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Animal Reproduction Science xxx (2008) xxx–xxx

Contents lists available at ScienceDirect

Animal Reproduction Science


journal homepage: www.elsevier.com/locate/anireprosci

Factors affecting progesterone production in corpora lutea


from pregnant and diestrous bitches
Lieta Marinelli a, Ada Rota b,∗, Paolo Carnier c, Laura Da Dalt a,
Gianfranco Gabai a
a
Dipartimento di Scienze Sperimentali Veterinarie, Università di Padova, Viale dell’Università 16, 35020 Legnaro (PD), Italy
b
Dipartimento di Patologia Animale, Università di Torino, Via Leonardo da Vinci 44, 10090 Grugliasco (TO), Italy
c
Dipartimento di Scienze Animali, Università di Padova, Viale dell’Università 16, 35020 Legnaro (PD), Italy

a r t i c l e i n f o a b s t r a c t

Article history: Factors affecting the characteristics of corpora lutea (number,


Received 9 June 2008 left/right ovary origin, weight, DNA and progesterone content)
Received in revised form 24 September
were studied in 73 healthy bitches divided into two classes of age
2008
(≤2.5 vs. >2.5 years; mean ± S.E. = 3.6 ± 0.3 years; range: 0.7–10
Accepted 3 October 2008
Available online xxx years), weight (≤20 vs. >20 kg; mean ± S.E. = 16.2 ± 1.2 kg; range:
5–45 kg), reproductive status (pregnancy vs. diestrous; pregnant
bitches N = 41 and diestrous bitches N = 32), stage of luteal phase
Keywords:
Bitch (20–40 vs. 41–55 days) and ovulation rate (≤7 vs. >7). Two differ-
Corpus luteum ent assessments were performed: (a) comparison of luteal tissue
Age characteristics and progesterone content between pregnant and
Body size diestrous bitches and (b) investigation of the effect of animal age,
Progesterone weight and ovulation rate on individual corpus luteum (CL) param-
eters. None of the luteal parameters differed between pregnant and
diestrous bitches, even when the stage of the luteal phase was con-
sidered. Age and weight of the bitch significantly influenced luteal
tissue characteristics: heavier bitches had more and heavier CLs
(P < 0.001) and carried more foetuses (P < 0.01), while older bitches
had a higher number of CLs (P < 0.001). In pregnant animals, the
rate of foetuses to Cls was 78.4%. Luteal progesterone content was
significantly affected by the ovulation rate (P < 0.01). A significant
individual effect (P < 0.0001) was present on all the parameters in
the single CL, with the right ovary carrying a higher CL number
(P < 0.01), with greater DNA (P < 0.01) and P4 content (P < 0.001).

∗ Corresponding author. Tel.: +39 011 6709051; fax: +39 011 6709097.
E-mail address: ada.rota@unito.it (A. Rota).

0378-4320/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2008.10.001

Please cite this article in press as: Marinelli, L., et al., Factors affecting progesterone pro-
duction in corpora lutea from pregnant and diestrous bitches. Anim. Reprod. Sci. (2008),
doi:10.1016/j.anireprosci.2008.10.001
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2 L. Marinelli et al. / Animal Reproduction Science xxx (2008) xxx–xxx

CLs of younger bitches showed a diminished efficiency of P4 pro-


duction (P4/mg, P4/DNA) with a significant effect of the interaction
between age and reproductive condition of the bitch on DNA and
progesterone content (P < 0.0001). These findings indicate that ani-
mal weight and age have a major influence on the characteristics of
canine corpora lutea.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction

The basic mechanisms that control luteal function in the canine species are poorly understood in
comparison to other domestic mammals. In the bitch, the corpus luteum (CL) is the unique source of
circulating progesterone during the oestrous cycle and pregnancy (Concannon et al., 1989) and has
the peculiarity to show an apparently equivalent functionality in pregnant and nonpregnant animals
(Concannon et al., 1977; Onclin and Verstegen, 1997), except that pregnant animals reach baseline
progesterone concentrations earlier, owing to the sharp prepartum decline (Hoffmann et al., 2004).
Nevertheless, it has been suggested that, in order to appreciate a possible different rate of proges-
terone secretion between pregnant and nonpregnant bitches, plasma progesterone levels should be
corrected for blood volume, which increases after the third week of pregnancy (Concannon et al., 1977).
A pregnancy-specific increase in progesterone production has been demonstrated between days 26
and 45 after ovulation by measuring fecal progesterone concentrations (Gudermuth et al., 1998), which
has been postulated to be less influenced by the different blood volume of pregnant animals. However,
fecal progesterone concentrations are highly affected by both hepatic clearance and fecal excretion,
which are likely to be different in pregnant and nonpregnant bitches. Hence, the presence of a local
mechanism supporting luteal progesterone secretion after implantation is still an open possibility that
deserves investigation.
Apart from pregnancy, the extent of variation in circulating progesterone levels among bitches
is supposed to be associated to the multiple and variable number of ovulations and corpora lutea
observed (Concannon et al., 1977). It has been reported in many species that plasma progesterone
concentrations increase as ovulation rate and number of CLs increase (Guthrie et al., 1974; Cahill et al.,
1981; Knox et al., 2003). To our knowledge, there are no studies investigating the effect of ovulation
rate on progesterone production by the CL in the bitch.
Data about factors affecting ovulation rate in the bitch have been mostly extrapolated from litter
size. An early study reported a positive correlation between litter size and weight of the dam, suggesting
that body weight could influence the number of CLs in the bitch, as is the case in a variety of mammals
(Robinson, 1973). This hypothesis seems to be confirmed by the influence of breed on litter size, with
large breeds having more pups than small breeds (Kelley, 2002). The same approach has been used to
evaluate the effect of age. Few retrospective studies on reproductive parameters registered by kennel
clubs of different breeds report the significant effect of age and parity of the dam on litter size, with
smaller litter sizes in older bitches and in bitches after the fifth pregnancies (Mutembei et al., 2002;
Gresky et al., 2005; Bobic Gavrilovic et al., 2008).
Concerning young age, data are limited and more controversial: in a retrospective study on
Dachshund bitches, animals younger than 2.5 years had significantly less pups than older bitches
(Gresky et al., 2005); both Beagle and Drever bitches had significantly smaller litter sizes at first par-
turition (Kelley, 2002; Bobic Gavrilovic et al., 2008). However, in the study of Kelley (2002) the “age
of peak litter size” was reported to be between 1 and 3–4 years for most of the breeds examined,
suggesting that the prolificity observed at the first pregnancy was about average. Since litter size is
the result of ovulation rate, conception rate and embryo survival, the above-mentioned studies have
not directly assessed either the effect of bitch characteristics on ovulation rate or analysed what bitch
characteristics could affect CL function.
The aims of the present research were: (1) to compare luteal tissue progesterone content/production
in pregnant and diestrous bitches during the mid and late luteal phase and (2) to investigate the effects
of animal age and weight on ovulation rate, luteal characteristics and progesterone production.

Please cite this article in press as: Marinelli, L., et al., Factors affecting progesterone pro-
duction in corpora lutea from pregnant and diestrous bitches. Anim. Reprod. Sci. (2008),
doi:10.1016/j.anireprosci.2008.10.001
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2. Materials and methods

2.1. Animals

The study was carried out on 73 mongrel (n = 52) or pure-breed (n = 21) healthy bitches, aged
0.7–10 years (mean ± S.E. = 3.6 ± 0.3 years) and weighing 5–45 (mean ± S.E. = 16.2 ± 1.2 kg), that were
subjected to elective ovariectomy or ovariohysterectomy at the Department of Veterinary Clinical Sci-
ences of the University of Padua. On the day of presentation at the clinic, the age and weight of each
animal were registered, and each bitch was assigned to a relative class of age (young ≤ 2.5 years old
and adult > 2.5 years old) and weight (light ≤ 20 kg and heavy > 20 kg). The stage of the reproductive
cycle was assessed on the basis of history, vaginal cytology and blood progesterone concentration. In
case of pregnancy, observation of foetal development was also used to establish the gestational stage
(Christiansen and Schmidt, 1982).
Before surgery, a blood sample was collected from each animal and plasma was stored frozen
(−25 ◦ C) until assayed for progesterone. The experimental protocol was in accordance with the Italian
legal requirements regarding animal welfare (DL no.116, 27/1/1992), and was approved by the Ethics
Committee of the Faculty of Veterinary Medicine at the University of Padua.

2.1.1. Experiment 1
Experiment 1 examined the hypothesis that in mid and late pregnancy, canine luteal tissue produces
more progesterone than during the same phases of diestrus. Moreover, Experiment 1 evaluated the
hypothesis that animal age and weight affect ovulation rate, luteal characteristics and progesterone
production. Corpora lutea from 21 diestrous (average age ± S.E. = 4.1 ± 0.7 years; range: 0.8–10 years;
average weight ± S.E. = 14.6 ± 2.3 kg; range: 5.0–45.0 kg) and 24 pregnant (average age ± S.E. = 3.1 ± 0.4
years; range: 0.7–7 years; average weight ± S.E. = 17.7 ± 2.1 kg; range: 5.0–38.8 kg) animals were used.
Corpora lutea collected from a single animal were pooled and analysed as whole individual luteal tissue
(LT). Each individual LT was assigned to two stages of luteal phase: 20–40 days (mid luteal phase; MLP)
and 41–55 days (late luteal phase; LLP) post-ovulation respectively, assuming that ovulation occurs on
the first days of an oestrous phase of an average length of 8–10 days (Tsutsui, 1989).

2.1.2. Experiment 2
In Experiment 2, the same hypothesis of Exp. 1 were investigated on the single CL, to verify if
pregnancy, animal weight and age affect single CL characteristics and progesterone production. Iso-
lated CLs from 17 pregnant (average age ± S.E. = 3.1 ± 0.4 years; average weight ± S.E. = 20.5 ± 2.6 kg;
average post-ovulation days ± S.E. = 38.3 ± 2.5) and 11 diestrous (average age ± S.E. = 4.4 ± 1.1 years;
average weight ± S.E. = 17.8 ± 3.4 kg; average post-ovulation days ± S.E. = 31.0 ± 2.4) animals were pro-
cessed separately. The ovarian origin of the CL (left vs. right ovary) was recorded and CLs were classified
in four groups according to weight (≤100 mg, n = 54; 101–150 mg, n = 91; 151–200 mg, n = 62; >200 mg,
n = 30).

2.2. Tissue collection and homogenisation

The right and left ovaries were recovered at surgery and, in the case of pregnancy, the num-
ber of foetuses was registered. Collected ovaries were weighed and the corpora lutea of each
ovary were isolated through accurate dissection from ovarian stroma. The number of CLs of each
ovary and CL weight were recorded before processing. The pooled CLs of each animal (Experi-
ment 1) or each single CL (Experiment 2), after a preliminary gross mincing, were suspended
in phosphate buffered saline (pH 7.4; 1 ml/mg wet weight) at 4 ◦ C, and homogenised using a
rotor-stator homogeniser (Ultra-Turrax) followed by a glass-glass tissue grinder to obtain a fine sus-
pension. Homogenised samples were then aliquoted and stored at −25 ◦ C for progesterone and DNA
determinations.

Please cite this article in press as: Marinelli, L., et al., Factors affecting progesterone pro-
duction in corpora lutea from pregnant and diestrous bitches. Anim. Reprod. Sci. (2008),
doi:10.1016/j.anireprosci.2008.10.001
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2.3. DNA determination

Aliquots of the homogenate were used for DNA determination as an index of luteal cells content.
The measurements were performed in triplicate, based on the Burton diphenylamine method (Burton,
1956) with minor modifications. Briefly, the sample and standards (0.25–32 ␮g/300 ␮l) were prepared
by dissolving 25 ␮l of homogenate and a DNA standard preparation (calf-thymus DNA, Sigma–Aldrich,
Milan, Italy), respectively, in 0.5N HClO4 . Buffer 0.5N HClO4 was used as a blank. Samples and standards
(300 ␮l) were then heated at 90 ◦ C for 30 min, and 600 ␮l of DRP reagent (270 mmol l−1 sulphuric
acid 99.9%, 88 mmol l−1 diphenylamine, 3 mmol l−1 acetaldehyde in acetic acid 99.5%; Sigma–Aldrich,
Milan, Italy) was then added to each tube. The reaction mixture was kept overnight (16 h) at 30 ◦ C
and the resulting blue colour was measured by reading the absorbance at 595 nm and comparing the
values obtained with the standard DNA.
The assay showed a good degree of parallelism between the standard curve and unknown samples,
and a good level of recovery: the regression line obtained by the test of parallelism was y = 24.7x − 0.6
and the coefficient of regression (R2 ) was 0.99, whereas the regression line obtained by the test of
recovery was y = 0.75x − 0.22 and the coefficient of regression (R2 ) was 0.99. The results of the intra- and
inter-assay precision test, expressed as coefficients of variation (CV), were 5.3 and 7.2%, respectively.

2.4. Progesterone assays

Plasma progesterone concentrations were determined after petroleum ether extraction by


microtitre radioimmunoassay (RIA), previously validated for canine plasma (Rota et al., 2003). The
efficiency of the extraction was 66.4 ± 6.9%, and the intra- and inter-assay CVs were 9.3 and 11.5%
for control low (4.1 nmol l−1 ) and 7.8 and 13.5% for control medium (13.7 nmol l−1 ), respectively.For
progesterone determination in luteal tissue, 25 ␮l of luteal homogenate was extracted using 8 ml
petroleum ether for 15 min in a rotating mixer at room temperature. The efficiency of the extrac-
tion was 86.5 ± 5.4%. The extracted homogenate was frozen, and the supernatant was decanted into
glass tubes and dried at 37 ◦ C under nitrogen. The dry extract was resuspended in 1 ml phosphate
buffered saline 0.1% bovine serum albumin (Sigma–Aldrich, Milan, Italy), pH 7.4, and further diluted
100-fold in the same buffer. Due to the high variability of progesterone concentration in luteal tissues,
a second dilution (600-fold) was performed, and both dilutions of each sample underwent proges-
terone determination. The RIA was performed in triplicate as previously described (Rota et al., 2003).
The test of parallelism (y = 76.75x − 0.13; R2 = 0.99) and recovery confirmed (y = 0.84x + 3.45; R2 = 0.99)
the accuracy of the system in luteal homogenates. The intra- and inter-assay CVs were, respectively,
8.8 and 10.7% for control low (318 nmol l−1 ) 6.3 and 9.0% for control medium (1430 nmol l−1 ) and 8.0
and 11.4% for control high (3815 nmol l−1 ).

2.5. Statistical analysis

Since all luteal progesterone and DNA data exhibited frequency distributions that were markedly
skewed, a log-transformation was applied to these traits. Data were analysed using the general linear
model and the MIXED procedures of SAS® (Statistical Analysis System, 2001). For Experiment 1, the
linear model included the fixed effect of age (young vs. adult), weight (light vs. heavy), reproductive
condition (pregnant vs. diestrous) and ovulation rate (low: number of CLs ≤ 7, high: number of CLs > 7)
of the animal and the stage of luteal phase (middle vs. late). For Experiment 2, the model included
the fixed effect of age (young vs. adult), weight (light vs. heavy), reproductive condition (pregnant vs.
diestrous) of the animal, the two- and three-way interactions between these effects, the random effect
of the animal, the fixed effect of the ovarian origin (left vs. right) and of the class of CL weight, and
two-way interactions between reproductive condition and ovarian origin or class of CL weight, those
between age and ovarian origin or class of CL weight and the one between ovarian origin and class of
CL weight effects. Analysis of variance of data was based on the specified models. When appropriate,
differences between means were analysed by Bonferroni test. The existence of repeated observations
for each bitch in Experiment 2 was properly accounted for by using the mean square of the bitch effect
as an error term for statistical inference and hypothesis testing on age, weight, reproductive condition

Please cite this article in press as: Marinelli, L., et al., Factors affecting progesterone pro-
duction in corpora lutea from pregnant and diestrous bitches. Anim. Reprod. Sci. (2008),
doi:10.1016/j.anireprosci.2008.10.001
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effects and their interactions. Correlation between plasma progesterone level and its content and
concentration in individual luteal tissue was assessed by a two-tailed Spearman rank correlation test.
The significance level was set at P < 0.05. Unless otherwise noted, the results are reported as least
squares means ± S.E.

3. Results

3.1. Experiment 1

Table 1 shows the mean values of individual and luteal parameters recorded in pregnant and
diestrous bitches. Pregnancy did not affect CL total and mean weight, and none of the functional
characteristics of luteal tissue differed significantly between pregnant and diestrous bitches, even if
the stage of the luteal phase was considered. Moreover, plasma progesterone concentration was not
correlated with any of the luteal characteristics (progesterone content and concentration expressed
as P4/LT and P4/DNA). A weak but statistically significant correlation was found between plasma P4
and LT weight (R = 0.31; P = 0.041) and between plasma P4 and luteal DNA concentration (DNA/LT;
R = −0.46; P = 0.002).
The stage of luteal phase affected plasma progesterone concentration, which was significantly lower
in bitches 41–55 days after ovulation (P < 0.01) regardless of the reproductive condition (pregnancy vs.
diestrous). When considering luteal tissue, the difference in terms of P4 content of the whole LT and
P4 concentration (P4/LT and P4/DNA) did not reach statistical significance (Table 1).
In pregnant animals, a mean foetal number of 6.37 ± 0.66 corresponds to a mean CL number of
8.12 ± 0.55, indicating a mean ratio of 78.4%. When the age of the bitch was taken into account,
younger bitches (≤2.5 years) showed a higher ‘foetal number to CL number’ ratio (89.6%) than older
bitches (66.2%) (P < 0.001; Table 2). Age and weight of the bitch significantly influenced the number
of CLs (P < 0.001; Table 2): bitches older than 2.5 years (2.6–10 years; mean ± S.E. = 6.4 ± 0.5 years) and
heavier than 20 kg (21–45 kg; mean ± S.E. = 29.5 ± 1.9 kg) had more CLs than younger (0.7–2.5 years;
mean ± S.E. = 1.4 ± 0.1 years) and lighter animals (5–20 kg; mean ± S.E. = 10.2 ± 0.8 kg). In contrast, the
age of the bitch had no effect on the number of foetuses in pregnant animals, while the effect of
weight was still significant, with animals heavier than 20 kg carrying more foetuses than lighter ani-
mals (P < 0.01; Table 2). The weight of the bitch also showed a significant effect on LT weight, mean
CL weight and DNA content (P < 0.001; Table 2); bitches heavier than 20 kg had more and heavier CLs,
resulting in a greater quantity of total luteal tissue and a higher DNA content. None of the progesterone
parameters were affected by animal age or weight. Total luteal P4, P4/TL, and P4/DNA were significantly

Table 1
Individual and luteal characteristics (mean ± S.E.) recorded in the mid and late luteal phase of pregnant and diestrous bitches.
Different superscript letters within a row indicate statistically significant differences between phases of analogous reproductive
conditions (a vs. b and c vs. d; P < 0.01).

Reproductive condition Pregnant (n = 24) Diestrous (n = 21)

Weight (kg) 17.7 ± 2.1 14.6 ± 2.3


Ovarian weight (g) 2.58 ± 0.31 2.03 ± 0.29
Number of corpora lutea 8.12 ± 0.55 7.71 ± 0.64
Number of foetuses 6.37 ± 0.66 /
Weight of LT (mg) 1140.2 ± 117.8 941.01 ± 139.1
Mean CL weight (mg) 135.8 ± 11.4 111.1 ± 10.8

Luteal phase MLP (n = 15) LLP (n = 9) MLP (n = 12) LLP (n = 9)


−1
Plasma P4 (nmol l ) 36.4 ± a
4.9 14.8 ± b
3.1 38.4 ± c
4.2 15.7 ± 1.1d
Total P4 (␮g) 47.2 ± 9.6 26.0 ± 6.7 40.8 ± 8.7 29.3 ± 9.9
P4/LT (ng/mg) 43.9 ± 5.3 20.4 ± 5.3 43.0 ± 6.1 35.0 ± 8.7
P4/DNA (ng/␮g) 13.6 ± 2.9 5.3 ± 2.8 12.4 ± 2.4 8.4 ± 0.5
DNA/LT (␮g/mg) 4.8 ± 0.7 6.1 ± 0.7 4.4 ± 0.4 5.0 ± 0.8

LT = individual luteal tissue; MLP = mid luteal phase; LLP = late luteal phase; P4 = progesterone; and total P4 = individual luteal
content of progesterone.

Please cite this article in press as: Marinelli, L., et al., Factors affecting progesterone pro-
duction in corpora lutea from pregnant and diestrous bitches. Anim. Reprod. Sci. (2008),
doi:10.1016/j.anireprosci.2008.10.001
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Table 2
Mean (±S.E.) luteal characteristics in bitches according to age and weight. Numbers of foetuses are referred to pregnant animals
only, which number is reported between brackets for each group.

Age Weight

≤2.5 years (n = 27) >2.5 years (n = 18) ≤20 kg (n = 30) >20 kg (n = 15)

Number of corpora lutea 7.25 ± 0.48 9.22 ± 0.54 ***


6.50 ± 0.50 9.99 ± 0.50***
Number of foetuses 6.5 ± 0.9 (n = 15) 6.1 ± 0.9 (n = 9) 5.3 ± 0.7 (n = 15) 8.1 ± 1.1 (n = 9)**
Weight of LT (mg) 1001.6 ± 101.2 1129.7 ± 114.1 650.8 ± 104.5 1480.5 ± 107.9***
Mean CL weight (mg) 125.7 ± 10.5 121.2 ± 11.8 100.4 ± 10.8 146.5 ± 11.2***
Plasma P4 (nmol l−1 ) 30.0 ± 4.0 34.3 ± 3.8 30.8 ± 3.1 33.4 ± 6.0
Total DNA (␮g) 5130.4 ± 669.6 4603.6 ± 850.0 3781 ± 493.9 7226.7 ± 973.3***
DNA/LT (␮g/mg) 5.2 ± 0.4 4.3 ± 0.5 5.1 ± 0.4 4.5 ± 0.5

P4 = progesterone; LT = individual luteal tissue; total DNA = individual luteal content of DNA.
**
P < 0.01.
***
P < 0.001.

affected by the ovulation rate (P4, P < 0.01; P4/LT, P < 0.05; P4/DNA, P < 0.05), while neither plasma P4,
luteal DNA content nor concentration reached statistical significance (Table 3).

3.2. Experiment 2

A significant individual effect (P < 0.0001) was detected for all the parameters that were studied in
the single CL, namely DNA content and concentration (DNA/CL), P4 content and concentration (P4/CL
and P4/DNA). As shown in Table 4, other factors that significantly affected CL characteristics were CL
weight and age of the bitch. Heavier CLs had a higher P4 and DNA content (P < 0.001). CLs from bitches
younger than 2.5 years contained less P4 than CLs from older bitches (P < 0.05), and their luteal cells
showed a lower efficiency in P4 production (P4/CL, P4/DNA; P < 0.01).
Moreover, while the reproductive condition (pregnancy vs. diestrous) had no significant effect on
the parameters of the single CL per se, it became significant when age of the animal was considered.
CLs from younger diestrous bitches (n = 53) had a lower DNA content and concentration (P < 0.0001;
Fig. 1) than CLs from younger pregnant bitches (n = 92), although they showed a higher efficiency
(P < 0.0001) in P4 synthesis (P4, P4/CL, P4/DNA; Fig. 2). In contrast, CLs from adult diestrous bitches
(n = 39) had significantly more DNA than CLs from adult pregnant bitches (n = 53) (Fig. 1), even though
the efficiency of P4 production was higher in pregnancy (Fig. 2).
Finally, the right ovary showed a significantly higher CL number (P = 0.01), CLs with higher DNA
(P = 0.0016) and P4 content (P = 0.001), and higher P4 production (P4/CL, P = 0.0003; P4/DNA, P = 0.04)
compared to the left ovary.

Table 3
Mean (±S.E.) luteal characteristics and plasma P4 in bitches according to ovulation rate.

Ovulation rate

≤7 CLs (n = 22) >7 CLs (n = 23)

Weight of LT (mg) 656.4 ± 74.7 1421.0 ± 118.2


Mean CL weight (mg) 112.9 ± 9.7 140.3 ± 11.7
Plasma P4 (nmol l−1 ) 22.6 ± 2.7 29.9 ± 4.8
Total P4 (␮g) 24.4 ± 3.0 53.5 ± 8.4**
P4/LT (ng/mg) 36.5 ± 4.0 41.4 ± 5.5*
P4/DNA (ng/␮g) 8.6 ± 1.3 13.7 ± 2.5*
Total DNA (␮g) 3147.2 ± 455.3 6402.2 ± 752.5
DNA/LT (␮g/mg) 4.9 ± 1.8 4.9 ± 2.4

LT = individual luteal tissue; P4 = progesterone; total P4 = individual luteal content of progesterone; and total DNA = individual
luteal content of DNA.
*
P < 0.05.
**
P < 0.01.

Please cite this article in press as: Marinelli, L., et al., Factors affecting progesterone pro-
duction in corpora lutea from pregnant and diestrous bitches. Anim. Reprod. Sci. (2008),
doi:10.1016/j.anireprosci.2008.10.001
ANIREP-3709;
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doi:10.1016/j.anireprosci.2008.10.001
duction in corpora lutea from pregnant and diestrous bitches. Anim. Reprod. Sci. (2008),
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Table 4
Effect of age and CL weight on progesterone and DNA parameters evaluated in single CLs (N = 237). Different superscript letters indicate statistically significant different mean values
between classes of CL weight (Bonferroni test; P < 0.05). Significant differences between class of age are indicated by asterisks correspondent to P values.

Age CL weight

<2.5 years (n = 144) >2.5 years (n = 93) ≤100 mg (n = 54) 101–150 mg (n = 91) 151–200 mg (n = 62) >200 mg (n = 30)

P4 content (␮g) 4.4 ± 0.3 9.8 ± 0.3* 5.6 ± 0.6a 5.5 ± 0.3a 6.2 ± 0.5a 11.2 ± 0.7b
P4/CL (ng/mg) 26.9 ± 2.3 56.9 ± 2.4** 52.6 ± 4.0a 43.4 ± 2.2ab 37.9 ± 3.1ab 33.9 ± 4.7b
P4/DNA (ng/␮g) 8.3 ± 1.1 16.6 ± 1.2** 14.9 ± 1.9 12.1 ± 1.1 11.8 ± 1.6 10.9 ± 2.4
DNA content (␮g) 747.5 ± 34.6 729.4 ± 35.7 476.1 ± 59.8a 626.2 ± 33.6ab 715.0 ± 47.1b 1136.6 ± 71.7c
DNA/CL (␮g/mg) 4.9 ± 0.2 5.0 ± 0.2 5.1 ± 0.3 5.1 ± 0.2 4.7 ± 0.2 4.9 ± 0.4

P4 = progesterone; P4 content = CL total content of progesterone; and DNA content = CL total content of DNA.
*
P < 0.05.
**
P < 0.01.

7
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Fig. 1. Effect of the interaction between reproductive condition and age on DNA content (A; P < 0.0001) and concentration (B;
P < 0.0001) of individual CLs.

4. Discussion

The present study did not detect any differences in luteal characteristics between pregnant
and diestrous bitches, and luteal tissue weight, DNA and progesterone content/concentration were
not affected by pregnancy. Our results do not support the hypothesis of Concannon et al. (1977)
who indirectly invoked a higher rate of progesterone secretion in pregnancy, and also disagree
with the findings of Gudermuth et al. (1998) who measured higher fecal concentrations of sexual
steroids in pregnant bitches. However, both of these studies had only indirect evidence for their
conclusions, regardless of differences in progesterone clearance during pregnancy, and the effects
of animal age, parity and ovulation rate were only partly taken into account by Concannon et al.
(1977).
Our findings show that factors like animal weight and age have a greater influence on luteal
dimension, cell number (fresh mass weight and DNA content) and progesterone production than the
reproductive condition per se. In fact, adult bitches carried a higher number of CLs with a higher effi-
ciency in P4 synthesis than younger bitches, suggesting that luteal endocrine activity modifies from
young age to adulthood, undergoing a maturation process. This hypothesis is further supported by the
effect of the interaction between age and reproductive condition, which revealed a difference in DNA
content and P4 production efficiency among young and adult pregnant/diestrous bitches. Neverthe-
less, it should be pointed out that the effects of age and parity cannot be analysed separately in the
present study as it involved privately owned dogs, for 25% of which the history of past pregnancy was
unknown.

Please cite this article in press as: Marinelli, L., et al., Factors affecting progesterone pro-
duction in corpora lutea from pregnant and diestrous bitches. Anim. Reprod. Sci. (2008),
doi:10.1016/j.anireprosci.2008.10.001
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Fig. 2. Effect of the interaction between reproductive condition and age on progesterone content (A; P < 0.0001) and concen-
tration (B and C; P < 0.0001) of individual CLs.

Whereas previous observations demonstrated the effect of the age of the bitch on litter size (Kelley,
2002; Mutembei et al., 2002; Gresky et al., 2005; Bobic Gavrilovic et al., 2008), we found no effect of
age on foetal number, but only on ovulation rate, which was higher in older bitches. Analogous results
on ovulation rate were reported by Rocha et al. (2006) who concluded that middle-aged bitches are
likely to yield more oocytes of higher quality. In our study, older bitches showed a lower ‘number of
foetuses to CL number’ ratio, but we have no means of determining whether the exceeding CLs are the
result of unfertilised oocytes or embryo-loss.

Please cite this article in press as: Marinelli, L., et al., Factors affecting progesterone pro-
duction in corpora lutea from pregnant and diestrous bitches. Anim. Reprod. Sci. (2008),
doi:10.1016/j.anireprosci.2008.10.001
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10 L. Marinelli et al. / Animal Reproduction Science xxx (2008) xxx–xxx

The relationship between weight of the dam and litter size has already been hypothesised
(Robinson, 1973; Kelley, 2002), and the present data confirm this directly at the ovarian level, where
the number of CLs was significantly higher in heavy bitches. Our results regarding the effect of
weight of the bitch on total luteal tissue open an interesting field of investigation: the relation-
ship between body size and total luteal weight is well known among other species, and the size of
the mature CL is relatively constant within a species under physiological circumstances (Reynolds
and Redmer, 1999). However, experimental manipulation of the CL number (i.e. superovulation or
unilateral ovariectomy) in both monoovulatory and polyovulatory species leads to a compensatory
modification of the size of the mature CL, that is the reduction of size when an increased number
is induced and vice versa (Reynolds et al., 1994). Our data confirm the effect of body size on total
luteal weight and CL number in the canine species, where a greater CL number did not lead to a
reduction in mean CL size, and heavier CLs exhibited a higher progesterone and DNA content. Fur-
ther studies will be required to understand the mechanisms that adjust luteal function to body size
in the canine, a species displaying the widest range of body weight among domestic mammals. Since
there is evidence that the GH/IGF-I axis is an important determinant in adult body size among dog
breeds (Eigenmann et al., 1984a,b; Nap et al., 1992; Favier et al., 2001) and GH/IGF-I are known to
stimulate CL growth and function in many species (Niswender et al., 2000; Chandrashekar et al.,
2004), the role of these molecules in adapting luteal function to body size in the bitch needs further
investigation.
Luteal tissue weight has been correlated with circulating progesterone concentration in ruminants
(Niswender et al., 1986) and sows (Ricke et al., 1999), and a similar pattern between luteal and plasma
progesterone concentration during the oestrous cycle has been reported in gilts (Ricke et al., 1999) and
ewes (Jablonka-Shariff et al., 1993). In our study, plasma progesterone concentration was only weakly
correlated with luteal tissue weight and DNA concentration, and not with luteal tissue progesterone
content or luteal tissue P4 concentration. Nevertheless, a similarity in patterns of progesterone con-
centration between luteal tissue and plasma was present in our data, despite the fact that statistical
significance was not reached. Again, the wider range of ovulation rate and luteal tissue weight in the
canine species may increase the variability of luteal progesterone parameters.
Luteal progesterone concentration does not strictly reflect plasma progesterone concentration
(Bjersing et al., 1970; Mann et al., 2007), and the decline in plasma progesterone concentration in
the late luteal phase could be due to a reduction in ovarian perfusion, while local synthesis decreases
at a slower rate. In fact, it is well known that canine CLs undergo a slow regression and that proges-
terone synthesis is sustained until late diestrous and peripartum (Luz et al., 2006a), while intraovarian
blood flow decreases during the same phases according to the plasma progesterone concentration
(Köster et al., 2001).
The effect of ovulation rate on progesterone production is consistent with reports in other species
(ewes, Cahill et al., 1981; superovulated cows, Saumande et al., 1985; gilts, Knox et al., 2003; goats,
Simões et al., 2007), even if this effect does not reach statistical significance on plasma progesterone
concentration in the bitches in the present study. However, when mixed-breed instead of Beagle
bitches were examined, as in our case, a larger inter-individual variation had to be expected in P4
plasma profiles (Luz et al., 2006b); it is possible that if the same study is repeated in homogeneous
experimental groups the effect of ovulation rate might become significant on plasma progesterone
concentration.
Besides, a recent work, although on a limited number of animals, reported a strong individual daily
variation in plasma progesterone concentration (Linde Forsberg et al., 2008).
The higher functionality of the right ovary has been occasionally mentioned in different species,
such as bovine (Reece and Turner, 1938), ovine (Casida et al., 1966) and mouse (Wiebold and Becker,
1987). Our data also show the same effect in the canine species, for which previous observations
are controversial. Shimizu et al. (1990) found an equal CL distribution between the left and right
ovaries in about 200 bitches, while a higher CL number was reported in the right ovary following
ultrasonographic (Hayer et al., 1993) and direct (Reynaud et al., 2005) observations. Furthermore, a sig-
nificant difference in blood flow parameters was found between ovaries in the same bitch (Köster et al.,
2001), a condition hypothesised to cause uneven functionality between ovaries in the cow (McDonald,
1980).

Please cite this article in press as: Marinelli, L., et al., Factors affecting progesterone pro-
duction in corpora lutea from pregnant and diestrous bitches. Anim. Reprod. Sci. (2008),
doi:10.1016/j.anireprosci.2008.10.001
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L. Marinelli et al. / Animal Reproduction Science xxx (2008) xxx–xxx 11

5. Conclusion

Our findings highlight that the characteristics of canine corpus luteum and progesterone production
are influenced by many factors that have received little scientific attention to date. Due to the high vari-
ability among and within breeds, most of the literature on canine reproductive endocrinology has been
obtained with highly homogeneous experimental groups of Beagles. While such an approach has led to
important progress in understanding basic physiological mechanisms, more particular characteristics
of the species, such as the huge variability in body size, have not yet been given due consideration.

Acknowledgements

The present study was supported by the University of Padova. Our thanks are due to all the owners of
the dogs for the invaluable cooperation and to Mr Tommaso Brogin for his skilled technical assistance.

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