Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 56 issue 12 (december 2022) : 1462-1467

Induction of Oestrus and Fertility Response using Bromocriptine, Cabergoline and eCG Plus hCG Treatment Protocols in Female Dogs

Abhisek Bisen1,*, S.N. Shukla1, R.P.S. Baghel2, Aditya Mishra3
1Department of Veterinary Gynecology and Obstetrics, College of Veterinary Science and Animal Husbandary, Jabalpur-482 001, Madhya Pradesh, India.
2Nanaji Deshmukh Veterinary Science University, Jabalpur-482 004, Madhya Pradesh, India.
3Department of Veterinary Physiology and Biochemistry, College of Veterinary Science and Animal Husbandary, Jabalpur-482 001, Madhya Pradesh, India.
Cite article:- Bisen Abhisek, Shukla S.N., Baghel R.P.S., Mishra Aditya (2022). Induction of Oestrus and Fertility Response using Bromocriptine, Cabergoline and eCG Plus hCG Treatment Protocols in Female Dogs . Indian Journal of Animal Research. 56(12): 1462-1467. doi: 10.18805/IJAR.B-4208.

ABSTRACT

Background: Reproductive pattern of dog is quite different from other mammalian species. A major difference between canine and other mammalian species is that in dog oestrus period is followed by long inter-oestrus interval (period of sexual quiescence), described as monocyclic animal. There are several reproductive problems encountered by the dog breeders and pet owners causing prolonged whelping interval. Anoestrus is thought to be one of the undiagnosed ovarian disorders responsible for prolonged oestrus interval in canine. Anoestrus of variable duration (2 to 10 months) following each oestrus cycle in female dogs is frequently observed. The approaches for the management of reproductive problems due to ovarian disorders encountered by the dog breeders and pet owners needs to be explored to optimize whelping interval. Hence, the current study aimed to study the Induction of oestrus and fertility response using bromocriptine, cabergoline and eCG plus hCG treatment protocols in female dogs.

Methods: The study was conducted in 28 apparently healthy female dogs at TVCC college of veterinary science and Animal Husbandry Jabalpur and at door step of pet owners or dog breeders during the period of 2018-19. Dogs used in the study were divided into small, medium and large breeds. Pomeranian and Pug breeds included under small breeds; German shepherd, Labrador, Siberian husky, Dalmatian, ND and Doberman under medium breeds whereas Great dane, Rottweiler, Bullmastiff and Saint Bernard included under large breeds. The animals were selected irrespective of breeds with the history of anoestrus up to two month or more after whelping. The selection of anoestrus dog was made based on the ratio of cells in vaginal cytology and serum progesterone estimation.

Result: Our results  shows that the oestrus and fertility response following hormonal treatments in anestrous female dogs was significantly (p<0.01) higher than the oestrus induction in all treatment groups (71.48% in Group-I, 85.70% in Group-II and 71.48% in Group-III) as compared to control (14.28%).The breeding was recorded in 83.33% of dogs in cabergoline treatment followed by 100% in eCG plus hCG and 80% in bromocriptine with the conception rates of 100%, 80% and 75%, respectively in three groups. Serum progesterone (P4) profile were statistically significant (P<0.05) in all the treatment groups (I, II and III), but not in control group and the exflotive veginal cytology were found significantly (P<0.05) higher in induced than the non-induced animals of same group.

INTRODUCTION

Reproductive pattern of dog is quite different from other mammalian species. A major difference between canine and other mammalian species is that in dog oestrus period is followed by long inter-oestrus interval (dioestrus and anoestrus or period of sexual quiescence), for which it is described as monocyclic animal. There is a great individual variation among bitches at different geographical condition in different breeds. The domestic female dog (Canis familiaris) is considered as monoestrous, spontaneous ovulator and non-seasonal breeder (Concanon, 1991). However, the seasonal effect on canine reproduction is still contradictory. Few studies favor (Mutembei et al., 2000; Gavrilovic et al., 2008), while others deny seasonal effect on the dog reproduction (Bouchard et al., 1991; Chatdaroing et al., 2007; Ortega-Pacheco et al., 2007).
       
There are several reproductive problems encountered by the dog breeders and pet owners like delayed puberty, missed oestrus, anoestrus, pseudopregnancy and or repeat breeding causing prolonged whelping interval. Anoestrus is thought to be one of the undiagnosed ovarian disorders responsible for prolonged inter-oestrus interval in canine. Bouchard et al., (1991) and Concannon et al., (1993) reported anoestrus of variable duration (2 to 10 months) following each oestrus cycle in female dogs.
       
Feldman and Nelson (2004) reported that anoestrus in dogs could be either primary (bitches that never had ovarian cycle) or secondary (bitches that had one or more ovarian cycle but subsequently failed to cycle). It was suggested that secondary anoestrus could occur after the onset of endocrine and non-endocrine disorders. Dogs with prolonged average inter-oestrus interval and delayed puberty create problem in efficient breeding program. 
       
Controlled breeding in canine reproduction is difficult especially in dogs of countries like India due to availability of different breeds of dog (pure breed, crossbred and mongrel) in different parts of country. Perhaps breeding/reproductive pattern is not very clear among them hence creating problem among the breeders. During each oestrous cycle, the bitch has a prolonged follicular and luteal phase as compared to farm animals. The life span of corpora luteal in non-pregnant female dog is similar to that in pregnant female dog because the uterus of bitch does not seem to exert a perceptible role in the regression of corpus lutea of the cycle.
       
Such ovarian activity is unique only in female dog among domestic animals and creates problem in efficient breeding program. Cost of maintaining anoestrus bitches for prolonged period makes commercial dog breeding most expensive. The approaches for the management of reproductive problems due to ovarian disorders encountered by the dog breeders and pet owners needs to be explored to optimize whelping interval. Hence, controlled breeding protocol may be standardized in female dogs for management of such disorders and better clinical applicability.

MATERIALS AND METHODS

The study was conducted in 28 apparently healthy female dogs at TVCC college of veterinary science and Animal Husbandry Jabalpur and at door step of pet owners or dog breeders during the period of 2018-19. Dogs used in the study were divided into small, medium and large breeds. Pomeranian and Pug breeds included under small breeds; German shepherd, Labrador, Siberian husky, Dalmatian, ND and Doberman under medium breeds whereas Great dane, Rottweiler, Bullmastiff and Saint Bernard included under large breeds. The animals were selected irrespective of breeds with the history of anoestrus up to two month or more after whelping. The selection of anoestrus dog was made based on the ratio of cells in vaginal cytology and serum progesterone estimation. The more numbers of parabasal cells and less superficial cells in cytology and less than 0.5 ng/ml serum progesterone indicated anoestrus in dogs. The selected dogs were randomly divided into four equal groups, each comprising seven animals. Group I was kept as untreated control, while the rest three groups were treated as per the protocols summarized in Table 1.
 

Table 1: Group wise treatment protocols in anoestrus dogs.


       
The fertility response post treatment in all the groups was studied in terms of oestrus induction rate, time taken for induction, conception rate and length of pro-estrus. The litter size and whelping percent were also observed.
       
Serum progesterone profile was studied on day 0, 5, 10, 15 and at pro-oestrus for validation of the results. The quantitative determination of progesterone concentration in serum was done using Enzyme-Linked Immuno Sorbent Assay (ELISA) kit, manufactured by Biochem diagnostics, Canada. Exfoliative vaginal cytology was studied on day 0, 3, 5, 7, 9, 11, 15 and on day of pro-oestrus after start of treatment using Papanicolaou’s Stain Kit (RAPID-PAP, Biolab Diagnostics, Pvt. Ltd). 
       
Data was analyzed using chi square test to compare data pertaining to oestrus induction and fertility response where as other comparisons were drawn by analysis of variance using standard statistical software Systat version 11.

RESULTS AND DISCUSSION

Fertility response
 
The results of oestrus and fertility response following hormonal treatments in anestrous female dogs revealed significantly (p<0.01) higher oestrus induction in all treatment groups (71.48% in Group-I, 85.70% in Group-II and 71.48% in Group-III) as compared to control (14.28%) (Table 2). The breeding was recorded in 83.33% of dogs in cabergoline treatment followed by 100% in eCG plus hCG and 80% in bromocriptine with the conception rates of 100%, 80% and 75%, respectively in three groups. In control group, the spontaneous oestrus induction was very low (14.28%) where only one animal was bred and conceived. The mean interval from start of treatment to pro-oestrus was recorded non significantly short in group I (10±0.70 days) as compared to group II (12±0.81 days) and group III (12±1.08 days). The length of pro-oestrus was also found to be 8 to 10 days in various treatment groups. Similarly, the mean litter size was 6, 4, 5.2 and 5 in control, group I, group II and group III respectively. However, whelping was found normal in all groups.
 

Table 2: Fertility response to various treatments in anoestrus female dogs.


       
The better induction of oestrus in cabergoline treated group in this study was almost similar to the finding of Ajitkumar et al., (2010). They found induction rate of 83.34 and 75.00% within 13.5 and 21 days by using respectively two doses of cabergoline @ 5 µg/kg and 1 µg/kg b.wt. However, they reported 20% more side effects in higher dose. Verstegen et al., (1999) and Gunay et al., (2004) has also reported almost similar induction of pro-oestrus and oestrus (80-100%) using higher dose of cabergoline i.e. 5 µg/kg b.wt. with 0-100% pregnancy rate. The induction of oestrus and conception rate in dogs with bromocriptine in present study was almost similar to findings of Jones et al., (1988). They reported 100% oestrus induction within 13 to 15 days with 83% pregnancy in anoestrus dogs using same dose. However, in few studies the induction of pro-oestrus achieved was up to 80-100% in anoestrus dogs, without any conception (Van- Haaften et al., 1989; Concannon, 1993).
       
The anti-prolactin drugs like cabergoline and bromocriptine cause suppression of prolactin secretion and thus shortens the duration of anoestrus or induce oestrus in case of prolonged anoestrus (Concanon, 1985). It has been observed that prolactin inhibition is necessary for oestrus induction (Concanon, 1993) and suggested that inhibition of prolactin secretion may regulate the induction of pro-oestrus. Kim-Jeong et al., (2000) reported that follicle development and resulting oestrus induction with bromocriptine was associated with an increased plasma FSH concentration without constant increase in plasma LH concentration and it has been found that in normal cyclic bitches prolactin concentration do not change prior to onset of pro-oestrus.
       
There are several reports on the use of eCG with hCG in different doses and schedules for induction of oestrus and fertility response in anoestrus bitches with varying degree of success. The higher dose in comparison to present study, i.e., eCG @ 44 IU I/M, SID for 9 days and hCG @ 25-50 IU/kg I/M single dose had also resulted almost similar induction of oestrus (80-100%) and conception (60%) (Archbald et al., 1980). However, few studies reported 100% induction of pro-oestrus with 35-50% pregnancy using lower dose of eCG i.e. 20 IU/kg for 5-10 days and hCG @ 500 IU/dog (Arnold et al., 1989; Weilenmannn et al., 1993). The literature is lacking regarding the effect of eCG and hCG in the dose and schedule as used in this study. In the present study, the dose of eCG was selected based on type of breed i.e. small, medium and large which may be easy to calculate in clinics. Here the number of injections required is less and alternate day dosing resulted in minimal side effects with the maintenance of potent action on induction of oestrus.
       
In anoestrus dogs, serum FSH is found increased while LH concentration is low (Concannon, 1993). However, FSH and LH are follicotropic as administration of pharmacological dose of FSH and LH alone induces oestrus (Shille et al., 1984; Concannon, 1993). Thus, oestrus induction protocol with combined FSH and LH is designed to resemble the gradual increase of endogenous FSH coincidentally with the increased LH during pro-oestrus (Shille et al., 1984). However, this protocol was not successful. The non- pituitary gonadotropins like eCG, hCG and hMG have also been used for oestrus induction in bitches (Arnold et al., 1989). Most widely studied protocol for oestrus induction in dog is eCG with daily to weekly injections using either subcutaneous or intramuscular routes. However, premature luteal failure with subsequent shortening of dioestrus and pregnancy loss has been reported following use of eCG (Jones et al., 1988; Weilenmann et al., 1993).  
 
Serum progesterone profile
 
Serum progesterone (P4) profile revealed that progesterone concentration was at the basal level ranging from 0.31±0.01 to 0.42±0.34 ng/ml on day 0 and then started to increase gradually on day 5th, 10th and 15th in all the groups (Table 3). The differences were statistically significant (P<0.05) in all the treatment groups (I, II and III), but not in control group. Segregation of the data of progesterone profile for oestrus induced and non-induced animals on different days revealed that mean serum progesterone level was significantly (P<0.05) increased in induced animal as compared to non-induced animals in all the groups (Table 4).
 

Table 3: Serum progesterone profile (ng/ml) before and after treatment in anoestrus bitches.


 

Table 4: Serum progesterone profile (ng/ml) before and after treatment in induced and non-induced bitches.


       
The initial progesterone concentration in all the experimental animals ranged from 0.3 to 0.4 ng/ml indicating anoestrus. Galabova et al., (2003) also reported 0.41±0.024 ng/ml serum progesterone (P4) level during early anoestrus. Similarly, Kim Joeng et al., (2000) found plasma P4 concentration less than 1.0 ng/ml during anoestrus in dogs. In this study, after various treatments the serum P4 concentration increased gradually from day 0 to 15 or at early pro-oestrus. This may be due to effectiveness of treatments in all the groups. Olson and Husted (1982) have also reported mean serum P4  levels of 0.34, 0.49, 0.66, 0.68 and 1.37 ng/ml, respectively on day 4, 3, 2, 1 and 0 before LH peak.
 
Exfoliative vaginal cytology
 
The changes in the cellular pattern of vaginal cytology in anoestrus bitches using different protocols of oestrus induction on the day of start of treatment (day 0) and during treatment (day 3, 5, 7, 9, 11 and at onset of pro-oestrus) revealed that the superficial cells before start of treatment (day 0) were ranging from 20.00±2.18 to 21.43±4.04, intermediate cells 24.29±2.02 to 38.57±4.04 and parabasal cells 44.29±5.28 to 55.71±6.12 per cent. Thereafter, on day 3 almost similar patterns were observed but from day 5 the cellular pattern started to change where superficial cells were found to increase and parabasal cells decreased. However, intermediate cells remained stable up to day 11, after that the cellular pattern changed at pro-oestrus in treatment groups where the superficial cells were the maximum (55.71±5.28 to 64.29±5.71 per cent) and the parabasal cells were the minimum (11.43±4.04 to 18.57±4.59 per cent) with almost constant intermediate cells i.e. 24.29±2.02 to 28.57±1.43 per cent. On segregation of values of cellular distribution between induced and non-induced bitches in various groups, the superficial cells were found significantly (P<0.05) higher in induced than the non-induced animals of same group. In contrast, the parabasal cells were also found significantly (P<0.05) low in number in induced animals as compared to non-induced animals in different groups (Table 5).
 

Table 5: Per cent distribution of cells on vaginal cytology in induced and non induced bitches.


       
The findings of vaginal cytology following induction protocols in canine were found similar to the results of Arun (2001) and Reddy et al., (2011). The changes in exfoliative vaginal cytology during pro-oestrus confirmed the fertility response of treatments given in the experiment. The increase percentage of superficial cells and the lowest parabasal cells are indicative of pro-oestrus and oestrus. It may be due to increase blood estradiol concentration which causes thickening of the vaginal mucosa and proliferation of cell layers. As mucosa thickened, the surface cells change in size, shape and staining characteristics thus became larger, irregularly shaped and ultimately became anuclear (England, 1998). The neutrophils could not enter into the vaginal lumen but abundant RBCs could enter into lumen by diapedesis due to estrogenic effect Feldman and Nelson 1996. During dioestrus, the progesterone levels remained at high concentration resulting in sloughing of vaginal epithelium. The numbers of cell layers decreased, deeper cells were uncovered as a result percentage of anuclear cells decreased (England, 1998) with higher numbers of neutrophils.

​CONCLUSION

It was concluded that the better fertility response in terms of oestrus induction and conception was recorded with cabergoline treatment followed by eCG and hCG treatment and the least in bromocriptine treatment group. The suggested treatments may be used for induction of oestrus in anoestrus female dogs after clinical trials in the more number of animals.

ACKNOWLEDGEMENT

The authors are extremely grateful to the authorities of College of Veterinary Sciences and Animal Husbandry, NDVSU, Jabalpur for their support and providing facilities to conduct the research work.

DISCLOSURE STATEMENT

No potential conflict of interest was reported by the authors.

REFERENCES


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