Introduction

Veterinary practitioners and specialist veterinarians providing reproductive services for small animals are aware of the aspects of reproduction that are unique or nearly unique to the dog. Bitch in estrus generally allows multiple mating due to the prolonged estrus period and this ensures that viable spermatozoa are invariably present in the oviduct, around the time which is most appropriate to result in conception. The breeder invariably confines the pets and segregates the sexes during the fertile period to result in limited accessibility to each other for breeding purpose. Many studies have established the fertile period of the bitch is short, commencing from a time shortly before the LH surge and ending with the degeneration of ova in the oviduct at 5 to 7 days later (Goodman, 1998) and many of the planned mating outside this period invariably results in poor conception rate. Identification of the correct time of mating which can result in pregnancy therefore becomes important. Vaginal exfoliative cytology has been described as a useful diagnostic tool in staging estrus and in the determination of the optimum time to present a bitch to stud dogs for mating (Olson et al., 1984; Wright and Perry, 1989). Several studies recommend that breeding should commence as soon as the vaginal cornification reaches 80 per cent or more and continued thereafter till there is abrupt decline in the percentage of cornification. However, most owners of the male dogs are willing to provide only limited number of services of their studs and many of the planned breeding by the breeders may occur outside the fertile period of the bitch resulting in poor conception rate and therefore improper timing of mating is generally considered as the most important cause of breeding failure in the bitch (Concannon, 1986; Concannon et al., 1989). Therefore breeding throughout the period of cornification may not be practically feasible.

The conception rate with the limited number of mating carried out at different intervals following optimum vaginal cornification have not been investigated and such a study may provide the information on the best time to mate a bitch after the onset of vaginal cornification.

Materials and Methods

The study was carried out in 60 female dogs presented during proestrus when they are presented to the outpatient unit of Department of Veterinary Gynaecology and Obstetrics, Veterinary College, Bangalore. On presentation the behavioural signs of the bitches were obtained from each animal to assess the reproductive status of bitch. Each animal was subjected to vaginal exfoliative cytological examination. The samples for the study were collected from the anterior vagina using sterile plastic bovine artificial insemination sheath. The first examination was made on day 5 of the proestrus bleeding and in each animal the procedure was repeated alternate day until cent per cent cornification index was recorded. The day on which cent vaginal cornification was recorded, considered as the first day of estrus. The exfoliated vaginal epithelial cells were classified as parabasal, intermediate and superficial and cornified cells.

Following cent per cent cornification, the nature of the vulva (relaxed or oedematous), vaginal discharge (haemorrhagic or serosanguinous), and presence or absence of standing signs of oestrus (flagging of the tail, upward deflection of the tail, rigidity of the tail on palpation of the perineum) were recorded on the day on which the peak vaginal cornification was first observed to assess the relationship between the behavioural signs of estrus and vaginal cornification.

After the peak vaginal cornification, the animals were randomly allotted to the following three groups of 20 animals each. out of which Group I consisted of 20 female dogs in which bitches were advised to be mated on the same day on which the peak vaginal cornification and then two days later and The Group II animals were advised to be mated on the same day on which the animals were presented (day three after the onset of peak vaginal cornification) and second mating another two days later (day five after the peak vaginal cornification) and The Group III animals were advised to get their animals mated on the same day on which the animals were presented (day five after the onset of peak vaginal cornification) and second mating was advised another two days later (day seven after the onset of peak vaginal cornification). All the bitches which were recommended for mating at different intervals following optimum vaginal cornification were subjected to pregnancy diagnosis at 30 to 35 days later by abdominal palpation or ultrasonography examination.

The data generated for all groups were tabulated and results were drawn using mean and standard error. The comparison between conception rates observed between the three groups was analyzed by one way ANOVA analysis as per the procedure detailed by Snedecor and Cochran (1989).

Results and Discussion

Table 1 presents the data related to genital and behavioral changes recorded in Group I animals which were recommended to be mated on the day of peak vaginal cornification was first observed.

Table 1: Genital and behavioral changes recorded on the first day of mating in the group of recommended to be mated at different intervals after the onset of peak vaginal animals cornification (n=20)

It was observed that in this group of animals which were recommended to be mated on the first day of peak vaginal cornification, the vulva was relaxed in only 10 per cent of the animals, the vaginal discharge was serosanguinous in only 5 per cent of the animals and only 10 per cent animals exhibited the standing estrus. The Group II was results revealed that on the day of recommended mating (day three after the onset of peak vaginal cornification) about 75 per cent of the animals exhibited a relaxed nature of the vulva. A similar per cent of animals also exhibited behavioral signs of standing estrus. However, 75 per cent of the animals continued to have a serosanguinous vaginal discharge and in Group III animals results that on the day of recommended mating (day 5 after the onset of peak vaginal cornification). About 60 per cent had a relaxed vulva and another 50 per cent of the bitches exhibited a serosanguinous discharge. Further, 50 per cent bitches exhibited standing signs of estrus.

It was observed that as in the previous two groups, very few number of animals exhibited relaxation of vulva, serosanguinous vaginal discharge and presence of standing signs of estrus on the first day of peak vaginal cornification. Further, on the first day mating on which was day five after the peak vaginal cornification, only 60 percent animals had a relaxed vulva, another 50 per cent of the animals exhibited standing signs of estrus and the discharge was serosanguinous in only 50 per cent of the animals with the rest having a very dark blackish red vaginal discharge. However, a majority of the owners reported that while first mating was successful, either the female was not receptive for the second mating or that the male did not show interest for the mating which was advised for the second time on day seven after the onset of peak vaginal cornification. This observation suggested that most of the animals were nearly at end of estrus phase between day 5 and 7 after the onset of peak vaginal cornification. Following mating, these animals were subjected for pregnancy diagnosis between 30 to 35 days after the last mating by abdominal palpation and were further confirmed by Trans abdominal ultrasonography. Conception rates recorded in bitches mated at different intervals following the peak vaginal cornification. The conception rate was 75 percent in group of which were mated immediately after onset of peak vaginal cornification. A conception rate of 85 percent was also obtained Group III in animals which were recommended mated on day three and five after onset of peak vaginal cornification. However, conception rate was lowest 35 percent when the animals were mated for the first time on day five after the onset of peak vaginal cornification in group III. Fifteen (75%) out of 20 animals were diagnosed as pregnant and the conception rate in animals mated once on the day of peak vaginal cornification and second time two days later was much higher than 30 per cent as reported by Okken et al. (1985). Suyash Vardhan Bhal (2002) also reported that a conception rate of only 50 per cent was obtained in his study when animals were mated at a time when the vaginal cornification was around 80 per cent. The conception rate recorded in the present study appears to be closer to the conception rate recorded in free roaming dogs which are mated throughout the period of estrus (Friedman, 1957).

Table 2: Conception rate of bitches bred at various intervals days after onset of peak vaginal cornification

Mean values with different superscripts within a column are significantly different (p< 0.05)

Following mating, 17 (85%) animals were diagnosed to be pregnant. This conception rate was 10% higher (85%) as compared to the conception rate observed when the animals were mated on day one and three after the onset of peak vaginal cornification. and 7 (35%) animals were diagnosed to be pregnant. and this conception rate was significantly lower than the conception rate recorded with the matings carried out on day 1 and 3 or day 3 and 5 after the onset of peak vaginal cornification.

Parabasal cells, 40×, Giemsa stain	Intermediate cells, 40×, Giemsa stain
Superficial cells, 40×, Giemsa stain

Conclusion

The ideal time of mating is recommended on the basis of vaginal exfoliative cytology that would be on day two and four when two matings are planned and mating is suggested on day three when a single mating is planned after the onset of peak vaginal cornification.

References

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