Aglepristone efficiency with and without the canine pyometra cloprostenol

Objective. Evaluate the effectiveness of aglepristone with and without cloprostenol for treating canine pyometra. Materials and methods. Ten female French Bulldogs between 3-8 years of age diagnosed with pyometra were divided into two groups, one receiving a 10 mg/kg dose of aglepristone subcutaneously on days 1, 2, 8, 14 and 28, and the other an equal dose on days 1, 3, 8 and 15, as well as a 1 µg/kg dose of cloprostenol subcutaneously on days 3 and 8. Both groups received 5 mg/kg of enrofloxacin orally every 12 hours for 28 days. Hematic profiles were generated on days 1 and 28 and counted days until the next heat, expelling purulent material and of clinical recuperation. Results. There was no statistical difference in the variables of the line red blood cells between times and protocols. The line of white blood cells showed a statistical difference between days of treatment but not between the two groups. Both treatments were effective resulting in complete clinical recuperation. The combination of aglepristone with cloprostenol acted more quickly, decreasing the number of days of secretion, abdominal pain, and the recuperation of normal activity and appetite. All 10 females were bred during their first heat. Three females from the aglepristone group and one from the combined aglepristone with cloprostenol group were impregnated. Conclusions. Both protocols, combined with enrofloxacin, produced complete clinical recuperation in bitches with pyometra, but it was achieved more rapidly when given the combination of both aglepristone and


INTROODUCTION
Pyometra is a uterine pathology of bacterial origin stimulated by progesterone during consecutive heat cycles, and by an exogenous estrogen and/ or progesterone treatment (1).It is reported principally in females over six years of age (2,3).
It is a pathological process that usually begins with cystic endometrial hyperplasia (CEH), and that permits the proliferation of vaginal bacteria, accumulating purulent material inside the uterus (3)(4)(5).It is considered one of the most frequent pathological alterations in canine reproduction (4,6).It begins during the estrous phase of the reproductive cycle, by morphological changes induced by progesterone (P4) and estrogen (E2), along with an abnormal response in the uterus during the luteal phase, which combined with bacterial action results in pyometra (5,7).Two clinical forms are distinguished: closed pyometra, whose signs are very severe and open pyometra.Some of the common signs are: vomiting, polydipsia, polyuria and anorexia (2,3,5).
Treatment of pyometra should be urgent and aggressive, including the extraction of contents, which is generally surgical in 80% of cases (8)(9)(10).None the less, pharmacological treatment is also possible (11)(12)(13).The effectiveness of the use of natural prostaglandin F2α has been traced in 55% of cases of open pyometra and 24% of closed pyometra, respectively (14)(15)(16).It was found least effective in cases of closed pyometra (24%), which brought about the use of progesterone antagonists like aglepristone (15,17), which improve efficacy when combined with prostaglandins, above all, in order to carry out therapies in bitches of high economic value and where surgery is not an option (18).
The principal cause of canine pyometra, in 90% of cases, is Escherichia coli in 90% of cases (2,6,19), which penetrates the uterus during estrus (5,19,20).For this reason treatment should always be accompanied by antibiotics or antimicrobials, especially with a spectrum against Gram-negative microorganisms such as gentamicin, amikacin, enrofloxacin, ciprofloxacin, among others (19).
Females treated jointly with progesterone a n t a g o n i s t s a n d n a t u r a l o r s y n t h e t i c prostaglandins F2α were 75% of the time cured (11,15,17), while patients that only received prostaglandins only reached 60% (13).This is due to the fact that the canines presented less uterine response to the use of prostaglandins (11,160); other treatments have been mentioned as having similar results (8,12,18).Among these Corrada et al (14) describe the effectiveness of using dopamine agonists (Cabergoline) in treating canine pyometra.
Various studies demonstrated the cure of pyometra with only a single use of 10 mg/kg of antiprogestin aglepristone subcutaneously on days 1, 2 and 8 of the diestrus, combined with cloprostenol, which induces the opening of the cervix and the resulting drainage of the uterus (13)(14)(15).Other protocols with an efficacy of 95% are: 10 mg/kg of aglepristone subcutaneously on days 1, 3, 8, and 15 of the diagnosis, combined with cloprostenol 1 µg/kg subcutaneously on days 3 and 8, or both combined on days 3, 5, 8, 10, 12 and 15 (13,18).Only Molina (18) describes using aglepristone as a singular therapy in a report on the case of a French Bull Dog with pyometra.However, the efficacy of aglepristone as a singular medicine for this treatment has not been determined in a comparative study.
The objective of the present study was to value the efficacy of the antiprogestin aglepristone with and without cloprostenol in treating pyometra in female canines.

MATERIAL AND METHODS
Study location.Veterinary Clinic of Antioquia, Medellín Colombia, from 2011 to 2013.Animals. 10 female French Bulldogs, between 3-8 years of age, diagnosed with closed pyometra.
All animals used were of high breeding value because of which their owners sought alternative therapy to conventional ovariohysterectomy.All bitches had closed pyometra, with clinical signs of polydipsia, polyuria, vomiting, lack of energy and anorexia; as well as their last heat being 45-60 days prior.
Experimental design and treatments.The females were randomly assigned to one of the two treatment groups, one with a 10 mg/kg dose acompañado de antibióticos o antimicrobianos, en especial con espectro contra microorganismos Gram negativos como lo son la gentamicina, amikacina, enrofloxacina, ciprofloxacina, entre otros (19).
The pyometra diagnosis in all patients was made by means of a clinical exam, blood profile, as cited later on, and confirmation by echography.
Evaluation of the bitches.The hematological evaluation was carried out starting with blood samples from the jugular vein, gathered in tubes with EDTA on days 1 and 28 of the diagnostic.The samples were processed on Abacus junior vet ® (Diatron Ltda, Austria) equipment according to the following parameters: hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin concentration (MCHC), erythrocytes, leukocytes, neutrophils (N), eosinophils (E), lymphocytes (L), lemniscus (B), monocytes (M), platelets (PL) and plasma proteins.
The evaluation of uterine contents was made with an abdominal echography using a Midray DP 3300 B/N ® system every 8 days, during the 28 days of treatment.In addition, the followup included: time until the next proestrus (by vaginal smear and progesterone measurements above 8 mg/ml), the days of expulsion uterine content and the time of clinical recuperation of each patient, measured by the absence of clinical signs; among which were evaluated: absence of vomiting, absences of abdominal pain through palpation, measured according to the Universidad de Melbourne (UMPS) pain scale, return of appetite and normal activity.The evaluation of the patient was ambulatory until resolving the symptoms.
The females were paired and naturally mated on days 11, 14 and 16 from the initial day of proestrus.For this study only the presence or absence of gestation was evaluated by echography 23 days after copulation; no further monitoring was carried out on the non-pregnant females.

Statistical analysis.
The variables were subject to ANOVA, with Tukey HSD and Chi 2 tests for qualitative variables and with a non-parametric test (Mann Whitney test).
Análisis estadístico.Las variables fueron sometidos a ANOVA, con el test de HSD Tukey y Chi 2 para las variables cualitativas y con test no paramétrico (Mann Whitney test).committee, by means of resolution 001 on October 2013.

RESULTS
Ten female French Bulldogs were evaluated, of which there was not found a significant difference between groups and protocols (p≥0.05).In the aglepristone group the average age was 4.2±1.92years and in the combined aglepristone and cloprostenol group 3.2±1.30years.
In the hemogram evaluation during the two periods, day 0 and 28, no variation in the red line was observed, neither before nor after the treatment (Table 1), there was no significant difference between inter or intra treatments (p≥0.05).
Las 5 hembras de cada protocolo se aparearon en el primer celo.De ellas, el 40% (3 de 5) del grupo de aglepristone quedo gestante, con diagnóstico ecográfico a los 23 días pos monta, mientras que sólo el 20% (1 de 5) del grupo aglepristone y cloprostenol quedo preñada.Todas las hembras In the leukogram there was no statistical difference found between aglepristone and aglepristone in addition to cloprostenol (p≥0.05),but there a difference was found between the values on day 0 and 28.For this reason the information from both treatment groups was analyzed together for each variable and time measurement.In this analysis it was found that all variables analyzed by the leukogram showed lesser post treatment values (Table 2).These results show that both treatments decreased the number of white blood cells, neutrophils and lemniscus', compared with the values before treatment (p≤0.05).
There was no significant difference (p≥0.05) between both protocols in regards to days of recuperation for the following clinical variables: days until the next proestrus (138 days), days until a normal echography (15 days), days until absence of vomiting (6.2 days) and days until an absence of polydipsia/polyuria (5.6 days) (Table 3).However, variables such as the days of absence of abdominal pain, days of vaginal secretion, days until normal appetite and days of normal activity, showed statistical differences (p≤0.05),being less for the combined aglepristone and cloprostenol group (Table 3).
All 5 females in each protocol paired during their first heat.Of them, 40% (3 out of 5) of the aglepristone group became pregnant, with a diagnostic echography on day 23 post mating, while only 20% (1 out of 5) of the aglepristone and cloprostenol group were impregnated.All pregnant females carried their pregnancy to full term.The present study did not evaluate the number of puppies or the vitality and malformation of the neonates.None of the bitches showed adverse clinical signs or local effects associated with the treatments.

DISCUSSION
The presence of the disease does not coincide with population data found by other authors, and there is no particular prevalence according to breed, thus, the French Bulldog was chosen in particular because of its high economic value.The table refers to females greater than four years of age, as mentioned by other authors (2,3), which does not coincide with the present work where the age was 4.2 years for the aglepristone group and 3.2 years for the aglepristone and cloprostenol group, indicating that they were young females, one cannot infer that the French Bulldog as a breed is the explanation, since pyometra has been found in young animals of different breeds (3).
The results indicate that treatments with aglepristone or aglepristone with cloprostenol probably have similar results as regards to the values of the full blood count.It is evident that the increase in the number of white blood cells, neutrophils and lemniscus' are characteristic to canine pyometra, as mentioned by many authors (15,21).However, the aglepristone and the aglepristone with cloprostenol treatments, gestantes presentaron una gestación a término.En el presente estudio, no se evaluó el número de cachorros, vitalidad y malformación de los neonatos.Ninguna de las perras mostró signos clínicos adversos o efectos locales asociados con los tratamientos.
Los resultados indican que probablemente los tratamientos con aglepristone o aglepristone con cloprostenol, tienen resultados similares en cuanto a los valores del hemoleucograma.Es evidente que los valores aumentados de los glóbulos blancos, neutrófilos y bandas son característicos del piómetra canino, como los describen muchos autores (15,21).Pero el tratamiento con aglepristone y aglepristone con cloprostenol, combinados con antimicrobianos, permitieron la recuperación de las variables del leucograma alteradas, lo que concuerda con resultados de otros estudios (19,21), donde la leucocitosis y neutrofilia sede al uso de ambos protocolos, debido a la eliminación del contenido piógeno.Además, el uso de enrofloxacina, demostró ser eficaz, en el control de la infección,  combined with antimicrobials, allowed the recuperation of the altered leukogram variables, which agrees with the results of other studies (19,21), where the use of both protocols mitigates leukocytosis and neutrophil by eliminating pyogenic material.Moreover, the use of enroflaxacin proved to be effective in the control of the infection, similar to those already mentioned for gentamicin in another study (19).
All bitches in this study presented uterine contents in the ultrasound evaluation, from 15 days on average which coincide with the results of other authors (13,15,18).Also, both treatments showed a similar behavior in regards to days of recuperation of the clinical variables: days of proestrus, days until absence of vomiting and days until absence of polydipsia/polyuria (15,16).Treatment with a combination of aglepristone and cloprostenol showed an advantage in regards to days of secretion, with an average of 7.2 days compared with 10 days for treatment with only aglepristone.This coincides with other studies where the greater effect of this combination is confirmed (22); none the less, this data differ from the 3 days of secretion reported by those who used at least one protocol of aglepristone with cloprostenol similar to that of this study (17).In vitro studies have demonstrated that the progesterone antagonist induced by aglepristone improves contractile activity of the myometrium induced by prostaglandins (16), which clinically implies assistance in the elimination of pyogenic material in less time, facilitating recuperation.
In this study the lowest average number of days until absence of pain, and days to normal activity and appetite associated with the combined treatment of aglepristone with cloprostenol, is related to the lowest number of days of uterine discharge and improvement of clinical evaluation, which can be related to studies that indicate a higher contractile capacity of the myometrium from the combination of aglepristone with cloprostenol than only aglepristone (15,18).However, the present study did not determine values according to the UMPS scale, it was only used to determine presence or absence of pain and this was observed when values decreased, indicating improvement.
None of the bitches in this study had a recurrence of the disease, at least not in the estrous cycle subsequent to this study, which does not rule out the occurrence of the disease in the rest of the subsequent estrous cycles, because they could not be evaluated in this study.The group of bitches in this study were young, none of which passed 7 years of age, which coincides with previous studies where there was no recurrence similar a los ya descritos para la gentamicina en otro estudio (19).
Ninguna de las perras de este estudio presentaron recidiva de la enfermedad al menos en un ciclo estral posterior a este estudio, lo que no descarta la presencia de esta enfermedad en los demás ciclos posteriores, que para este estudio no pudieron ser evaluado.El grupo de perras de este estudio fue joven pues ningún animal superó los 7 años de edad y coincide con estudios previos en donde no hubo reincidencia de la enfermedad en el siguiente ciclo en animales menores de 5 años y de donde se concluye que se obtienen mejores resultados en preservar la fertilidad de la hembras tratadas en animales jóvenes (23).
of the disease in the following cycle in animals less than 5 years of age and from which it is concluded that better results in preserving female fertility is obtained in young animals (23).
In the present study no side effects associated with the use of prostaglandins was found, such as: vomiting, sialorrhea, nausea, diarrhea, hypotension, tachycardia, shock and death, as is described in handling doses of 2.5 µg/kg (16).Similar circumstances were reported by other canine studies (13).This could be due to the use of low doses of cloprostenol in this study.Further, neither the aglepristone treatment reported side effects, which compares with the results of previous studies (24).
These results should continue to be analyzed, even though there is clinical evidence that the combination of aglepristone with cloprostenol can generate better results, as are describes by Gobello et al (13), Fieni (15) and Jena et al (21), in curing patients.The present study demonstrates that only using aglepristone is sufficient for guaranteeing the recuperation of the patient, similar to what is described by Molina (18).The analysis should be guided toward not only guaranteeing future fertility, but also to avoid side effects and risk to the life of the female, through the use of synthetic prostaglandin (16,25).
In addition, to dispute the concept that pyometra is a reproductive disease in female canines that requires surgical treatment (3,5,26), due to the pathogenicity of its etiologic agent and its death rate (2,3,21), it is known that procedures like the ovariohysterectomy, which eliminates possible recurrence (11), leave breeding females without the possibility of progeny.None the less, surgical treatments have their limits when the surgical risk increases or the owner refuses OVH because they are patients of high breeding value, in which the possibility of sterilization is considered expensive (20).
It is at this point where progesterone antagonists play an important role, especially the use of aglepristone, which due to its medical advantage in facilitating the contractibility of the myometrium, permits the elimination of pyogenic material (12,15,26), relaxing the uterine neck, due to its competitive effect against progesterone (17).
The therapeutic response to the use of aglepristone was excellent with no side effects.In addition, the presence of two treatment protocols allows the veterinarian the opportunity to manage medication depending on the severity of the case.
In conclusion, treatments with aglepristone and a combination of aglepristone with cloprostenol, along with enrofloxacin, permit the recuperation of the animal with pyometra in the variables of the full blood count on day 28; nonetheless, the combination of aglepristone with cloprostenol obtained better results in the recuperation of the treated females.
To the Veterinary Clinic of Antioquia for allowing us to use their facilities and patients for the present study.To the Corporación Universidad de Sabaneta and to Caequinos for providing the technical personnel for the present study.paciente.Mientras que el uso complementario de aglepristone con prostaglandina F2α, alcanzó el 90% de efectividad (7,15).Logrando la recuperación dentro de las 3 primeras semanas después de iniciado el tratamiento; con una tasa de curación exitosa de corto plazo de 92.3%, el uso de aglepristone como única herramienta es ciertamente justificable.

Agradecimientos
A la colaboración de la Clínica Veterinaria de Antioquia, por facilitar las instalaciones y pacientes para la presente investigación.A la Corporación Universitaria de Sabaneta y a Caequinos por proveer el personal técnico para el presente estudio.
of secretion; DP: Days of proestrus DNE: Days to normal echography; DAV: Days until absence of vomiting; DAP: Days until the absence of pain; DNA: Days to normal activity; DNA: Days to normal appetite; DAPP: Days until the absence of polydipsia/polyuria. a, b The numbers with a different superscript for each column implies a significant difference (p≤0.05).
a None of the variables had significant difference (p≥0.05).

Table 2 .
Average values (±DS) of the leukogram for French Bulldog bitches with pyometra and treated with aglepristone alone or combined with cloprostenol.