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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Author contributions
  9. References

This study investigated the duration of the interval between the onset of vulval bleeding at pro-oestrus and ovulation estimated from the plasma progesterone concentration in a large number of beagle bitches. The influence and association of individual variation, ageing and duration of the oestrous cycle were also investigated. The mean time of ovulation after the onset of vulval bleeding was 11.1 ± 0.2 days, but it widely ranged from 3 to 31 days. This timing was not influenced by age or duration of the oestrous cycle, and within-individual variation was small. As there has been no previous report in which the ovulation day was investigated by the age, these data may be very valuable.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Author contributions
  9. References

The studies to clarify canine reproductive physiology are valuable for researchers and dog breeders because many reproductive physiological characteristics of bitches are different from those of other animal species.

Bitches are mono-oestrous animals, and the duration of a single oestrous cycle is long. The canine oestrous cycle is divided into four stages: pro-oestrus, oestrus, di-oestrus and anoestrus. The duration of canine pro-oestrus varies among individuals, ranging from 3 to 27 days (mean: 8.1 ± 2.9 days) (Tsutsui and Shimizu 1973). This period corresponds to follicular development, and male dogs are not accepted. Oestrus starts when females accept mating, and ovulation occurs on approximately day 3 of oestrus (Tsutsui 1973). Follicular development reaches the maximum when the cycle enters oestrus, and an LH surge is induced by positive feedback to the pituitary, inducing ovulation. The duration of oestrus also varies among animals, ranging from 5 to 20 days (mean: 10.4 ± 2.7 days), that is, acceptance of males continues after ovulation, and this is a characteristic of canine reproductive physiology not noted in other animal species. It is known that the time of ovulation after the onset of vulval bleeding at pro-oestrus markedly varies in bitches because of variation in the duration of pro-oestrus. However, generally, many dog breeders calculate the mating date from the onset of vulval bleeding at pro-oestrus and mate dogs 10–14 days after the onset. Although canine ova are ovulated in an immature state, they remain fertile for many days after ovulation and because sperm retain fertilizing capacity for approximately 5 days, the duration of the period when copulation may achieve conception is long: approximately 7 days, starting from 48 h before ovulation until 108 h after ovulation (Tsutsui 1989). Thus, the above mating schedule does frequently result in fertility. But, mating too early or late may reduce the fertilization rate (the rate of the number of implanted ova to the number of ovulated ova), and, of course, it is desirable to mate dogs in the optimum mating period, for which a method to accurately identify the ovulation day by measuring the plasma LH or progesterone (P4) concentrations has been established. However, simultaneously, it is interesting to know the characteristics of individual variation of the time of ovulation (days), that is, individual variation of the follicular phase. The variation of length from vulval bleeding to LH peak within bitches has been previously characterized by one report (England et al. 1989). They showed that the mean calculated day of LH peak was 10.8 ± 2.8 days after the onset of pro-oestrus, but there was great variation between bitches and between cycles in several of the bitches. But the individual variation of the time of ovulation after the onset of bleeding in dogs has not been well characterized, and there has been no previous report in which the ovulation day was investigated by the age.

In this study, we investigated the duration of the period between the onset of vulval bleeding at pro-oestrus and ovulation (ovulation day) in a large number of beagle bitches. The ovulation day was estimated from plasma P4 concentrations and the influence and association of individual animal variation, age and duration of the oestrous cycle were investigated. This information would be important for the purposes of making decisions on planned breeding at clinical practice and may be very valuable basic knowledge for research of canine reproductive physiology.

Materials and Methods

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Author contributions
  9. References

Animals

The test dogs were 102 female beagles aged 0.75–11 years maintained over a period of 4 years at our laboratory. The animals were housed in 160 × 75 × 65 (high)-cm stainless cages at two dogs per cage and given commercial dog food (Hill's Maintenance Dry, Topeka, KS, USA) in the morning and drinking water three times a day in the morning, afternoon and evening. The study conformed with the animal study guidelines of Nippon Veterinary and Life Science University.

Determination of onset day of pro-oestrus and ovulation day

The vulva was observed once a day, and the first day on which vulval bleeding was confirmed was regarded as the onset day of pro-oestrus. Bitches were then monitored daily for signs of oestrus (perineal enlargement, tail deviation and standing for a male). When vulval bleeding was unclear, a cotton swab was inserted into the vulva and examined for the presence of bloody material.

Blood was collected every other day from the 4th day after the onset of vulval bleeding, and the ovulation day was estimated from the plasma P4 concentrations measured using EIA (Hase et al. 2000) or a fluorescent enzyme immunoassay (FEIA, Spotchem Vidas SV-5010, Arkray). The day on which the concentration exceeded 2 ng/ml on EIA was regarded as the ovulation day. The concentration measured using FEIA was strongly correlated with that using EIA (r2 = 0.97247), and the day on which the concentration exceeded 4 ng/ml was estimated to be the ovulation day (Tsutsui, unpublished data). The intra-assay and inter-assay coefficients of variation were 7.8% and 11.4% in EIA methods and 0.9% and 2.2% in FELA methods, respectively. The sensitivity of this immunoassay method was 0.25 pg/well in EIA methods. Blood (3 ml) was collected from the cephalic vein. Serum was separated by centrifugation at 4°C and stored at −30°C until measurement.

Statistical analysis

The interval between ovulation after the onset of vulval bleeding at pro-oestrus (ovulation day) and its relationship with age (initial oestrus and 1–3, 4–6, and 7–11-year-old groups) were analysed employing Student's t-test, and a p-value < 5% was regarded as significant. Age-related changes in the ovulation day in individual animals, and the correlation between the ovulation day and duration of the previous oestrous cycle (from the onset of bleeding to the following bleeding at next pro-oestrus) were investigated.

Results

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Author contributions
  9. References

Time of ovulation after onset of vulval bleeding at pro-oestrus (ovulation day)

The ovulation day was investigated in a total of 390 oestrous cycles in 102 bitches. The rates of animals included in the groups of various ovulation days are shown in Fig. 1. The earliest and latest ovulation days were 3 and 31 days, respectively, showing a wide distribution, and the mean was 11.1 ± 0.2 (SE) days. The ovulation day was 8–14 days in majority of cycles (82.8%). Split oestrus was noted in three oestrus cycles (0.8%).

image

Figure 1. The distribution of time of ovulation after onset of vulval bleeding at pro-oestrus (ovulation day) (n = 390)

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Ovulation day by age

The ovulation day is shown by age in Fig. 2. The ovulation day was 5–31 days (mean: 10.6 ± 0.8 days) in the initial oestrus group (29 bitches), 3–28 days (mean: 11.0 ± 0.2 days) in the 1–3-year-old group (136 cycles in 60 bitches), 5–22 days (mean: 11.0 ± 0.2 days) in the 4–6-year-old group (164 cycles in 51 bitches), and 4–25 days (mean: 11.0 ± 0.6 days) in the 7–11-year-old group (61 cycles in 27 bitches). There was no significant difference among the age groups.

image

Figure 2. The distribution of ovulation day by age (n = 390)

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Influences of changes in individual animals and ageing on ovulation day

To investigate changes because of variations in individual animals and ageing, six or more oestrous cycles were observed in 26 bitches. The ovulation days in these animals are shown in Table 1. The ovulation day was 8–14 days in all 6–9 oestrous cycles observed in 13 animals (50%). In seven animals (26.9%), the ovulation day was earlier than the mean in 1–2 cycles in four animals, and later than the mean in two cycles in 2 (B15 and B18) and five cycles in one (B20). In the remaining six animals (23.1%), ovulation days earlier and later than the mean were noted within the individual animals. In animals with a mean ovulation day earlier than the mean in all animals, the ovulation day was early in most cycles, whereas the ovulation day was late in most cycles in animals with a late mean ovulation day, clarifying that the within-individual variation was small. No constant tendency with ageing, such as becoming earlier or later, was noted in any animal.

Table 1. The days from vulval bleeding to ovulation in 26 bitches that were observed six or more oestrous cycles
image

Relationship between the ovulation day and duration of the oestrous cycle

In 248 cycles in which the duration of the period before ovulation was clear excluding the initial oestrus, the relationship between the ovulation day and duration of the oestrous cycle was investigated, but no correlation was noted (Fig. 3).

image

Figure 3. Relationship between the ovulation day and duration of the oestrous cycle (n = 248)

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Discussion

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Author contributions
  9. References

The ovulation day in beagles in our colony was 8–14 days in majority of cycles (323 of 390 cycles, 82.8%), and the mean was 11.1 ± 0.2 days, but it ranged widely from 3 to 31 days. This was consistent with that similarly estimated from the plasma P4 level in 10 female Labrador Retrievers in a report (Wright 1991), where the range was also considerable at 8–18 days after onset of vulval bleeding, suggesting that the variation in pro-oestrus length is not specific to beagles but common to all dog breeds (England et al. 1989). However, in the investigation of within-individual variation of the ovulation day, for example, six oestrous cycles were observed in B8 at 2–5 years old, and the ovulation day was 8 days in all cycles, whereas it was late (14–17 days) in many cycles in B20 in which seven oestrous cycles were observed. The variation in the ovulation day was within ± 3 days on average in most cases, and variation was noted only in 23.0%, suggesting that within-individual variation of the ovulation day is small in beagles. The within-individual variation in this study was smaller than that previously reported (England et al. 1989). It may be caused by differences in how the ovulation day was determined or because many breeds were used in previous study. It was also clarified that the ovulation day was not related to the duration of the oestrous cycle. Although the data are not shown, the duration of the oestrous cycle is constant to some extent within individual animals (Tsutsui, unpublished data). Although the reason for an almost constant ovulation day within individual animals was unclear, clinically what is important is that at least in beagles it may be determined within individuals.

Changes with ageing were surveyed in the data because it was plausible that age-related hormone differences might influence the ovulation day at an advanced age. The ovulation day appeared relatively widely distributed in the 7–11-year-old group, compared to the distribution in the initial oestrus, 1–3-year-old, and 4–6-year-old groups, in which the data were more normally distributed centring on the mean. There was, however, no tendency for changes in the ovulation day with ageing, and no change in variability with ageing was noted in any animal in which six or more oestrous cycles were observed. However, the data were derived from bitches aged up to 8 years old, and we could not investigate ovulation in very old bitches. It is assumed that cessation of cyclicity does not occur in bitches because normal corpora lutea are present in the ovary in bitches aged 10 years or older with pyometra. To clarify the reproductive physiological background of dogs, it will be interesting to identify the ovulation day after the onset of bleeding in elderly bitches aged 10 years or older, for which further studies are necessary.

These findings in dogs contrast with those in women where follicular phase length decreases with age (Lenton et al. 1984) and is highly variable within individuals in over a third of women. Interestingly, in women, short follicular phases are associated with higher FSH concentrations at the start of the follicular phase and involve a shortening of the period prior to the rapid increase in oestrogen (Miro et al. 2004). Whether or not in bitches length of pro-oestrus is similarly an inverse function of FSH concentrations in late anoestrus or the start of pro-oestrus is not known. Likewise, whether a short pro-oestrus reflects delayed response to increased oestrogen or a short follicular phase associated with initially high concentrations of FSH and a foreshortened period of increasing oestradiol had not been studied. These potential associations merit further study.

Split oestrus was noted in three of the 390 cycles observed. Split oestrus is defined as oestrus in which pro-oestrus or short oestrus is observed, but no ovulation occurs, followed by normal oestrus after 1–2 months (Meyers-Wallen 2007). In the three cases, subsequent normal oestrus was observed after 18, 60 and 67 days, respectively, in which normal ovulation occurred, and the ovulation day was 10–13 days, showing no abnormality. As split oestrus occurred in bitches aged 6–8 years old, further study is necessary to clarify the relationship between split oestrus and ageing-associated abnormality of hormone secretion (particularly FSH).

In conclusion, the mean time of ovulation after the onset of vulval bleeding at pro-oestrus in beagles was 11.1 ± 0.2 days, but it widely ranged from 3 to 31 days. The results demonstrate that follicular phase lengths in beagle bitches are not only less variable within bitches than among bitches, and apparently do not change with age or the duration of oestrous cycle, but that they also have a low variability within the majority of bitches. As there has been no previous report in which the ovulation day was investigated by the age, these data may be very valuable. The results may be comparable across most or all breeds although only one breed was considered in this study. If the ovulation day after onset of vulval bleeding of bitches intended for breeding can be grouped into an early or late ovulation group, it may help planning matings and allow reduction in the frequency and number of matings for fertility.

Conflicts of interest

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Author contributions
  9. References

None of the authors have any conflicts of interest to declare

Author contributions

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Author contributions
  9. References

T Hori, T Tsutsui contributed to designing the study. T Hori, Y Amano carried out the experiment and collected samples (data).

T Hori, T Tsutsui, PW Concannon contributed to analysing data and drafting the paper. All authors contributed to finalizing the manuscript.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Author contributions
  9. References
  • England GCW, Edward Allen W, Blythe SA, 1989: Variability of the time of calculated LH release in 218 canine pregnancies. Vet Rec 125, 624625.
  • Hase M, Hori T, Kawakami E, Tsutsui T, 2000: Plasma LH and progesterone levels before and after ovulation and observation of ovarian follicles by ultrasonographic diagnosis system in dogs. J Vet Med Sci 63, 243248.
  • Lenton EA, Landgren BM, Sexton L, Harper R, 1984: Normal variation in the length of the follicular phase of the menstrual cycle: effect of chronological age. Br J Obstet Gynaecol 91, 681684.
  • Meyers-Wallen VN, 2007: Unusual and abnormal canine estrous cycles. Theriogenology 68, 12051210.
  • Miro F, Parker SW, Aspinall LJ, Coley J, Perry PW, Ellis JE, 2004: Relationship between follicle-stimulating hormone levels at the beginning of the human menstrual cycle, length of the follicular phase and excreted estrogens: the FREEDOM study. J Clin Endocrinol Metab 89, 32703275.
  • Tsutsui T, 1973: Studies on the physiology of reproduction in the dog. II. Observation on the time of ovulation. Jpn J Anim Reprod 18, 137142.
  • Tsutsui T, 1989: Gamete physiology and timing of ovulation and fertilization in dogs. J Reprod Fertil Suppl 39, 269275.
  • Tsutsui T, Shimizu T, 1973: Studies on the physiology of reproduction in the dog. I. Duration of estrus. Jpn J Anim Reprod 18, 132136.
  • Wright PJ, 1991: Practical aspects of the estimatioon of the time of ovulation and of insmination in the bitch. Aust Vet J 68, 1013.