Background: Real time RT-PCR (qPCR) is a useful and powerful tool for quantitative measurement of gene expression. The proper choice of internal standards such as reference genes is crucial for correct data evaluation. In female dogs, as in other species, the reproductive tract is continuously undergoing hormonal and cycle stage-dependent morphological changes, which are associated with altered gene expression. However, there have been few attempts published so far targeted to the dog aimed at determining optimal reference genes for the reproductive organs. Most of these approaches relied on genes previously described in other species. Large-scale transcriptome-based experiments are promising tools for defining potential candidate reference genes, but were never considered in this context in canine research. Results: Here, using available microarray and RNA-seq datasets derived from reproductive organs (corpus luteum, placenta, healthy and diseased uteri) of dogs, we have performed multistudy analysis to identify the most stably expressed genes for expression studies, in each tissue separately and collectively for different tissues. The stability of newly identified reference genes (EIF4H, KDELR2, KDM4A and PTK2) has been determined and ranked relative to previously used reference genes, i.e., GAPDH, β-actin and cyclophillin A/PPIA, using RefFinder and NormFinder algorithms. Finally, expression of selected target genes (luteal IL-1b and MHCII, placental COX2 and VEGFA, and uterine IGF2 and LHR) was re-evaluated and normalized. All proposed candidate reference genes were more stable, ranked higher and introduced less variation than previously used genes. Conclusions: Based on our analyses, we recommend applying KDM4A and PTK2 for normalization of gene expression in the canine CL and placenta. The inclusion of a third reference gene, EIF4H, is suggested for healthy uteri. With this, the interpretation of qPCR data will be more reliable, allowing better understanding of canine reproductive physiology.

To evaluate the expression of AMH and its receptor AMHRII, ovaries of 33 p cats were investigated by western blot and immunohistochemistry. After ovariohysterectomy, the cats were grouped according to pregnancy stages and ovarian/placental endocrine activity: group I (n = 3, 24–29 days), II (n = 8, 32–40 days), III (n = 4, 41–46 days), IV (n = 6, 53–61 days) and according to cycle stages: V (n = 6, interestrus) and VI (n = 6, estrus). Serum progesterone- and AMH-concentration was measured. Follicle numbers did not differ between groups. The number of corpora lutea was higher in pregnant cats than in the non-pregnant cats. Serum AMH concentration was at maximum between day 30 and 50 of gestation, and was higher than in non-pregnant cats, then decreased towards term (p < 0.05). In the ovaries, AMH immunopositivity was observed in granulosa cells of secondary and antral follicles, and in interstitial cells of corpora lutea; highest percentage of immunopositive areas was detected in group III (p < 0.05). A positive correlation between the number of corpora lutea and the positive AMH signals in ovarian tissue was determined (r² = 0.832, p < 0.05); however, only during mid-gestation (group II). Expression of AMHRII was in close co-localization with AMH and strong in the interstitial cells surrounding follicles undergoing atresia. AMHRII expression did not differ between pregnant groups but was higher compared to estrus cats (p ˂ 0.05). We conclude that AMH and AMHRII expression in the feline ovary is comparable to other species. The high serum AMH concentration and ovarian AMHRII expression between day 30 and 50 of gestation are probably related to ovarian activity and follicular atresia.

The search for an alternative approach of estrus control (induction or suppression) in dogs is an important issue and the use of slow GnRH agonist-releasing implants has been the subject of frequent research in recent years. Studies to date demonstrate that the short- and long-term effects of deslorelin implants applicated at different time points of the prepubertal period are similar to those of adult dogs; however, there are important differences. The age of the prepubertal bitch and the dosage appear to be the main determinants of the response to deslorelin, as well as the individual metabolism of the bitch. Recent studies reported that the deslorelin-mediated long-term delay of puberty does not have negative carry-over effects on subsequent ovarian functionality, serum steroid hormone concentrations, uterine health, and fertility; however, more molecular studies are needed to determine the effects of application time of GnRH agonists on hormone concentrations and peripheral receptor expression. Furthermore, the long-term effects of delay of puberty with deslorelin on joint health, tumor development, the immune system, and social behavior deserve further investigations.

The canine corpus luteum (CL) is the main source of reproductive steroids during dioestrus in the dog and remains active even in the absence of pregnancy (non‐pregnant dioestrus, physiological pseudopregnancy). Whereas the biological effects of 17β‐oestradiol (E2) in the canine CL remain unclear, the transcriptional availability of oestrogen receptors, ESR1 and ESR2, as well as other modulators of local availability of E2, for example, HSD17B7 (converts oestrone into oestradiol), SULT1E1 (inactivates E2 binding capacity to its own receptors through sulphonation) and STS (reverts E2 sulphonation), were previously detected in the CL of non‐pregnant bitches. The aim of the present work was to evaluate the mRNA amounts of these factors involved in luteal sensitivity and metabolism of E2 in the canine CL during the course of non‐pregnant dioestrus (days 10, 20, 30, 40, 50 and 60 post‐ovulation, n = 5/group) and at different stages of pregnancy (n = 4‐6/group): pre‐implantation (days 8–12), post‐implantation (days 18–25), mid‐gestation (days 35–40) and prepartum luteolysis. During pregnancy, the availability of ESR1, HSD17B7, SULT1E1 and STS decreased from mid‐pregnancy to prepartum luteolysis. The main findings during non‐pregnant dioestrus were as follows: increased ESR2:ESR1 ratio on days 40 and 50 after ovulation, decreasing during luteal regression (day 60); increased STS at day 30 when SULT1E1 levels decreased; increased availability of SULT1E1 transcripts during luteal regression; and decreased amounts of HSD17B7 mRNA in early dioestrus, increasing towards later stages. These results suggest that E2 signalling and biologically active local concentrations could diverge in response to time and pregnancy status of the bitch.

The values of luteal blood flow (LBF), total corpus luteum (CL) area (TAR), and progesterone (P4), during and after OvSynch (OvS) protocol in comfort (CP; n = 40) and hot periods (HP; n = 40) were compared. We investigated how low and high P4 values obtained before the application affected the parameters above during CP and HP periods. Blood samples were collected before the OvS application on day 0 (OVSd0), day 9 (OeG), and day 18 (9th day after OeG: OvSd9). The P4 (ng/mL) values of the animals exhibiting dominant follicles were between 0.12–0.82 in HC and 0.1–0.88 in CP (P4-2: 4.36–4.38 and P4-3: ≥7.36 ng/mL). The LBF values were measured on days 7 (OvSd7) and 9 (OvSd9) after the OeG. The P4 mean values at day 0 (OvSd0) were classified as low (P4-1), medium (P4-2), and high (P4-3). The LBF and the TAR values in the P4-2 and P4-3 on OeG day 9 were higher than in HP (p < 0.05; 0.001), but there was no significant difference in the P4-1. In conclusion, when the OvS program was initiated with low P4 values, no difference was observed between HP and CP in terms of LBF values; however, when the program was started with high P4 values, there were significant increases in LBF and TAR values in the CP compared to the HP.