Introduction

In dogs, detailed endocrine interactions and their consequent physiological changes throughout pregnancy and parturition are still unknown. The steroid hormones (progesterone and estrogen) play an important role in the maintenance of pregnancy and onset of labor, through the constant modulation of myometrium excitability and contractility (Mesiano et al., 2002). In some animal species, the production of estrogen during the course of pregnancy occurs at various tissue sites. Estrogen action is opposed to progestational stimuli and contributes to uterine growth, which is of utmost importance for embryonic and fetal development (Dobson et al., 1993; Wood, 1999; Kindahl, 2002). The main endocrine event during parturition refers to the rapid increase in estrogen/progesterone ratio in the placenta. The decrease in progesterone and the increasing estrogen concentration possibly trigger detachment of the placenta, dilatation of the cervix and increase in uterine contractility (Concannon, 1986). Estrogen stimulates the onset of labor by enhancing the expression of genes related to myometrium excitability and contraction. Thus, estrogen increases uterine sensitivity to oxytocin progressively during pregnancy (Fuchs, 1983). However, until now no research has been undertaken in order to demonstrate such mechanisms in dogs. The objective of this study was to correlate estrogen and oxytocin profiles throughout pregnancy and parturition.

Materials and Methods

46 pregnant bitches at the reproductive age of 1 to 6 years of distinct breeds, clinically healthy and without history of reproductive or obstetric problems were selected. Bitches were allocated into 4 groups according to gestational age, determined by ultrasound examination and reproductive history. Group 1 (n = 11), consisted by females at the initial phase of pregnancy (up to 20 days), Group 2 (n = 12), consisted by females at 20 and 40 days of pregnancy, Group 3 (n = 12) represented by females at gestational age superior to 40 days and Group 4 (n = 11) with females presenting signs of first stage of labor. Blood samples were collected by jugular venous puncture, centrifuged at 1500 xg for 10 minutes and the serum stored at - 20 °C until processing. Estrogen concentration was measured by radioimmunoassay using a commercial kit DSL-4400 (Diagnostic Systems Laboratories, Webster, Texas, USA), previously validated for dogs. The sensitivity of the estrogen assay at 95% binding was 1.85 pg/mL and the low and high intra-assay coefficients of variation were 7.32% and 0.05%, respectively. For the oxytocin assay, 1 mL of serum was added to petroleum ether and acetone and the concentration of oxytocin was measured by radioimmunoassay, as described by Elias et al. (1997). The sensitivity of the oxytocin assay was 0.9 pg/mL and the low and high intra-assay coefficients of variation were 7.0% and 12.6%, respectively. Data were tested using the analysis of variance for repeated measures (ANOVA) followed by the Tukey test for multiple comparisons. The significance level considered was 5%.

Results

In relation to estrogen assay, Group 4 presented statistically higher concentration compared to Groups 1 and 2 (Figure 1). No statistical difference among Groups 1, 2 and 3 and between Groups 3 and 4 were verified. Oxytocin concentration did not differ statistically throughout pregnancy (Groups 1, 2 and 3) and parturition (Group 4) (Figure 2).

Figure 1. Estrogen concentration in Groups 1, 2, 3 and 4. a,b represent significant difference among Groups (p < 0.05).

Figure 2. Oxytocin concentrations in Groups 1, 2, 3 and 4. a,b represent significant difference among Groups (p < 0.05).

Discussion and Conclusions

In dogs, research related to endocrinology and physiology of pregnancy and parturition includes the localization of the main tissue sites involved in the synthesis, secretion and action of reproductive hormones. Effects and changes arising from hormone interaction during these events are fairly known. Thus, the use of hormonal treatment for diseases associated with pregnancy and parturition is empirically performed. The increased level of estrogen verified in this study from 40 days of pregnancy onwards is in agreement with previous research (Luz et al., 2006). The increase in estrogen concentration at the end of pregnancy may be related to the stimulus of oxytocin receptors at the endometrial site and consequently release of prostaglandin F2α. In ruminants, this mechanism triggers luteolysis and the onset of labor (Spencer et al. 1995). Thus, future research should be carried out in order to accurately relate those events in the canine species. In women, plasmatic concentration of oxytocin remains low throughout pregnancy and parturition. However, treatment with estradiol may enhance oxytocin circulating levels (Amico et al., 1981, Mitchell et al., 1998). Recently, Klarenbeek et al. (2007) showed that oxytocin concentration at the end of pregnancy in bitches remains low. However, it may increase only at the second stage of labor, but not necessarily at the time of fetal expulsion. The results of the present study can be corroborated by previous reports, as oxytocin profile remained constant throughout pregnancy and parturition. Despite the recognized placental production of oxytocin as a mechanism to induce luteolysis (Chan et al., 1993), it is known that circulating levels of this hormone do not change throughout pregnancy. This suggests that oxytocin profile in dogs has minimal influence in the onset of parturition. Hence, we can infer that the activation of oxytocin receptors in the myometrium may be a result of oxytocin paracrine action from placental origin. The information that arose from the present research can serve as a contribution to the practical application of hormones during pregnancy and parturition. However, future studies should be conducted to explain endocrine interactions and standardize endocrine profile for the scientific knowledge in the canine species.

References

1.  Mesiano S, Chan EC, Fitter JT, Kenneth K, Yeo G, Smith R. Progesterone withdrawal and estrogen activation in human parturition are coordinated by progesterone receptor A expression in the myometrium. The Journal of Clinical Endocrinology e Metabolism, v. 87, n 6, p. 2924-2930, 2002.

2.  Dobson H, Rowan TG, Kippax IS, Humblot P. Assessment of fetal number, and fetal and placental viability throughout pregnancy in cattle. Theriogenology, v. 40, p. 411-425, 1993.

3.  Wood CE. Control o f parturition in ruminants. Journal of Reproduction and Fertility Supplement., v. 54, p. 115-126, 1999.

4.  Kindahl H, Kornmatitsuk B, Königsson K, Gustafsson H. Endocrine changes in late bovine pregnancy with special emphasis on fetal well-being. Domestic Animal Endocrinology, v. 23, p. 321-328, 2002.

5.  Concannon PW. Canine Pregnancy and Parturition. Veterinary Clinics of North America Small Animal Practice., v. 16, n. 3, p. 453-475, 1986.

6.  Fuchs AR, Periyasamy S, Alexandrova M, Soloff MS. Correlation between oxytocin receptor concentration and responsiveness to oxytocin in pregnant rat myometrium: effects of ovarian steroids. Endocrinology, v. 113, p. 742-749, 1983.

7.  Luz MR, Bertan CM, Binelli M, Lopes MD. Plasma concentrations of 13, 14-dihydro-15-keto prostaglandin F2 alpha (PGFM), progesterone and estradiol in pregnant and nonpregnant diestrus cross bred-bitches. Theriogenology, v.66, p. 1436-1441, 2006.

8.  Spencer TE, Becker WC, George P, Mirando MA, Ogle TF, Bazer FW. Ovine interferon-tau regulates expression of endometrial receptors for estrogen and oxytocin but not progesterone. Biology of Reproduction, v. 53, p. 732-745, 1995.

9.  Amico JA, Seif SM, Robinson AG. Oxytocin in human plasma: correlation with neurophysin and stimulation with estrogen. J Clin Endocrinol Metab, v. 52, n. 5, p. 988-93, 1981.

10. Mitchell BF, Fang X, Wong S. Oxytocin: a paracrine hormone in the regulation of parturition? Journal of Reproduction and Fertility, v.3, p. 113-122, 1998.

11. Klarenbeek M, Okkens AC, Kooistra HS, Mol JA, Bevers MM, Taverne MAM. Plasma oxytocin concentrations during late pregnancy and parturition in the dog. Theriogenology, v. 68, p. 1169-1176, 2007.

12. Chan WY, Chen DL, Manning M. Oxytocin receptor subtypes in the pregnant rat myometrium and decidua: pharmacological differentiations. Endocrinology. v. 132, p. 1381-1386, 1993.