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Reprod Dom Anim 44 (Suppl. 2), 279–282 (2009); doi: 10.1111/j.1439-0531.2009.01455.

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ISSN 0936-6768

Concentrations of Prolactin, LH, Testosterone, TSH and Thyroxine in


Normospermic Dogs of Different Breeds
C Urhausen1, A Seefeldt1, FM Eschricht1, A Koch1, HO Hoppen2, M Beyerbach3, C Möhrke, SJ Dieleman4 and AR Günzel-Apel1
1
Small Animal Clinic; 2Department of Endocrinology; 3Department for Biometry, Epidemiology and Information Processing, University of
Veterinary Medicine Hannover, Hannover, Germany; 4Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University,
Utrecht, The Netherlands

Contents compared with cross-bred and German Shepherd dogs.


Concentrations of prolactin (PRL), LH, testosterone (T), TSH The objective of the present study was to compare basal
and thyroxine (T4) were determined before and at 20, 120 and concentrations of hormones of the hypothalamo–pitui-
180 min after a single iv injection of thyrotropin-releasing tary–gonad and HP–thyroid axis, including PRL, in male
hormone (TRH) in eight Beagles, eight Fox Terriers, six dogs of different breeds confirmed to be normospermic
Labrador Retrievers and five Great Danes that were normo- and also to characterize their response to a single iv
spermic. Mean basal PRL concentrations were lower in the injection of TRH.
Fox Terriers compared with the Great Danes (p < 0.05).
Mean LH concentrations were higher in the Fox Terriers than
in the Beagles, and T was lower in the Fox Terriers at some Materials and Methods
times but not others (p < 0.05). Thyroid Stimulating Hor-
Experimental animals and experimental design
mone (TSH) concentrations did not differ among breeds, while
mean basal T4 values were lower in Fox Terriers compared A total of 27 male dogs including eight Beagles (1–6 years,
with Labrador Retrievers and Great Danes (p < 0.05). median 3.5 years; body weight: 14.8–16.2 kg, median
Stimulation of T4 secretion 120 and 180 min after iv TRH 15.3 kg), eight Fox Terriers (1–10 years, median 2 years;
injection was most pronounced in the Beagles and less in the body weight: 8.5–11.5 kg, median 9.6 kg), six Labrador
Fox Terriers (p < 0.05). The results of the present study Retrievers (1–5.5 years, median 1.5 years; body weight:
indicate that potential breed differences in circulating concen- 30.5–40 kg, median 33.8 kg) and five Great Danes
trations of PRL, LH, T, TSH and T4 in male dogs with
(age: 2.5–4 years, median 2.5 years; body weight:
apparently normal fertility can be encountered, but further
studies are needed to determine whether the observed differ- 58–69.3 kg, median 68 kg) were used in the study. All
ences are typical features of these breeds, reflect subsets of dogs were physically healthy and were normospermic
dogs within breeds, or are in part because of possible based on recent semen collections and analyses (‡60%
uncontrolled parameters such as sample timing, ambient motility; total sperm per ejaculate: for body weight
photoperiod, housing conditions or diet. £10 kg: 450 · 106; 11–20 kg: ‡800 · 106; 21–40 kg:
‡1200 · 106 > 40 kg: ‡1500 · 106; sperm with intact
plasma membrane: ‡85%; morphological intact sperma-
Introduction tozoa ‡70%) (Günzel-Apel 1994). The Beagles were kept
The role of prolactin (PRL) and the physiology of its in groups of four in covered outdoor kennels provided
secretion in intact male dogs have been the subject of a with two shelter huts each. All Fox Terriers belonged to
number of investigations in the last five years. As a basis one owner, six of them had a close genetic relationship
for assessing pathological conditions, a reference range with each other, and were kept in groups of three to five
(mean ± 2 SD) for serum PRL concentrations in male dogs in indoor kennels and had free access to an outdoor
dogs was proposed to be between non-detectable and kennel 24 h a day, while the individual Labrador
6 ng ⁄ ml based on results for 65 male dogs of different Retrievers and the Great Danes lived in homes of different
breeds (Corrada et al. 2006). As in other mammalian breeders. All dogs were kept under natural light
species, pulsatile pituitary LH release has been demon- conditions and were fed with different types of commer-
strated to stimulate the production and secretion of cial dry food. Animal housing, care and experimentation
testosterone (T) from testicular Leydig cells in the male complied with the animal welfare regulations in
dog (Günzel-Apel et al. 1990). Physiological PRL secre- Germany.
tion is also required to support T biosynthesis (Barañao In each dog, blood samples were collected immediately
et al. 1982). The predominant regulatory signal is the before (0 min) and 20, 120 and 180 min after iv injection
inhibition of PRL secretion by the neurotransmitter, of 10 lg ⁄ kg TRH (Protirelin, TRH-Ferring, Ferring
dopamine, from neurons in the hypothalamus (Lopez Arzneimittel GmbH, Kiel, Germany) as described by
et al. 1989). Prolactin secretion can be stimulated by Hoppen et al. (1997). Thyrotropin-releasing hormone
thyrotropin-releasing hormone (TRH) (Meij et al. 1996), tests were performed 12–16 h post-prandial between 8.00
which is primarily responsible for regulation of thyroid and 12.00 a.m., in the Fox Terriers, Labradors and
function. Such stimulation of PRL release by TRH was Great Danes from September 2005 to February 2006.
demonstrated in intact male Beagles in our previous study Thyrotropin-releasing hormone tests in the Beagles had
(Koch et al. 2006). Breed differences in PRL secretion been part of a previously published study (Koch et al.
have been detected by Corrada et al. (2006) with signif- 2006). Blood serum was separated by centrifugation
icantly higher mean PRL concentrations in Beagles at 1700 · g and stored at 20C until hormone analyses.

 2009 The Authors. Journal compilation  2009 Blackwell Verlag GmbH


280 C Urhausen, A Seefeldt, FM Eschricht, A Koch, HO Hoppen, M Beyerbach, C Möhrke, SJ Dieleman and AR Günzel-Apel

Hormone analyses (a)


20
Blood serum samples of the Fox Terriers, Labradors before TRH 20 min 120 min 180 min after TRH
18
and Great Danes were analyzed within the same assay
16
run. Identical assays were used for hormone analysis in
14
all four breeds.

prolactin ng/ml
Serum PRL concentrations were determined with a 12

previously validated heterologous radioimmunoassay 10

(RIA) (Okkens et al. 1985). Intra- and inter-assay 8


a
coefficients of variance (CVs) were 3.5% and 11.5%, 6
a
respectively. Assay sensitivity was 0.8 ng ⁄ ml. Serum LH 4
concentrations were determined by means of a heterol- 2
ogous RIA (Nett et al. 1975), with the following 0
modification: a rabbit antiserum against ovine LH Beagle (n = 8) Foxterrier (n = 8) Labrador R. (n = 6) Great Dane (n = 5)
(CSU-204), radioiodinated NIA-MDD-bLH-4 and ca- (b)
nine pituitary standard LER 1685-1 were used in this 14
before TRH 20 min 120 min 180 min after TRH
assay. Intra- and inter-assay CVs for values above 12
0.5 ng ⁄ ml were 2.3% and 10.5%, respectively. Assay
sensitivity was 0.3 ng ⁄ ml. Testosterone analysis was 10
a
performed by RIA as described by Günzel-Apel et al.
LH ng/ml
8
(1990). Intra- and inter-assay CVs were 1.1% and 0.5%,
respectively. Assay sensitivity was 20 pg ⁄ ml. 6
a
The concentrations of TSH and (thyroxine) T4 were
4
measured by homologous solid-phase chemiluminescent
immunometric assays (Immulite Canine TSH and 2
Immulite Canine Total T4; Siemens Medical Solutions
0
Diagnostics, Bad Nauheim, Germany) according to the Beagle (n = 8) Foxterrier (n = 8) Labrador R. (n = 6) Great dane (n = 5)
instructions of the manufacturer. Thyroid stimulating
(c)
TSH intra- and inter-assay CVs were 6.3% and 8.2% at 5
before TRH 20 min 120 min 180 min after TRH
TSH concentrations of 0.16 and 2.8 ng ⁄ ml, respectively.
Assay sensitivity was 0.03 ng ⁄ ml. Thyroxine intra- and 4
inter-assay CVs was 13.8% and 6.8% at concentrations
Testosterone ng/ml

of 0.65 and 3.84 lg ⁄ dl, respectively. Assay sensitivity


3
was 0.12 lg ⁄ dl. a b

Prolactin, LH and T were analyzed in all four samples


2
(before TRH, and 20, 120 and 180 min after TRH).
TSH and T4 concentrations were measured according to a b
the routine procedure of a TRH stimulation test for 1
assessing thyroid function (TSH before and at 20 min
after TRH, T4 before and at 120 as well as 180 min after 0
TRH; Hoppen et al. 1997). Beagle (n = 8) Foxterrier (n = 8) Labrador R. (n = 6) Great dane (n = 5)

Fig. 1. Mean (±SD) concentrations of prolactin, LH and testosterone


Statistical evaluation in normospermic Beagles, Fox Terriers, Labrador Retrievers and
Great Danes in serum samples obtained at selected times before and
Basal hormone levels in the four groups were defined as after a single iv injection of TRH at 10 lg ⁄ kg (arrow). Corresponding
the respective concentrations in serum samples collected means with the same letter designation (e.g. b and b) denote a
before administering TRH. The response to TRH was difference between those breeds (p < 0.05) for values at that time
point
evaluated by comparing the hormone concentrations
measured at each sampling time. The Wilcoxon two-
sample test for independent samples (Wilcoxon’s signed- however, a significant difference was found between the
rank test) was used to compare the differences between lowest (Fox Terrier) and highest mean basal PRL
breeds regarding (i) overall levels of LH and T and (ii) concentration (Great Danes; Fig. 1a). In all dogs TRH
hormone concentrations at corresponding times relative injection was followed by a sharp PRL increase (p <
to TRH injection for each hormone. The results are 0.05) to 9.1 ± 5.5 ng ⁄ ml in Beagles, 10.9 ± 6.4 ng ⁄ ml in
presented as mean ± SD (p < 0.05 was considered Fox Terriers, 8.5 ± 2.3 ng ⁄ ml in Labrador Retrievers
significant). and 6.9 ± 1.7 ng ⁄ ml in Great Danes by 20 min and a
subsequent nearly equivalent decrease (p < 0.05) at
120 min.
Results Mean basal LH concentrations were higher in Fox
Mean PRL concentrations pre-TRH (Beagles Terriers (8.2 ± 4.7 ng ⁄ ml) in comparison to Beagles
3.8 ± 0.9 ng ⁄ ml; Fox Terriers: 3.2 ± 0.6 ng ⁄ ml, Labra- (4.6 ± 2.7 ng ⁄ ml; Fig. 1b). In all breeds, LH secretion
dor Retrievers 3.3 ± 0.7 ng ⁄ ml; Great Danes: did not show any clear patterns in relation to TRH
4.5 ± 0.9 ng ⁄ ml) did not vary widely among breeds, injection.

 2009 The Authors. Journal compilation  2009 Blackwell Verlag GmbH


Role of PRL and its Secretion Physiology in Dogs 281

In the Fox Terriers, the mean T concentrations were potential breed differences in T and T4 were detected.
all low, at 0.8 ng ⁄ ml (Fig. 1c) and those concentrations The low hormone concentrations found in the Fox
at 120 and 180 min after TRH injection were signif- Terriers may be due to a close genetic relationship in the
icantly lower than the corresponding values of the majority of dogs in this group rather than to breed
Beagles (p < 0.05). Mean T concentrations of the specificity. However, whether or not any of the other
Beagles, Labradors and Great Danes varied inconsis- differences detected as statistically significant are bio-
tently during the TRH test. logically meaningful is difficult to determine. Further-
There were no differences among breeds in TSH more, the possibility that some of the apparent breed
concentrations before TRH injection (Fig. 2a). Thyrot- differences observed may reflect differences in times of
ropin-releasing hormone injection was followed by a blood collection, ambient photoperiod, diets, or other
marked increase in TSH after 20 min in all dogs. Mean parameters or experimental variables not closely con-
TSH after TRH did not differ among breeds. trolled in this preliminary, essentially clinical study must
Lowest basal T4 values were detected in the Fox be taken into account. It is interesting, however, to note
Terriers (0.9 ± 0.4 lg ⁄ dl) and were lower (p < 0.05) that an effect of breed on PRL concentrations was
than those in the Labradors (1.3 ± 0.3 lg ⁄ dl) and in described by Corrada et al. (2006) with higher PRL
the Great Danes (1.4 ± 0.4 lg ⁄ dl; Fig. 2b). Mean T4 values in Beagles compared with cross-breeds and
concentrations, both at 120 min and 180 min after TRH German Shepherds (p < 0.05). In contrast, in the
in Beagles, were higher (p < 0.05) than in the Fox present study, mean PRL in Beagles did not differ from
Terriers (Fig. 2b). values in the other breeds studied.
The increase in PRL in response to a single iv
injection of TRH in all breeds in the present study is in
Discussion agreement with the responses described by Hoppen
Basal PRL values measured in the 27 healthy dogs with et al. (1997) in euthyroid dogs, demonstrating the PRL-
normospermia in this study were within the reference stimulating efficiency of TRH.
range of non-detectable to 6.0 ng ⁄ ml suggested by The observation in the Fox Terriers of comparatively
Corrada et al. (2006), albeit using a different PRL assay high basal LH concentrations that were accompanied by
and different canine PRL standard. In our study, breed lowest T concentrations may be biologically meaningful
differences in peripheral PRL concentrations, as well as and reflect increased LH suppression by T negative
feedback (Günzel-Apel et al. 1990). The low T in Fox
Terriers may indicate decreased Leydig cell activity;
however, all eight dogs of this breed had normal semen
(a)
1.0 quality and normal fertility and this finding may have no
before TRH 20 min after TRH clinical relevance. T secretion is in pulses and it may be
0.8 that all animals in this group were sampled at or near
nadir T concentration and their normal T pulses were
0.6
not included in the sampling. It is not known whether or
TSH ng/ml

not the inclusion of a GnRH or Human chorionic


Gonadotropin (hCG) administration test would have
0.4
allowed the detection of normal post-challenge T
concentrations in the Fox Terrier dogs. That there was
0.2 no effect of TRH on LH is in agreement with observa-
tions of Koch et al. (2006) that secretion of LH and
0.0 T was not influenced by TRH injection.
Beagle (n = 8) Foxterrier (n = 8) Labrador R. (n = 6) Great dane (n = 5) Basal TSH and T4, and changes in TSH and T4 after
(b) TRH injection were in most instances within the normal
4
before TRH 120 min 180 min after TRH
range and in agreement with previous observations
(Hoppen et al. 1997). However, both basal and stimu-
3
lated T4 concentrations in the Fox Terriers were below
b
our laboratory’s reference range of 1.7–4.0 lg ⁄ dl.
Thyroxine µg/dl

a Nevertheless, we conclude that primary or secondary


2 hypothyroidism can be excluded in these dogs, as basal
a b
d TSH concentrations were at physiological levels and
c
cd
TRH injection induced a clear TSH increase in all dogs
1 of this breed. Normal semen quality, in contrast, is not a
relevant parameter as regards thyroid assessment, as
Johnson et al. (1999) reported that no relation exists
0
Beagle (n = 8) Foxterrier (n = 8) Labrador R. (n = 6) Great dane (n = 5) between hypothyroidism and disturbed fertility in male
dogs.
Fig. 2. Mean (±SD) concentrations of TSH and thyroxine in In conclusion, the results of the present study indicate
normospermic Beagles, Fox Terriers, Labrador Retrievers and Great potential breed differences in the secretion of hormones
Danes in serum samples obtained at selected times before and after a
single iv injection of TRH at 10 lg ⁄ kg (arrow). Corresponding means
related to the pituitary–gonadal and –thyroid axis. The
with the same letter designation (e.g. a and a) denote a difference determination of the extent to which these are either
between those breeds (p < 0.05) for values at that time point consistently or biologically significant would require

 2009 The Authors. Journal compilation  2009 Blackwell Verlag GmbH


282 C Urhausen, A Seefeldt, FM Eschricht, A Koch, HO Hoppen, M Beyerbach, C Möhrke, SJ Dieleman and AR Günzel-Apel

evaluation of larger groups of dogs in more controlled Hoppen HO, Lohmann P, Schlote S, Günzel-Apel AR,
situations as regards possible effects of season, environ- Müller-König A, Grünau B, Hämmerling R, Leidinger K,
mental lighting, diet and other potential variables. Morisse B, Nolte I, 1997: Die Messung von caninem TSH
Importantly, the present results also demonstrate that zur Diagnostik der Hypothyreose des Hundes. Prakt Tier-
arzt 78, 13–17.
healthy dogs with normospermia may at times have what
Johnson CA, Olivier NB, Nachreiner RF, Mullanay T, 1999:
are typically considered low or subnormal serum concen- Effect of 131J-induced hypothyroidism on indices of repro-
trations of T and T4. Therefore, any clinical suspicion of ductive function in adult male dogs. J Vet Intern Med 13,
causality between T or T4 status and fertility status 104–110.
requires careful and individual evaluation. Koch A, Hoppen HO, Dieleman SJ, Kooistra HS, Günzel-
Apel AR, 2006: Effects of the dopamine agonist cabergoline
on the pulsatile and TRH-induced secretion of prolactin,
Author contributions LH, and testosterone in male beagle dogs. Theriogenology
CU carried out the experiments and wrote the manuscript. AS was 65, 1666–1677.
involved in the experiments and was the assistant supervisor. FE and Lopez FJ, Dominguez JP, Sanchez-Franco F, Negro-Vilar A,
AK were involved in the experiments. HOH conducted the hormone 1989: Role of dopamine and vasoactive intestinal peptide in
analyses. MB was the statistics supervisor. CM was involved in the the control of pulsatile prolactin secretion. Endocrinology
experiments and was the owner of the Foxterrier dogs. SD conducted 124, 527–535.
the prolactin analyses. AGA was responsible for the organization and Meij BP, Mol JA, Hazewinkel HAW, Bevers MM, Rijnberk A,
supervision.
1996: Assessment of a combined pituitary function test in
beagle dogs: rapid sequential intravenous administration of
four hypothalamic releasing hormones. Domest Anim
Conflicts of interest
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estradiol and progesterone in serum during the estrous cycle
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tragung Beim Hund. Verlag Gustav Fischer, Jena, pp. 11–
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Günzel-Apel AR, Brinckmann HG, Hoppen HO, 1990: tionsmedizinische Einheit der Kliniken, Stiftung Tierärzttiche
Dynamik der LH- und testosteron-sekretion bei beagle- Hoschschule Hannover, Bünteweg 15, 30559, Hannover, Germany.
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 2009 The Authors. Journal compilation  2009 Blackwell Verlag GmbH