CLINICAL USE OF LONG-TERM GnRH AGONIST IN PREPUBERTAL BITCHES: EFFECT ON P4, E2 AND MELATONIN SERUM CONCENTRATION BEFORE AND DURING FIRST ESTRUS

The aim of this study was to determine the length of the contraceptive period after the application of deslorelin implant (4.7 mg) in juvenile, prepubertal bitches and assess the effects on the hormonal status (P4, E2 and melatonin), as well as on the occurrence of normal physiological estrus. Twelve crossbred prepubertal female dogs (4 months of age) were divided into the two groups, the treated group (DESLO) and control (placebo) group (C). Hormone assays, vaginal microbiological and cytological smears and the measurements of electrical resistance of cervical mucus were performed once in 3 weeks until the ﬁ rst signs of proestrus, and every day during estrus. GnRH implant signi ﬁ cantly postponed time of onset of estrus (633 ± 30,38 days vs. 143.80 ± 52,30 days, P<0.001). In estrus, DESLO group showed statistically higher E2 (34.58 pg/mL vs. 20.59 pg/mL, P<0.001) and melatonin serum concentration (45.86 pg/mL vs. 18.85 pg/mL, P<0.001) compared to the control group. The average P4 serum concentration had no statistically signi ﬁ cant differences between groups. In conclusion, deslorelin implants do not cause “ ﬂ are up” effect in perpubertal bitches, however, increase of E2 and especially melatonin serum concentrations show the need to explore the consequences and mutual in ﬂ uence of melatonin and deslorelin implants in the future.


INTRODUCTION
In modern veterinary practice, many of the problems faced by veterinarians, and of great concern for dog owners, are related to reproduction and reproductive control options.For many years, surgical castration was the only reliable and permanent method of contraception in dogs.Ovariectomy, ovariohysterectomy (OHC) and orchidectomy are the most common surgical procedures routinely performed in small animal practice [1,2].Prepubertal ovariectomy (or OHC) is defi ned as the surgical castration of immature female animals between 6 to 14 weeks of age, and it is often associated with some long-term behavior and health effects [3][4][5][6].Therefore, discussions and controversies about surgical spaying are ongoing in many countries, emphasizing the associated benefi ts and risks in prepubertal animals as undergo physical and physiological changes to become reproductively viable [3,7].
In recent years, numerous contraceptive drugs have come onto the market, many of which have more than one indication and application.They have direct or indirect effect on the sexual cycle of animals.Therefore, their use requires experience, correct and timely dosing and knowledge of side effects and contraindications [8][9][10].The chosen method should be acceptable to the owner (single application of injectable medications, uncomplicated oral application, and low cost) [10].
Application of procedures to suppress estrus in female and reduce libido in male dogs are common in small animal practice [11].Many studies have investigated the use of GnRH agonists as hormonal reversible sterilization in males and its effi cacy has been well established [12][13][14][15], therefore the commercially available products are registered for use only in male dogs [10].In the past decades, gonadotropin-releasing hormone (GnRH) agonists had a great perspective in the management and control of estrus in bitches [11,[16][17][18].Implants are subcutaneous insertions of slow-release GnRH, such as the 4.7 mg and 9.4 mg deslorelin acetate and represent an excellent alternative for reversible control of ovarian activity in diestrus bitches [11].The fi rst step in the mechanism of action of GnRH agonists is the "fl are-up" effect, which is characterized by an increased synthesis of gonadotropins.This stimulating effect is more pronounced in bitches than in males.Estrus induction by this method can be observed in most bitches in anestrus, regardless of the phase of anestrus [16,19].Most authors have confi rmed that all adult bitches respond in the same way regardless of size and age; however, depending on the phase of the cycle, it is possible that the response to the applied implant may be absent [14].After the "fl are-up" effect, the natural pulsatile releases of hypothalamic and pituitary hormones are blocked.This leads to a basal release of gonadotropin, after which synthesis and release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are reduced and consequently prevent follicular development, i.e., it leads to a temporary long-term delay of the estrus cycle [7].
Melatonin (N-acetyl-5-methoxytryptophan) is a neurohormone secreted by the pineal gland and has a circadian rhythm, with low secretion levels during day and high during night.In short-day breed animals, melatonin controls the seasonal biological and reproductive rhythm, onset of estrus and improves sperm quality [5,20].Melatonin synthesis and secretion play one of the main roles in the complex process of GnRH synthesis, and it is of most importance to determine to what extent deslorelin implant affects the endocrine status in dogs.
There are insuffi cient data on the length of action of GnRH implants applied to prepubertal bitches and its infl uence on the appearance of estrus and hormonal status after the discontinuance of action, as well as the possibility of complications.The aims of this study were to determine the length of the contraceptive period after the application of GnRH agonist in juvenile, prepubertal bitches and assess the effects on the hormonal status (progesterone, estrogen and melatonin), as well as on the occurrence of normal physiological estrus.

Animals
Twelve healthy, crossbred prepubertal female dogs were used in this study, divided into two groups, the treated group (DESLO) and control (placebo) group (C).All dogs were on average 4 months of age, similar constitution, and body weight (range 10-15 kg), and were held in the same housing conditions (indoor/outdoor).They were fed with same commercial granulated food in accordance with age category.Before being introduced into the study, a clinical examination was performed on each animal, blood analyses (hematological and biochemical parameters, hormonal status -progesterone and estrogen), as well as examination of the genital tract (ultrasound, vaginal cytological and bacteriological smear, the electrical resistance of cervical mucus -Draminski DOD2, Poland) with the aim of excluding puberty.This study was reviewed by the Ethics Committee of Ministry of Agriculture, Forestry and Water Management; Veterinary Administration (No. 323-07-07459/2020-05) and has been approved by the owners.

Treatment
Animals in the DESLO group (n=6) were treated with subcutaneous 4.7 mg deslorelin acetate implants (Suprelorin®, Virbac, France) in the umbilical region, and recorded data were submitted in the accompanying health record.The control group (n=6) received a placebo subcutaneous injection (2 ml sodium chloride 0.9%).After insertion, animals were monitored daily for the presence of allergic reactions and estrus by physical examination of the vulva, vaginal smears (Diff-Quick staining) and changes in sexual behavior for the fi rst 5 weeks.Hormone assays, vaginal cytological smears and the measurements of electrical resistance of cervical mucus were performed once in 3 weeks until the fi rst signs of proestrus, and every day during the estrus period.The changes in the cellular profi les obtained by vaginal cytological smears were monitored in order to detect proestrus: from the predominance of parabasal cells in anestrus (accompanied by varying numbers of neutrophils), to the successive alternation of medium-sized squamous cells to large intermediate cells, and then to large keratinocytes until they fi nally consist entirely (98-100%) of keratinocytes (cornifi ed) cells with an absence of neutrophils (Concannon, 2011).Gradual increase of electrical resistance of cervical mucus from 120 to 220 ohms was considered as the onset of proestrus (Concannon, 2011).Increase of E2 (during proestrus) and P4 (at the end of proestrus and estrus) serum concentrations indicated follicular growth and ovulation.

Hormone assays
For the hormone analyses blood samples were taken from the v. cephalica antebrachii and collected in test tubes without anticoagulants.Blood serum was separated after centrifugation at 3000 rpm during 15 minutes and stored at -20°C.Progesterone (P4) and 17-beta estradiol (E2) concentrations were determined by the chemoluminescent method, using an automatic hormone analyzer (Access 2 Beckman Coulter, USA) using commercial kits according to the manufacturer's instructions in special laboratory (VetLab, Veterinary laboratory for clinical diagnostics, Serbia).

Statistical analyses
The data were tested for normality using the Shapiro-Wilk's test.If the data were normally distributed (Shapiro-Wilk's test, P > 0.05), the unpaired t-test was used for the comparison of two groups.If the data were not normally distributed (Shapiro-Wilk's test, P < 0.05), the Mann-Whitney test was used for the comparison of two groups.Signifi cant difference was estimated at P < 0.05 level.Statistical analysis of the results obtained in the experiment was carried out using statistical software GraphPad Prism version 7 (GraphPad, San Diego, CA, USA).

Treatment
The implant procedure was done routinely, and no clinically detectable local or systemic side effects associated with the treatment were observed in any of the experimental groups.

Hormone assays
The values of the days to the fi rst signs of estrus showed homogeneity (CV<30%) in DESLO group (CV = 4.8%), while in the control group values showed heterogeneity (CV= 36.36%).The time of onset of estrus between the groups was signifi cantly different (P<0.001),whereby the control group had an average 143.80 ± 52,30 days and DESLO group had average 633 ± 30,38 days until fi rst signs of estrus, which was 4.4 times longer in DESLO group (Figure 1).Application of desloreline implants led to higher average blood serum E2 concentrations (P<0.01) in DESLO group one week before the start of estrus (10.13 pg/mL vs 4.95 pg/mL).It is important to note that serum E2 concentrations were slightly higher, with no statistical signifi cance, in DESLO group between 6-12 days after application of implants, while bitches in the control group showed no increase of E2 concentracion.In estrus, DESLO group showed a statistically higher E2 concentration (P<0.001) compared to control group (Figure 2) with average concentration of 34.58 pg/mL and 20.59 pg/mL, respectively, and E2 concentration in both groups displayed homogeneity (CV<30%).Two of 6 animals in DESLO group showed an increase of E2 serum concentration up to 36 and 40 pg/mL, while maximum E2 concentration in the control group was 23 pg/mL (2/6 animals).During the period of anestrus (monitoring) in the treated DESLO group, P4 serum concentration was under 0.6 ng/mL.The average P4 serum concentrations during estrus were higher in DESLO group (DESLO 13.69 ng/mL and C 10.07 ng/mL), but with no statistical signifi cances between groups, and the P4 concentrations in the control group displayed heterogeneity (CV= 41.99%).Melatonin serum concentrations in DESLO group were statistically signifi cantly higher (P<0.001)during estrus compared to the control group with average concentrations of 45.86 pg/mL and 18.85 pg/mL, respectively.In both experimental groups melatonin concentration displayed homogeneity (CV<30%), and highest values were in DESLO group 3 week after fi rst signs of estrus (52.16 pg/mL).In 2/6 bitches in the DESLO group dorsal vaginal wall hyperplasia (type 1) was noticed after the fi rst signs of estrus which regressed spontaneously and no longer occurred in the following estrus.In addition, 2/6 bitches in the DESLO group and one bitch in the control group developed mild juvenile vaginitis, between the 20th -30th week and at the 14th week, respectively.These conditions did not require any special treatment and the animals recovered spontaneously.

DISCUSSION
The aim of this study was to evaluate the long-term effects of GnRH deslorelin implants (4.7 mg) on endocrine status, anestrus duration and eventual visible side effects in medium-sized prepubertal bitches.Although the safety and effect of the GnRH agonist application has been proven in dogs [21][22][23][24][25] showing promising results of reversible contraception, these aims in bitches encountered a bit complicated physiological issues.Generally, the defi nition of puberty in dogs is highly variable since there are many breeds and it is independent on body weight [7,26].Defi nition of onset of puberty between male and female dogs is different and has its own timetable, where puberty in females probably starts earlier followed by change in follicular growth, vaginal cytology, and steroid hormones, while in males is marked with mounting and fi rst spermatozoa in the urine [27].
A limited number of previous studies have revealed that a visible response to the administration of deslorelin implants -the "fl are-up" effect -occurred in prepubertal bitches at more than 7 months of age, whereas prepubertal bitches at 4 months of age showed no signs of estrus after administration [28][29][30][31].After insertion of the implant, all bitches were monitored for the "fl are-up" effect using clinical examination, vaginal cytology, the electrical resistance of cervical mucus and P4 and E2 serum concentration.In DESLO group, like in the previous investigations with bitches at the age of 4 months there was no "fl are-up" effect, and the average time of onset of estrus was 633 days (90 weeks).This average period of reversible anestrus was longer than average time to puberty (82.7 weeks) in the investigation of Kaya et al. (2015) using animals of similar size and age.However, 4 animals in the mentioned study showed fi rst signs of estrus on average 101.5 weeks after the application.In favor of deslorelin effectiveness, Faya et al. (2018) treated newborn male and female puppies within 24h after birth and postponed puberty for an average of 76.8 weeks.
During the observation period, the animals from the DESLO group showed small variations in P4 and E2 serum concentrations, as previously explained in the study [31], in which the deslorelin implants showed individually variable P4 and E2 serum concentrations during the observation period of 8 to 19.5 months.The reason for these slight discrepancies in the results could be due to the high variability of factors in each study with similar or different designs and, more likely, individual differences in drug absorption or metabolism between the bitches, as only 1-2 of them showed these variations [7,30,31].These oscillations of P4 and E2 could be avoided using neonatal deslorelin administration since there is a lack of sexual hormonal receptors at the early neonatal period [32].
Serum P4 concentrations did not differ among the DESLO and control group as stated in previous studies [31,32] and the status of P4 concentration is in compliance with the conclusion that deslorelin does not have negative effects on ovarian functionality after long-term estrus delay in bitches [34].
Melatonin, as a regulatory neuropeptide, causes many responses to changes in the body, as well as the response of the organism to changes in the environment.For the fi rst time, was brought in connection the suppressive effect of deslorelin on the hypothalamus -pituitary gonadal axis and the status of melatonin serum concentration, since melatonin has one of the most important roles in the synthesis of GnRH.In addition, E2 serum concentration is in discrepancy compared to previous studies [28][29][30][31]34], since there are statistically higher E2 concentrations (P<0.001) in the DESLO group.Also, melatonin serum concentrations were statistically higher in the DESLO group after onset of estrus.One of the fi rst researches related to the infl uence of melatonin on endocrine status in castrated and intact male dogs [5,35], showed that increase of melatonin decreased progesterone and estrogen concentrations in the intact and castrated dogs.Since the research was done on male dogs [5,35], it remains questionable how much difference there is in the mechanisms of action and regulation of certain hormones between the sexes, or to be more precise, how estrus effects on melatonin status after deslorelin suppression.It is hypothesized that following deslorelin effect -estrus suppression in prepubertal females -a larger pool of follicles is recruited whose ovulatory capacity is questionable but which are capable of producing a higher concentration of estrogen.During anestrus, deslorelin implant is a source of GnRH, and the hypothalamus -pituitary gonadal axis is suppressed.After long-term reversible contraception effect of deslorelin, activity of melatonin follows the synthesis and release of GnRH, which, in our study, showed high serum concentrations.
In conclusion, deslorelin implants do not cause the "fl are up" effect in perpubertal bitches, however, increase of E2 and especially melatonin serum concentrations were observed.Since the positive effects of melatonin on endocrine and immunological development as well as its antioxidant, anticancer and cytotoxic effects have been demonstrated in decades of research [5,[35][36][37][38], it is of utmost importance to explore the consequences and mutual infl uence of melatonin and deslorelin implants in the future.