Factors Affecting Progesterone Production In Corpora Lutea From Pregnant And Diestrous Bitches

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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 b c

Dipartimento di Scienze Sperimentali Veterinarie, Università di Padova, Viale dell’Università 16, 35020 Legnaro (PD), Italy Dipartimento di Patologia Animale, Università di Torino, Via Leonardo da Vinci 44, 10090 Grugliasco (TO), Italy 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

Article history: Received 9 June 2008 Received in revised form 24 September 2008 Accepted 3 October 2008 Available online xxx Keywords: Bitch Corpus luteum Age Body size Progesterone

a b s t r a c t Factors affecting the characteristics of corpora lutea (number, left/right ovary origin, weight, DNA and progesterone content) were studied in 73 healthy bitches divided into two classes of age (≤2.5 vs. >2.5 years; mean ± S.E. = 3.6 ± 0.3 years; range: 0.7–10 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 (20–40 vs. 41–55 days) and ovulation rate (≤7 vs. >7). Two different assessments were performed: (a) comparison of luteal tissue characteristics and progesterone content between pregnant and diestrous bitches and (b) investigation of the effect of animal age, weight and ovulation rate on individual corpus luteum (CL) parameters. None of the luteal parameters differed between pregnant and diestrous bitches, even when the stage of the luteal phase was considered. 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: [email protected] (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 production in corpora lutea from pregnant and diestrous bitches. Anim. Reprod. Sci. (2008), doi:10.1016/j.anireprosci.2008.10.001

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CLs of younger bitches showed a diminished efficiency of P4 production (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 animal 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 progesterone 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 parturition (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 production 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 Sciences 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. Isolated 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 processed 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 number 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 (Experiment 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 suspension. Homogenised samples were then aliquoted and stored at −25 ◦ C for progesterone and DNA determinations.

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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 extraction 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 progesterone 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

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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 animals (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)

Weight (kg) Ovarian weight (g) Number of corpora lutea Number of foetuses Weight of LT (mg) Mean CL weight (mg) Luteal phase

17.7 2.58 8.12 6.37 1140.2 135.8 MLP (n = 15)

−1

Plasma P4 (nmol l Total P4 (␮g) P4/LT (ng/mg) P4/DNA (ng/␮g) DNA/LT (␮g/mg)

)

36.4 47.2 43.9 13.6 4.8

± ± ± ± ±

a

4.9 9.6 5.3 2.9 0.7

± ± ± ± ± ±

LLP (n = 9) 14.8 26.0 20.4 5.3 6.1

Diestrous (n = 21) 14.6 ± 2.3 2.03 ± 0.29 7.71 ± 0.64 / 941.01 ± 139.1 111.1 ± 10.8

2.1 0.31 0.55 0.66 117.8 11.4

± ± ± ± ±

MLP (n = 12) b

3.1 6.7 5.3 2.8 0.7

38.4 40.8 43.0 12.4 4.4

± ± ± ± ±

c

4.2 8.7 6.1 2.4 0.4

LLP (n = 9) 15.7 29.3 35.0 8.4 5.0

± ± ± ± ±

1.1d 9.9 8.7 0.5 0.8

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

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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

Number of corpora lutea Number of foetuses Weight of LT (mg) Mean CL weight (mg) Plasma P4 (nmol l−1 ) Total DNA (␮g) DNA/LT (␮g/mg)

Weight

≤2.5 years (n = 27)

>2.5 years (n = 18)

± ± ± ± ± ± ±

± ± ± ± ± ± ±

7.25 6.5 1001.6 125.7 30.0 5130.4 5.2

0.48 0.9 (n = 15) 101.2 10.5 4.0 669.6 0.4

9.22 6.1 1129.7 121.2 34.3 4603.6 4.3

***

0.54 0.9 (n = 9) 114.1 11.8 3.8 850.0 0.5

≤20 kg (n = 30) 6.50 5.3 650.8 100.4 30.8 3781 5.1

± ± ± ± ± ± ±

0.50 0.7 (n = 15) 104.5 10.8 3.1 493.9 0.4

>20 kg (n = 15) 9.99 8.1 1480.5 146.5 33.4 7226.7 4.5

± ± ± ± ± ± ±

0.50*** 1.1 (n = 9)** 107.9*** 11.2*** 6.0 973.3*** 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) Weight of LT (mg) Mean CL weight (mg) Plasma P4 (nmol l−1 ) Total P4 (␮g) P4/LT (ng/mg) P4/DNA (ng/␮g) Total DNA (␮g) DNA/LT (␮g/mg)

656.4 112.9 22.6 24.4 36.5 8.6 3147.2 4.9

± ± ± ± ± ± ± ±

74.7 9.7 2.7 3.0 4.0 1.3 455.3 1.8

>7 CLs (n = 23) 1421.0 140.3 29.9 53.5 41.4 13.7 6402.2 4.9

± ± ± ± ± ± ± ±

118.2 11.7 4.8 8.4** 5.5* 2.5* 752.5 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.

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4.4 26.9 8.3 747.5 4.9

± ± ± ± ±

0.3 2.3 1.1 34.6 0.2

>2.5 years (n = 93) 9.8 56.9 16.6 729.4 5.0

± ± ± ± ±

0.3* 2.4** 1.2** 35.7 0.2

≤100 mg (n = 54) 5.6 52.6 14.9 476.1 5.1

± ± ± ± ±

0.6a 4.0a 1.9 59.8a 0.3

P4 = progesterone; P4 content = CL total content of progesterone; and DNA content = CL total content of DNA. * **

P < 0.05. P < 0.01.

101–150 mg (n = 91) 5.5 43.4 12.1 626.2 5.1

± ± ± ± ±

0.3a 2.2ab 1.1 33.6ab 0.2

151–200 mg (n = 62) 6.2 37.9 11.8 715.0 4.7

± ± ± ± ±

0.5a 3.1ab 1.6 47.1b 0.2

>200 mg (n = 30) 11.2 33.9 10.9 1136.6 4.9

± ± ± ± ±

0.7b 4.7b 2.4 71.7c 0.4

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<2.5 years (n = 144) P4 content (␮g) P4/CL (ng/mg) P4/DNA (ng/␮g) DNA content (␮g) DNA/CL (␮g/mg)

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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.

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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 efficiency 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. Nevertheless, 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.

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Fig. 2. Effect of the interaction between reproductive condition and age on progesterone content (A; P < 0.0001) and concentration (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.

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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 relationship 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. Further 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 concentration 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 progesterone 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 significant 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).

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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 variability 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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