cattle and rats (Katongole et al., 1971; Fox et al., 1972; Purvis and Haynes, 1974).
Nevertheless, such alteration was not always obvious in rams during the
breeding and non-breeding season (Purvis et al., 1974; Gonzalez et al.,
1988a, b, 1991).
Semen collection is an essential step to achieve artificial insemination
(AI) and other reproductive techniques. In most domestic livestock species,
the collection of semen could be conducted throughout the year during
breeding and non-breeding season. However, semen can be collected from live
animals by using artificial vaginas, but this technique requires a previous
training period for males (Wulster-Radcliffe et al., 2001).
Electro-ejaculation (EE) is an alternative technique when males are not
trained to artificial vaginas. This method allowed the collection of high-quality semen from bulls and rams (Palmer, 2005; Garcia-Alvarez et al.,
2009) without noticeable differences from those collected by artificial
vaginas. Electro-ejaculation without anesthesia in humans and animals is
known to be painful; elevated blood pressure and cortisol levels have been
reported (Ortiz de Montellano et al., 2007; Damian and Ungerfeld, 2011). In
this respect, cortisol is often used in stress and welfare assessments
(Kannan et al., 2002), and it measures acute pain (Mosure et al., 1998). It is
worth noting that the values and the relation between cortisol and
testosterone secretion after exposure to stressful stimuli and copulation
process were not always clear in sheep species.
Awassi is the predominant sheep breed in Syria and the Middle East (Galal
et al., 2008). This breed is well adapted to harsh environmental conditions
and raised primarily for three types of products: meat, milk and wool
(Iniguez and Hilali, 2009). Although seasonality pattern of Awassi rams has
been previously reported (Taha et al., 2000; Kridli et al., 2007), little
information is available about the seasonal profile of both testosterone and
cortisol and the effects of electro-ejaculation and copulation process on
Awassi rams. Therefore, the objectives of the current experiments were
(1) to determine the seasonal pattern of testosterone and cortisol in Syrian
Awassi rams during a period of 1 year and (2) to investigate
electro-ejaculation and copulation effects on cortisol and testosterone
levels during the breeding and non-breeding season.
Materials and methodsSite description
The present study was carried out at the Division of Animal Production, Der
Al-Hajar Research Centre, located at 33∘21′ N,
36∘28′ E and 617 m above sea level. This area is dry
with an annual rainfall of about 140 mm. The climatological data of this
study were collected from a climatological station inside the centre. The
average longest and lowest day length was in June (14 h 22 min) and January
(10 h 14 min), respectively. For temperature, the average maximum day
temperature was recorded in August (40.77 ∘C) while the minimum day
temperature was recorded in January (14.50 ∘C).
Animals
At the start of the study, 10 sexually experienced Syrian Awassi rams aged
between 2 and 4 years (weighing mean ± SD; 90 ± 8.8 kg) were
used. Rams were maintained in an open-side barn, exposed to natural
photoperiod and isolated from any other animals. The 10 rams were fed a diet
based on concentrate and wheat straw, while water was available ad
libitum.
Experiments
Three experiments were conducted in this study; for all experiments,
blood samples were collected from the jugular vein using heparinized
evacuated tubes with minimal disturbance and immediately centrifuged at 2000×g
for 15 min. Plasma was prepared and stored at -20∘C until the
hormones assayed.
In the first experiment, a weekly collection of blood was done under the
same conditions at 09:00 LT (November–March: UTC +2, April–October:
UTC +3), and this experiment lasted for 1 year.
In the second experiment, electrical stimulation was applied with the aid of
an electro-ejaculator (ElectroJac 5, Ideal Instruments, USA) administrating
a series of 32-cycle pulses of short electrical stimuli with each cycle
(2 s on, then 2 s off) delivering a slightly higher
intensity until semen production. The entire procedure was performed in
approximately 2 min and repeated directly if the ram did not respond. The
rams were not previously subjected to electro-ejaculation process before the
conduction of this experiment. Animals were held in a standing position
during treatments. In this position, the penis of the rams was easily grasped
and held into the end of calibrated centrifuge tube. It must be noted that
all used animals were under veterinary care supervision after semen
collection, and animal's welfare was highly respected. Blood samples were
collected at 60 min before electrical stimulation as well as 0 (immediately at the
time of ejaculation), 20 and 60 min after electrical stimulation. This
experiment was replicated three times in July using the 10 rams (which is
normally considered as a part of the breeding season in Awassi sheep
according to previous results from our research group), and three times using
the same rams in February (which is also considered as a part of the
non-breeding season).
In the third experiment, nine Syrian Awassi ewes were synchronized to
exhibit oestrus, on three different dates (three ewes on each date) on a
weekly interval. Vaginal progestagen sponges (40 mg flourogestone acetate,
FGA) were inserted for 14 days. The three ewes to be used in the coupling
procedures were detected to be in oestrus by experienced Syrian Awassi rams
50 h after sponge withdrawal. Each ram was individually entered to the
females and evaluated by exposing it to two or three oestrus ewes for 15 min
on each test day. Blood samples were collected at 60 min before copulation
procedure, as well as 0 (immediately at the time of ejaculation or after 15 min if the
ram did not respond to estrous female), 20 and 60 min after ejaculation. It
must be noted that the males were isolated from females during the whole
study period, and the only visual and olfactory contacts were allowed at the
testing days. This experiment was replicated three times using the 10 rams
in July and three times using the same rams in February.
Radioimmunoassay analysis
All collected samples were assayed for testosterone and cortisol by the
radioimmunoassay technique using validated testosterone (REF: RK-61M, LOT:
10416AB) and cortisol (REF: RK-240CT, LOT: 10425C) [125 I] commercial
RIA KITS (Institute of Isotopes, Budapest, Hungary). The intra-assay
coefficients of variations were 9.28 and 8.38 %, while the inter-assay
coefficients of variations were 10.68 and 13.65 % for testosterone and
cortisol, respectively.
Statistical analyses
Statistical analyses were performed by using MINITAB 13.31 program (Minitab,
Coventry, UK). Monthly variations of testosterone and cortisol levels were
investigated by repeated one-way analysis of variance (ANOVA). Data regarding
electro-ejaculation stimulation and copulation process were subjected to
three-way ANOVA-3 using the general linear model procedure (GLM) including
the season and the time as fixed factors and replication as a random factor
(replication was done three times in each season). The analysis was followed by
multiple pairwise comparisons using a post hoc (Tukey) test. The threshold
of signification was set at P<0.05.
Results
The monthly changes in the concentration of testosterone and cortisol are
illustrated in Fig. 1. Mean testosterone values from all rams were
affected by month (P<0.001). From March to June, a sustained
testosterone rise was observed and the level was declined from June to
October. The highest testosterone level was recorded in June, while the
lowest was noted in December. In contrast to testosterone trend, cortisol
showed a very low level in June and the highest one was observed in December
with a significant month effect (P<0.001).
The outlines of electro-ejaculation and copulation experiences are shown in
Figs. 2 and 3. According to these findings, a sharp and significant (P<0.001) increase in cortisol level occurred at 0 and 20 min post EE
during both the breeding and non-breeding season. However, no difference was
found between the two seasons for the same two end time points. A
significant increase (P<0.001) in cortisol level can also be seen
in Fig. 3 following copulation process at 0 and 20 min point during the
non-breeding season. Anyhow, no differences were noted between -60, 0, 20
and 60 min pre- and post-copulation points in the breeding season.
During the breeding season, the level of testosterone significantly differed
at 60 min after EE stimulation than the one at -60 min. Nevertheless, no
significant differences (P>0.05) were noted between the four
time points during the non-breeding season. The level of testosterone
increased after copulation in the non-breeding season at 0 and 20 min points
reaching a significant level only at 60 min (P<0.001). However, no
differences were recorded in this hormone over the four time periods of this
experiment during the breeding season (P=0.146). Furthermore, clear
significant differences (P<0.001) were observed for each time
point (-60, 0, 20 and 60 min) between the two seasons.
Variations in plasma testosterone and cortisol level in samples
(mean ± SEM) collected monthly in Syrian Awassi rams (n=10).
Mean (± SEM) plasma testosterone and cortisol level prior to
and following electro-ejaculation (EE) stimulation during breeding and non-breeding season. Values for each hormone with different letters within breeding and within
non-breeding season (ABC) significantly differ (P<0.05).
Values for each hormone with different letters between breeding and
non-breeding season (ab) significantly differ (P<0.05).
Mean (± SEM) plasma testosterone and cortisol level prior to
and following copulation process during breeding and non-breeding season.
Values for each hormone with different letters within breeding and within
non-breeding season (ABC) significantly differ (P<0.05).
Values for each hormone with different letters between seasons (ab)
significantly differ (P<0.05).
Discussion
The results reported in this study on Syrian Awassi rams showed a very clear
and defined seasonal pattern. According to the climatological data, the
increase in plasma testosterone levels was associated with the increase in
photoperiod. In a clear contrast to our results, a higher level of
testosterone was found in rams during short days in comparison with long
days (Gundogan, 2007). However, the influence of the photoperiod depends on
sheep breeds and their location; those originating from temperate climates in
mid and high latitudes are seasonal breeds, their breeding season
being limited to autumn (Rhim et al., 1993); while, in tropical and subtropical
climates, some breeds are almost or completely aseasonal. The latitudinal location
of the current experiment (33∘ N) could be partly responsible
for shifting the breeding season in summer rather than in autumn. A
pronounced increase in testosterone level in spring and summer seasons has
been recorded in studies conducted by Olster and Foster (1988) and Taha et
al. (2000). The previous authors suggested that in mature rams the
stimulation of the hypothalamic–pituitary axis begins in spring when
photoperiod increases. Anyhow, cyclic reproductive behaviour in rams could
be influenced by environmental and hormonal factors other than photoperiod
(e.g. nutrition, social interactions, temperature, and blood growth hormone)
(Hamidi et al., 2012). In addition, a high percentage of Awassi females
showed oestrus activity during June and July (Zarkawi, 1997) leading to a
lag time in gonadal activity between males and females. This fact allows the
synchronization of the first ovulations in the females with the maximum
fertilization capacity of spermatozoa. Thus, the increase of testosterone
secretion in Awassi rams which occurs in April and May followed by a clear
peak in June may be necessary to succeed the breeding activity process of
this breed between males and females later.
Cortisol and testosterone are both critical endocrinal variables that affect
stress responses and sexual activity. However, the dearth of information
concerning cortisol level in the ram throughout the year made it interesting
to show the profile of the two hormones together. In the current study,
cortisol level showed an inverse relation with testosterone and this was
obvious in June and December. The high levels of cortisol in December and
January may be partly related to the low temperature during these months of
the year. Elevated circulating glucocorticoid levels as a response to
cold stress were clearly documented in farm animals (Dantzer and Mormede,
1983). In contrast, the high temperature during summer did not affect
cortisol level in Syrian Awassi rams, and this could be a result of an
adaptation to harsh environmental conditions during this season. Although
the seasonality pattern of cortisol in rams could be more related to the
environmental factors, however, this matter needs additional research
focusing on the endocrinal aspects.
The current study is the first to monitor the levels of cortisol and
testosterone before and after electro-ejaculation and copulation in Awassi
breed. The cortisol peak observed 20 min after EE represents a
significant rise from the pre-EE levels in the breeding and non-breeding
season. Similarly, criollo goats peaked 20 min post-EE stimulation but with
a level of no more than 30 nmol L-1 at this time point (Ortiz de Montellano et
al., 2007). Moreover, Damian and Ungerfeld (2011) reported that
electro-ejaculated rams had an important stress response including changes
in respiratory rate. Thus, welfare considerations should always be taken
into account by the administration of sedative and/or analgesic medications
after using such technique.
Testosterone levels in Awassi rams exposed to electrical stimulation
treatments during the breeding season appeared to decline over time. A
cause–effect relationship between adrenal hormones and testosterone levels
following EE has been suggested for certain species such as cattle (Welsh and
Johnson, 1981) and cats (Carter et al., 1984). On the other hand, during
stressful events in mice, the blood level of testosterone may be reduced due
to the deterioration of the blood flow in testicles (Kamiya et al., 2003).
However, the reason for the stability of testosterone level in the four
experimental times during the non-breeding season in this study may be
attributed to the normal basal level of testosterone already found during
this period.
It is well known that the presence of females and especially oestrus ones
may change ram plasma testosterone (Stellflug, 2006). The rams of our study
responded to oestrus ewes with an increase of 2.7 fold in testosterone
levels only in the non-breeding season. Gonzalez et al. (1988a)
noted a 3.5-fold increase in mean testosterone levels in rams after the
introduction of oestrous females during the non-breeding season. The
presence of oestrous females during the breeding season affected luteinizing hormone pulse
frequency and increased testosterone levels in rams (Gonzalez et al.,
1988b). Anyhow, sexual experience of Awassi rams and the period of
cohabitation may modify the testosterone response to copulation process.
Conclusions
Taken together, the findings of the present investigation suggest that, under
local Syrian conditions, Awassi rams have a distinct testosterone and
cortisol seasonality pattern. Testosterone and cortisol have obvious inverse
relation in June and December. Cortisol levels indicate an acute stress
response to EE treatment, especially at 0 and 20 min with no differences
between response according to breeding and non-breeding season. In Syrian
Awassi sheep, females have an effect on the testosterone of rams and cortisol
concentrations only during the non-breeding season.
Acknowledgements
The authors would like to thank the Director General and the Head of the
Department of Agriculture, Atomic Energy Commission of Syria for their
support while conducting the experiments.
Edited by: S. Maak
Reviewed by: two anonymous referees
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