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Seasonal variation in the estrous cycle of mares in the subtropics
Theriogenology
39:631-653,
1993
SEASONAL VARIATION IN THE ESTROUS OF MARES IN THE SUBTROPICS K.F. Dowsett, Department
L.M. Knott, R.A. Woodward
CYCLE
and D.A.V.
Bodero
of Farm Animal Medicine and Production The University of Queensland Queensland, Australia 4072
Received for publication: Accepted:
May 22, 1992 January
6,
1993
ABSTRACT Data on the estrous cycles and sexual receptivity scores of 7 maiden Australian Stock Horse mares were used to study seasonal variation from December until the following November. Mares were grazed in paddocks containing both native and improved tropical pasture The study was conducted in South-East Queensland (27O27’ species. There were no differences between either the sexual South latitude). receptivity or the lengths of natural (Mean=7.5 days, SEM =0.4) and PGF-induced estruses (Mean = 7.6 days, SEM =0.4). Plasma estrogens were not related to differences in sexual receptivity scores between mares. The lowest incidence of estrus occurred at the time of the winter solstice (June 22) in the Southern Hemisphere. Winter estruses (Mean =9.3 days, SEM =0.8) were longer than those of summer (Mean = 6.6 days, SEM =0.5) and autumn (Mean =6.6 days, SEM =0.9). Approximately 30% of estruses were anovulatory. Most of these occurred in autumn and winter (P
mare, season, estrus, diestrus,
anestrus,
anovulatory
Acknowledgments The authors wish to thank Mr. W.L. Arthur for his technical assistance and Mr. B.E. Wilson for permitting use of the mares for this study.
Copyright 0 1993 Butterworth-Heinemann
Theriogenology
632
INTRODUCTION Conventional breeding programs must contend with problems caused by the seasonal pattern of estrous behavior in the mare, inconsistencies in the duration of estrus, silent estrus, split estrus, anovulatory estrus and persistent corpora lutea which can extend the duration of diestrus from 16 or 17 days to 3 months (1). In addition to these problems, the conventional breeding season in Australia (September 8 to December 31) is out of phase with the physiological breeding season of the mare and stallion (2, 3). The stimulus of increasing daylength on the reproductive behavior and ovarian activity of mares is well documented (4-7). Differences in latitude have been suggested to influence ovarian function in mares. Hammond (8) concluded that the natural breeding season of mares in higher latitudes occurred during the spring and summer but was extended in the subtropics and tropics and that there was a tendency to have 2 breeding seasons per year. Gonzalez and Valencia (9) found that mares in Mexico had 2 peaks of sexual receptivity (April to June and September to October). However, they presented no data on the ovarian status of the mares, and the evidence to support these findings is inconclusive (2, IO). To gain information on the reproductive pattern of mares in a subtropical environment, a study was conducted on the sexual receptivity, length of estrus and diestrus, and frequency of ovulatory and anovulatory estrus in 7 mares during spring, summer, autumn and winter in South-East Queensland (27O28’ South latitude). Total plasma estrogens were related to sexual receptivity. Progesterone was used in conjunction with behavior and follicle palpation to distinguish between phases of the estrous cycle.
MATERIALS Animals
AND METHODS
and Management
Maiden Australian Stock Horses mares (n = 71, 3 to 4 years of age and weighing 425 to 475 kg, were used to study variation in the estrous cycle from December to the following November, as part of and in preparation for an embryo transfer program at the Equine Unit, Veterinary School Farm, The University of Queensland (1 1). The mares were grazed in paddocks containing a mixture of native and improved tropical pasture species. Adequate body condition was maintained in the mares by supplementation with lucerne hay and
633
Theriogenology
molasses when pasture quality and/or quantity deteriorated (June to October). The mares were also given 1.5 kg of horse pellets while in the stocks for physical examination. Sexual Receptivity Sexual receptivity of the mares was assessed each morning while they were in the presence of a teaser stallion for a period of 2 hours. Palpation of the ovaries, uterus and cervix as well as the behavior of the mares were used to categorize them according to the following scores: 1 - approached the teaser voluntarily, raised the tail, abducted the hind limbs, everted the clitoris and urinated; 2 - did not approach the teaser voluntarily, but exhibited a submissive stance, raised the tail, everted the clitoris but did not urinate; 3 - not positively receptive nor antagonistic to the teaser but was found to be in estrus by follicle, uterine and cervical palpation; 4 - was aggressive to the teaser, flattening the ears and intermittent kicking but had a follicle when palpated; 5 - indifferent to the presence of the teaser. Effect of Prostaglandin
on Sexual Receptivity
As part of the embryo transfer program which has been described elsewhere (1 l), injections of prostaglandin F2a (PGF) were used to induce Prostaglandin injections were given following embryo transfer estrus. collection attempts at 6 to 9 days post-ovulation. This ensured that the corpus luteum was susceptible to lysis by prostaglandin. A comparison of the patterns of sexual receptivity exhibited during induced estruses and those of natural estruses was made. Estrus Estrus was defined as the time within an estrous cycle when sexual receptivity scores for most of the mares were 1, 2 or 3 and plasma progesterone values were less than 1 .O ng/ml (12). Estrus may or may not have been associated with ovulation but was always followed by diestrus, with plasma progesterone values greater than 1 .O ng/ml. Estruses for purposes of the embryo transfer program were classified as either naturally occurring or PGF-induced. An estrus was classified as anovulatory if the largest follicle did not collapse but remained the same size and had a thickened wall when estrus had concluded. The lengths of natural, induced and anovulatory estruses were compared between and within seasons. The frequency of anovulatory estrus in both natural and induced estruses was compared within seasons.
634
Theriogenology
Diestrus The length of diestrus was taken as the time between 2 consecutive estruses when plasma progesterone levels were greater than 1.0 ng/ml (12). Diestrous periods classified as normal for statistical analyses were those not interrupted by prostaglandin injections. If the length of diestrus was longer than 30 days, approximately twice the normal range reported in other studies (13-l 5), it was classified as prolonged, due to the presence of a persistent corpus luteum. Estrous Cycle Length The duration of an estrous cycle was determined by the summation of the length of natural diestrus and the subsequent estrus. There were 5 estrous cycles which commenced in autumn and concluded in winter. These were included as autumn cycles for the purpose of statistical analyses. Blood Samples Jugular blood samples were taken from the mares daily when in estrus and 3 times per week when not. The samples were collected into lithium heparin tubes and centrifuged at 3000 rpm for 10 minutes. The plasma was separated and stored at -20°C until assayed for progesterone and total estrogens. Plasma Progesterone
Assay
Plasma progesterone concentrations were estimated using the method described by Cox et al. (16). The progesterone antibody was #334/240576 and was used at a final dilution of 1:7060. The tracer, 11,2,6,7,16,1 7-3Hl progesterone had a specific activity of 112 Ci/mM and was used at a concentration of 6000 cpm/lOO ~1. The nonspecific binding of the assay was found to be 2.4%, the intra-assay variability over the range of the standard curve was 4.7% while the inter-assay variability was 13.0%. The sensitivity of the progesterone assay was 0.08 ng/ml, and the major cross reactions were with 118 hydroxyprogesterone (27.0%), corticosterone (5.8%), and 11 -deoxycorticosterone (6.9%). Total Plasma Estrogen Assay Total plasma estrogens (conjugated and unconjugated) were estimated using a modification of the method developed by Palmer and Terqui (17). The antisera was used at a dilution of 1:400000. The tracer had a specific activity of 153 Ci/mM and was used at a concentration of
Theriogenology
635
8000 cpm/500 ~1. The concentrations of total plasma estrogen were expressed as picagram equivalents of estradiol 17-S per millilitre. The nonspecific binding of the assay was 4.0%, the intra-assay variability was 3%; the inter-assay variability was 11.4% at 163 pg/ml and 18.8% at 68 pg/ml. The major cross-reactions were with estrone (1%), estradiol 17~1 (I %) and estriol (1 %I, and the sensitivity of the assay was 31 pg equivalents of estradiol 17-S per ml. Statistical
Analyses
The data contained both continuous and discrete variables. Examples of the continuous variables are length of the estrous cycle and its components. Data analyzed as discrete variables were those recorded as frequencies, e.g. the occurrence of estrous types. Continuous variables were tested using analysis of variance and subsequent t-tests or paired t-tests. The standard errors reported were calculated from the within-group variation and are independent of the “t” value, which was calculated to determine the level of significance. Discrete variables were tested using Chi-square tests of dependence. Seasonal differences in the duration of naturally occurring and induced estruses, the duration of diestrus, the duration of the estrous cycle and the duration of ovulatory and anovulatory estruses were Within seasons, the duration of naturally compared using paired t-tests. occurring and induced estruses and the duration of ovulatory and anovulatory estruses were compared using paired t-tests. The maximum total plasma estrogen concentrations and sexual receptivity of mares in both naturally occurring and induced estruses were compared by analysis of variance. The occurrence of anovulatory estrus and natural or induced estrus was analyzed both within and between seasons. The occurrence of anovulatory estrus in individual mares within seasons and the influence of induced estrus on sexual receptivity were analyzed using Chi-square tests of dependence. Generalized log-linear modelling (I 8) was used to test the effects of mare, month, and season on distributions of estruses.
RESULTS Sexual Receptivity Sexual receptivity scores of individual mares were relatively consistent during successive estrous cycles. There were no significant
636
Theriogenofogy
differences between sexual receptivity scores of either natural or induced estrus within mares (P > 0.05; Table 1).
Table 1.
Sexual receptivity induced estrus
scores
of
mares
during
natural
and
Type of estrus
Mare Natural Receptivity score
Induced
No. of estruses per mare
Receptivity score
No. of estruses per mare
1
4 2
8 2
4 2
4 1
2
3 2
8 2
3 1
6 1
3
1
11
1
5
4
1
7
3 1
1 5
5
3 1
5 2
3 1
2 3
6
3 2 1
5 2 2
3 1
3 2
7
3 1
1 6
3 1
1 4
Total Plasma Estrogen Concentrations There were no significant differences between the maximum total plasma estrogen concentrations of mares displaying overt sexual receptivity (Scores 1 and 2) or covert sexual receptivity (Scores 3 and 4) The least squares means for total plasma estrogen during estrus. concentrations for overt and covert sexual receptivity during estrus were 13.29 and 13.25 pg/lOO/.A, respectively. Total plasma estrogen concentrations of natural and induced estruses were not significantly different. The least squares means of plasma estrogen concentrations for the 2 types of estrus were 13.13 and 13.41 pg/lOO/.A, respectively.
Theriogenology
637
Plasma Progesterone
Concentrations
Plasma progesterone concentrations of less than 1 ng/ml were obtained from mares when in either estrus or anestrus. Diagnosis of whether mares were in estrus or anestrus was made by assessing sexual receptivity and by palpation of the ovaries, cervix and uterus. Mares in diestrus had plasma progesterone concentrations of 1.8 to 10 ng/ml. Seasonal
Incidence
and Length of Estrus
The total number of estruses studied was 99. Of these, 61 were naturally occurring and 38 were induced with prostaglandin. The mean length of all estruses over the year was 7.6 days (SEM =0.3). The mean lengths of natural and induced estruses were 7.5 days (SEM=0.4) and The means and standard errors for 7.6 days (SEM =0.4), respectively. the length of natural and induced estruses within mares are presented in Table 2.
Table 2.
Means and standard errors for the induced estruses within mares
Mare
length
and
Induced estrus
Natural estrus No. estruses oer mare
of natural
Length (days) Mean
SEM
No. estruses oer mare
Length (days) Mean
SEM
1
10
7.7
0.4
5
6.8
0.8
2
10
7.5
1.1
7
5.6
0.9
3
11
6.2
0.6
5
8.2
1.7
4
7
7.3
0.9
6
7.5
0.8
5
7
9.0
1.6
5
9.0
0.4
6
9
8.7
1.5
5
7.8
1.5
7
7
6.7
0.9
5
9.2
1.5
There were 5 occasions involving 3 mares when the length of an induced estrus was 3 days or less. Four of these short estruses occurred between March and May (autumn). There were 9 induced estruses Four of these involving 5 mares which lasted for 10 or more days. occurred in February and March while 5 occurred in September and October (spring). The seasonal and monthly estruses are presented in Table 3.
distributions
of natural
and
induced
Theriogenology
638
Table 3.
Season
Summer
Autumn
Winter
Spring
Seasonal and monthly and induced estruses
distributions
Month
of natural
Type of estrus Natural
Induced
December
9
0
January
5
0
February
3
7
Total
17
7
March
4
9
April
3
6
May
7
1
Total
14
16
June
7
0
July
4
0
August
5
0
Total
16
0
September
6
4
October
3
6
November
5
5
14
15
Total
The lowest incidence of estrus occurred around the time of the winter solstice (June 22) in the Southern Hemisphere. There was also a decrease in the occurrence of estrus in January, which was attributed to 3 periods of prolonged diestrus, due to the presence of persistent corpora lutea in 3 of the 7 mares. The length of natural estrus in winter (Mean = 9.3 days; SEM =0.8) was significantly longer than the length of natural estrus in summer (Mean = 6.6 days; SEM =0.5; P
7
Spring
a,b
7
6
7
Autumn
Winter
Spring
Frequencies within columns test of dependence).
7
No. of mares
distribution estrus
different
14
16
14
17
different
of natural
superscripts
1ac
9b
7bc
0a
Anovulatory estruses
paired
different
7
0
7
6
(P
15
0
0.7
0.9
1.0
SEM
(days)
Chi-square
3b
0ab
1oa
2ab
Anovulatory estruses
8.0ab
6.6a
7.6ab
estrus
16
7
Length Mean
estrus
estruses
No. of estruses
Induced
estruses
t-test).
15
0
16
7
No. of estruses
No. of mares
anovulatory
(P
7
0
7
6
No. of mares
Induced
and prostaglandin-induced
are significantly
and prostaglandin-induced
No. of estruses
Natural
with
0.7
0.8
1.2
0.5
SEM
(days)
length
are significantly
8.2ab
9.3b
7.5ab
6.6a
Mean
Length
of natural
superscripts
14
16
14
17
No. of estruses
Summer
Season
Seasonal
different
6
Winter
with
7
Autumn
Means
7
Summer
Table 5.
ab
No. of mares
for the
estrus
errors
Natural
Means and standard per mare per season
Season
Table 4.
s 0 6 9
G
2 s *.
640
Theriogenology
Anovulatory
Estrus
Of the 99 estruses observed, there were 32 in which ovulation Anovulatory estrus occurred in 17 of the 61 natural failed to occur. estruses and in 15 of the 38 induced estruses. The seasonal distribution of anovulatory estrus, whether natural or induced, is presented in Table 5. Significantly more natural anovulatory estruses occurred during autumn and winter than during summer (PO.O5). There was considerable variation in the occurrence of anovulatory estrus between mares. Since there were no differences within seasons, the mare and seasonal data were combined for analysis. Mare 1 had significantly more incidences of anovulatory estrus than Mares 3, 4 and 7 All other mare differences were not significant (P>O.O5; (P
Table 6.
Mare
Occurrence of anovulatory between seasons Summer
Autumn
estrus
between
Winter
mares
and
Spring
No. of estruses
A0
No. of estruses
A0
No. of estruses
A0
No. of estruses
A0
la
3
1
4
4
4
4
4
1
2ab
5
1
5
4
3
3
4
1
3b
4
0
4
2
4
0
4
0
qb
2
0
5
0
2
1
4
0
5ab
3
0
5
3
0
0
4
1
gab
4
0
4
2
1
1
5
1
7b
3
0
3
2
2
0
4
0
A0 a.b
Occurrence of anovulatory estruses. Mares with different superscripts had significantly different occurrences of anovulatory estruses (P < 0.05; Chi-square test of dependence).
641
Theriogenology
There were no differences between the lengths of the ovulatory and anovulatory estruses within seasons (P~0.05). The length of ovulatory estrus in spring was significantly longer than that in summer (PO.O5; Table 7).
Table
7.
Means and standard errors for the length ovulatory estruses per mare per season
Season
of anovulatory
and
Type of estrus Ovulatory
Anovulatory No. of mares
Length
(days)
Mean
SEM
No. of mares
Length
(days)
Mean
SEM
Summer
2
4.5
0.5
7
7.1a
0.3
Autumn
6
6.2
1.2
6
6.7 ab
0.7
Winter
4
10.1
1 .o
3
7.8 ab
0.2
Spring
4
7.0
1.8
7
8.1b
0.5
0
Length
Means with different (P-CO.05; paired t-test).
superscripts
are
significantly
different
of Diestrus
There were 58 natural diestrous periods during the study. Five of these were greater than 40 days in length and were regarded as prolonged, due to the presence of a persistent corpus luteum. One diestrus lasted only 10 days, due to the occurrence of an acute but transient endometritis and for this reason was not included in the analysis. The influence of season on the mean length of diestrus per mare is presented in Table 8. Only diestrous periods of between 10 and 30 days were included in the data. The overall mean length of diestrus was 16.6 days (SEM = 0.3; n = 50). Season had no significant influence on the length of diestrus (P>O.O51. There were fewer periods of diestrus in summer than in the other seasons, due to the occurrence of persistent corpora lutea in 3 of the mares at this time. There were 5 diestrous
Theriogenofogy
642
periods which lasted from 20 to 24 days. July to October.
Table 8.
Season
Means and standard per season
No. of diestrus
Four of these occurred
from
errors of the length of diestrus per mare
No. of mares
Length (days) Mean
Summer
11
5
16.1
0.6
Autumn
15
7
16.7
0.4
Winter
13
5
16.8
0.6
Spring
13
7
15.5
0.5
The differences in the lengths of diestrus between mares were not significant when diestrous periods of 10 to 30 days were analyzed (P>O.O5). However, when the prolonged diestrous periods were included, significant differences in the mean length of diestrus between mares were obtained (P
Theriogenology
643
The mean estrous cycle length in winter was significantly greater than that in autumn (PO.O5). However, there was a trend that suggested the cycle length in winter was greater than that in summer. The difference between the lengths of the estrous cycles in autumn and spring approached the 5% significance level.
Table 9.
Means and standard errors for the length of normal and total diestrous periods (including prolonged ones1 in mares Classification Normal n
Mare ID
of diestrous period Total (normal plus prolonged)
Length (days) Mean
SEM
n
Length (days) Mean
SEM
la
9
16.6
1.0
10
20.aCd
4.3
2
9
17.4
1.0
9
17.4d
1.0
3
10
16.6
0.7
10
16.6d
0.7
4a
6
16.5
0.8
7
21.4Cd
5.0
5
7
17.3
0.8
7
17.3d
0.8
6
8
15.5
0.6
8
15.5d
0.6
7a,b
3
15.3
1.3
6
41 .oc
13.7
a b cd
Winter
Indicates the presence of a persistent corpus luteum during January. This mare had a persistent corpus luteum on 3 occasions and 1 short diestrus of 3 days duration. Means with different superscripts are significantly different (P < 0.05; t-test).
Anestrus
Four mares continued to cycle during winter while 3 mares (Mares 4, 5 and 6) were in anestrus for 59, 114 and 88 days, respectively. diestrus before the onset of true These mares entered a period of
Theriogenology
644
anestrus. Because of the high incidence of anovulatory estrus, anestrus was assumed to commence on the final day of the last estrus instead of on the day of the final ovulation of the season. Anestrus was assumed to terminate at the first ovulation of the following breeding season. This definition was used since only 3 of the 7 mares entered anestrus, and they failed to ovulate during the penultimate estrus of the breeding season. Two of these mares ovulated during their final estrus, while the other mare failed to ovulate during her final estrus. Progesterone concentrations of the mares were initially consistent with those of diestrous levels and then dropped to between 0.1 ng/ml and 0.3 ng/ml until the first ovulation of the next breeding season. Mares 5 and 6 failed to ovulate during their last estrus before the onset of anestrus.
Table 10.
a,b
Means and standard per mare per season
errors of the length of estrous
cycles
Season
No. of cycles
No. of mares
Summer
12
5
22.5ab
1.1
Autumn
17
7
24.2a
0.7
Winter
11
4
25.5b
1 .o
Spring
12
7
22.3ab
0.6
Means with different (P < 0.05; paired t-test).
superscripts
Cycle length (days)
are
significantly
different
Mare 5 was passive when teased until the first large follicle developed in spring. Then she displayed erratic sexual receptivity (Scores 2, 3 and 4) until the first ovulation of the breeding season. Mare 6 remained passive in the presence of the teaser until her first follicle developed in spring, when she exhibited overt sexual receptivity (Score I). Mare 4 exhibited overt sexual receptivity (Score 2) on 4 occasions that were randomly scattered throughout winter, when both ovaries were less than 4 cm in length. Following development of the first large follicle of the breeding season, she displayed positive signs of estrus (Score 1) which continued until ovulation. Anestrus terminated in the 3 mares with an ovulation within 2 weeks of each other in September. These 3 mares showed overt sexual receptivity for extended periods of 10, 11 and 18 days, respectively (spring estrus) before entering the ovulatory season. Table 1 1 presents data on the duration of the anestrous period.
Theriogenology
645
Mares 2 and 7 displayed anestrous behavior during July and August. However, Mare 2 had progesterone concentrations that were indicative of cyclical activity during winter but she did not ovulate until September. Both ovaries were at their smallest size (4 cm) in July and her peak progesterone concentration during winter was 2.8 ng/ml, which was well below that of her other diestrous concentrations (10.0 ng/ml) during the study. Another mare (Mare 7) was in prolonged diestrus from July 7 until August 20. Her plasma progesterone concentrations were elevated during this time and ranged from 1.7 ng to 10.0 ng/ml. She failed to ovulate during this period and her ovaries were only 4 cm in length. Table 12 presents progesterone and ovarian data on these mares over the winter season.
Table 11.
Duration of anestrus in 3 mares
Characteristics of winter anestrus
Mare 4
Mare 5
Mare 6
Start of anestrus
July 16
June 5
June 16
Time from last estrus to low progesterone concentration (< 1 .O ng/ml)
17 days
15 days
18 days
Appearance
Sept 6
Aug 18
Aug 9
Time from low progesterone concentration to the first estrus
31 days
81 days
60 days
Length of the first estrus
11 days
18 days
10 days
Date of the first ovulation
Sept 13
Sept 27
Sept 12
Length of anestrus
59 days
114 days
88
Table 12.
Mare
2 7
of first follicle
days
Plasma progesterone concentrations and ovarian data from 2 mares displaying anestrous behavior during July and August Progesterone secretion pattern
Peak progesterone concentration (ng/ml)
No. of ovulations
Ovarian length (cm)
Cyclical
2.8
0
4
10.0
0
4
Persistent
CL
Theriogenology
646 DISCUSSION
The results of this study show that the intensity of sexual receptivity is repeatable within individual mares, which supports the findings of McKenzie (19) and Hillman and Loy (20). Silent estrous behavior has been associated with estruses induced with prostaglandin (21-23). This was not apparent in these mares since the intensity of sexual receptivity within the mares was not affected by prostaglandin treatment. The incidence of silent estrous behavior in pony mares was reported to be 7% (1, 15, 24). Four of the 7 mares in our study frequently had silent estruses with indifferent sexual receptivity. Group teasing by a single stallion was not adequate for assessing these mares so they had to be teased individually and their ovaries palpated to detect estrus. The lack of normal signs of estrus in these mares was unrelated to prostaglandin treatment. No consistent relationship was found between total plasma estrogen concentrations of the mares and their sexual receptivity scores. These results conflict with those of Nelson et al. (23) but agree with those of Munroe et al. (251, who found no direct association between the intensity of sexual receptivity and either plasma estrogen or plasma androgen concentrations in the mare. Ganowicz (26) found no relationship between intensity of sexual receptivity and follicular development in mares. It is possible that mares with a low sexual receptivity score of 5 (indifferent) might have responded more strongly if the teasing time was extended or if they were permitted to be mounted by a teaser stallion (27). Dominance hierarchies and the social interactions at the time of puberty may be important factors for investigation when determining why some domesticated mares fail to exhibit normal sexual receptivity during estrus. A study of New Forest ponies showed that mutual grooming was part of the mating ritual between the stallions and young mares, which even though they were in estrus, often showed signs of fear. Mutual grooming did not occur between the stallions and the older mares (28). If these conditions could be simulated on equine stud farms the incidence of silent estrus could possibly be reduced. However, the risk of injury and the spread of venereal diseases by teaser stallions would have to be considered before such a practice was initiated. The mean length of naturally occurring estruses increased as the photoperiod decreased during the transition from summer to winter, which is similar to the results of other studies (1, 15, 29, 30). It is widely accepted that the length of estrus early in the season is prolonged
Theriogenology
647
(spring estrus) and shortens as the summer solstice approaches. The length of estrus in spring in our study (Mean = 8.2 days; SEM = 0.7) was greater than that in summer (Mean =6.6 days; SEM =0.5) but this difference was not significant (P>O.O5). This result was probably due to the number of mares that continued to cycle throughout the year. Mares in the other studies, except for that of Hughes et al. (I), had estruses that decreased in length as summer approached. These findings may be due to the relatively small difference in the photoperiod in the sub-tropics as compared with that at higher latitudes. Thirty-eight percent of the estruses of the mares in this study were induced by prostaglandin injections. However, there was no significant difference between the mean length of prostaglandin-induced and natural estruses. These findings are similar to those of Douglas and Ginther (31) but differ from those of Oxender et al. (32) and of Burns et al. (33), who reported that prostaglandin-induced estruses were significantly longer than natural estruses. Nelson et al. (23) on the other hand, found that induced estruses were shorter than those of control mares. The mean length of diestrus in mares in our study was 16.6 days, which is similar to that of other data (1, 34). There was no significant difference between the lengths of diestrus due to season of year. The coefficient of variation for the duration of estrus (38%) was more than twice that for the duration of diestrus (15%). These findings are similar to those of Hughes et al. (l), Back et al. (34), Ginther et al. (35) and Greenhoff and Kenney (36). Thus the relative uniformity of the length of diestrus between seasons appears to be a constant feature of the estrous cycle of mares in both hemispheres and at different latitudes. There were 5 periods of diestrus, ranging from 20 to 24 days during winter and spring, which were not regarded as prolonged. There were another 5 diestrous periods of 43 to 90 days duration which were due to the presence of persistent corpora lutea. The average duration of prolonged diestrous due to persistent corpora lutea was reported to be 2 Neely (37) and Neely et al. (38) demonstrated that months (12, 27). either a diestrous ovulation or the release of insufficient prostaglandin to cause complete luteolysis was the likely cause of persistent corpora lutea in the mare. The definition of the estrous cycle in this study included the possible occurrence of an anovulatory estrus. Even so, the mean estrous cycle length of the mares over all seasons was 23.7 days, which is similar to the composite mean of data (21.7 days) collated by Ginther (27) and the mean of his own data (22.7 days).
640
Theriogenology
A ‘relatively low occurrence of normal sexual receptivity was recorded in our study due to the prolonged diestrous periods in 3 of the mares. However, a seasonal trend was apparent in the length of estrous This cycles, with longer cycles occurring in winter than in summer. difference in estrous cycle length was due to seasonal differences in the lengths of the actual estrus, as was shown by several other workers (1, 30, 35). Hammond (8) and Gonzalez and Valencia (9) suggested that mares This was not in tropical regions had 2 breeding seasons per year. apparent in the mares in our study. They were found to be polycyclic as were mares in a study in the tropics (39). Most of the mares in our study continued to display estrus during autumn and winter but most of these estruses were anovulatory. Both studies showed a relationship between frequency of ovulation and season of year, with fewer ovulations occurring in winter. The occurrence of anovulatory estrus in the mare has been In our study there were 6 anovulatory reported to be rare (1, 15, 40). estruses involving 4 mares during the ovulatory season, while in autumn and winter, 26 of 46 estruses were anovulatory. However, this was more common in 2 of the mares. Ginther (27) stated that mares are very dependable ovulators, but that anovulatory estrus, which occurred most commonly in the transition from the ovulatory to the anovulatory season, was due to insufficient LH to stimulate final development of the preovulatory follicle. The proportion of mares in this study that continued to display estrus during winter was twice that reported by Osborne (2), Loy (41) and Saltiel et al. (39). Sharp (42) defined anestrus as the period when less than 25% of mares ovulated. The results of the present study, with only 42% of the mares not cycling and a high proportion of anovulatory estruses during winter, support Sharp’s definition of winter anestrus. However, Sharp’s (42) concept that the number of mares cycling is indicative of the number of mares ovulating was not applicable in this study, possibly due to the reduced variation in photoperiod at this latitude. Failure of the preovulatory follicle to develop and insufficient LH were found to be the reasons for ovulatory failure at the onset of anestrus in ponies (43). It has been shown that as winter approaches the ovulatory season in ponies ceases more quickly than in horses, and that while no ponies ovulate during winter, up to 25% of horses continue to do so (15, 44). It is possible that the hypothalamus of the horse is less sensitive to melatonin than that of the pony or that the pineal of the horse is less sensitive than that of the pony to changes in photoperiod.
Theriogenology
649
The low proportion of winter anestrus in the mares of our study may have been due to the relatively small changes in photoperiod which did not stimulate sufficient melatonin secretion to inhibit GnRH secretion. This may account for the number of mares which displayed estrus during winter and why there were more anovulatory estruses in autumn and winter than in spring and summer. It has been shown by Asa et al. (45) that it is common for pony mares to exhibit sexual receptivity during the anovulatory season, and, in fact, ovariectomized pony mares also display sexual receptivity. It was suggested by Asa et al. (46) that the adrenal gland, which is a source of estrogens and androgens, may be responsible for unseasonal sexual receptivity. This was confirmed by suppression of sexual receptivity in ovariectomized mares using dexamethasone.
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Hughes, J.P., Stabenfeldt, G.H. and Evans, J.W. Clinical and endocrine aspects of the estrous cycle of the mare. Proc. 18th Ann Conv. Am. Ass. equine Pratt. pp.1 99-148 (1972).
2.
Osborne, V.E. An analysis of the pattern of ovulation as it occurs in the annual reproductive cycle of the mare in Australia. Aust. Vet. J. 42:149-154 (1966).
3.
Dowsett, K.F. The breeding season and semen Reprod. Fertil. 32 (Suppl.):636-637 (1982).
4.
Burkhardt, J. Transition from anoestrus in the mare and the effects of artificial lighting. J. Agric. Sci. =:64-68 (1947).
5.
Arthur, G.H. An analysis of the reproductive function based on post-mortem examination. Vet. Rec. m:682-686
6.
Kooistra, L.H. and Ginther, O.J. reproductive activity and hair in x:1413-1419 (1975).
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Sharp, D.C., Vernon, M.W. and Zavy, M.T. Alteration of seasonal following superior cervical reproductive patterns in mares ganglionectomy. J. Reprod. Fertil. 27 (Suppl.):87-93 (1979).
8.
Hammond, J. Recent scientific problems. Trans. Yorks. Agric. Sot.
9.
Gonzalez, M.F. and Valencia, M.J. Estudio del comportamiento reproductive de la yegua en Mexico. Vet. Mex. 8: 19-22 (1977).
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Nyborg, R.G. Ovulationsuregelmaessigheder og et uheldigt valgt bedaeknings-og afprovningssystem som arsag til ufrugtbarhed hos hopper. Nord. Vet.-Med. 5:473-538 (1953).
11.
Dowsett, K.F., Woodward, R.A. and Bodero, D.A.V. A study of in the mare. transfer Theriogenology nonsurgical embryo 3l_:631-642 (1989).
12.
Hughes, J.P., Stabenfeldt, G.H. and Evans, J.W. The oestrous cycle of the mare. J. Reprod. Fertil. 23 (Suppl.): 161-I 66 (1975).
13.
Andrews, F.N. and McKenzie, F.F. Estrus, ovulation and related phenomena in the mare. University of Missouri, Agric. Exp. Sta. Res. Bull. =:1-l 17 (1940).
14.
Berliner, V.R. The estrous cycle of the mare. In: Cole, H.H. and Cupps, T.P. (eds), Reproduction in Domestic Animals. Academic Press, New York, 1959, p.267
15.
Ginther. O.J. Occurrence of anestrus, diestrus and ovulation over a 12-month period in mares. Am. J. Vet. Res. s:l 173-I 179 (1974).
16.
Cox, R.I., Hinks, N. and Waste, C. Assay of progesterone in plasma. Hormone assay development group, C.S.I.R.O., Blacktown, Sydney, N.S.W., Australia, 1977.
17.
Palmer, E. and Terqui, M. The measurement of total plasma estrogens during the follicular phase of the mares’s estrous cycle. Theriogenology 1:331-338 (1977).
18.
Baker, R.J. and Nelder, J.A. The GLIM System Release 3. Generalised Linear Interactive Modelling. Royal Statistical Society, London, 1978.
19.
McKenzie, F.F. Recent reproduction studies on equines. Ann. Mtg. Amer. Sot. Anim. Prod. pp. 98-102 (1940).
20.
Hillman, R.B. and Loy, R.G. Oestrogen relation to various reproductive states. (Suppl.):223-230 (1975).
Proc. 33rd.
excretion in mares in J. Reprod. Fertil. 23
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21.
Keifer, B.L., Roser, J.F., Evans, J.W., Neely, D.P. and Pacheco, C.A. Progesterone patterns observed with multiply injections of a PGF2a analogue in the cyclic mare. J. Reprod. Fertil. 27 (Suppl.):237-244 (1979).
22.
Loy, R.G., Buell, J.R., Stevenson, W. and Hamm, D. Sources of variation in response intervals after prostaglandin treatment in mares with functional corpora lutea. J. Reprod. Fertil. 27 (Suppl.):229-235 (I 979).
23.
Nelson, E.M., Kiefer, B.L., Roser, J.F. and Evans, J.W. Serum estradiol-17B concentrations during spontaneous silent estrus and after prostaglandin treatment in the mare. Theriogenology =:241-262 (1985).
24.
Cummings, J.N. A study of estrus and ovulation Anim. Sci. 1:309-313 (1942).
25.
Munroe, C.D., Renton, J.P. and Butcher, R. The control of oestrous behaviour in the mare. J. Reprod. Fertil. 27 (Suppl.):217-227 (1979).
26.
Ganowicz, M. Relationship between the intensity of the outward signs of oestrus, maturation of the Graafian follicle, and infection of the genital tract in mares during the breeding season. Acta. Ag. Silvest. Ser. Zootech. 5:53-57 (1965).
27.
Ginther, O.J. Reproductive Biology of the Mare: Basic and Applied Aspects. McNaughton and Gunn, Ann Arbor Ml, 1979, pp. 67141.
28.
Tyler, S. The behaviour and social organization ponies. Anim. Behav. Monogr. 5:87-l 96 (1972).
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29.
Day, F.T. Clinical and experimental the mare. J. Agric. Sci. X:244-261
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30.
van Niekerk, C.H. Pattern of the oestrous cycle of mares. II. The duration of the oestrous cycle and oestrous period. J. S. Afr. Vet. Med. Ass. =:299-307 (1967).
31.
Douglas, R.H. and Ginther O.J. Effect of prostaglandin F2a length of diestrus in mares. Prostaglandins 2:265-268 (1972).
observations (1940).
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Forest
in
on
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32.
Oxender, W.D., Noden, P.A., Bolenbaugh, D.L. and Hafs, H.D. Control of estrus with prostaglandin F2a in mares: minimal effective dose and stage of estrous cycle. Am. J. Vet. Res. 3&l 145-1147 (1975).
33.
Burns, S-J., Irvine, C.H.G. and Amoss, M.S. prostaglandin-induced oestrus compared to normal oestrus. J. Reprod. Fertil. 27 (Suppl.):245-250 (1979).
34.
Back, D.G., Pickett, B.W., Voss, J.L. and Siedel, G.E. Observations on the sexual behavior of nonlactating mares. J. Am. Vet. Med. Ass. 165:717-720 (1974).
35.
Ginther, O.J., Whitmore, H.L. and Squires, E.L. Characteristics of estrus, diestrus and ovulation in mares and effects of season and nursing. Am. J. Vet. Res. =:1935-1939 (1972).
36.
Greenhoff, G.R. and Kenney, R.M. status of non-pregnant mares. m:449-458 (1975).
37.
Neely, D.P. Studies on the Control of Prostaglandin Release in the Mare. Ph.D. California, Davis, 1979.
38.
Neely, D.P., Kindahl, H., Stabenfeldt, G.H., Edquist, L.-E. and Hughes, J.P. Prostaglandin release patterns in the mare: Physiological, pathophysiological and therapeutic responses. J. Reprod. Fertil. 27 (Suppl.):181-189 (1979).
39.
Saltiel, A., Caideron, A., Garcia, N. and Hurley, activity in the mare between latitude 15O and 22” Fertil. 32 (Suppl.):261-267 (I 982).
40.
Caslick, E.A. The sexual cycle and its relation to ovulation breeding records of the Thoroughbred mare. Cornell 27: 187-206 (1937).
41.
Loy, R.G. The reproductive managers’course, Lexington,
42.
Sharp, D.C. Environmental influences on reproduction in horses. Vet. Clinics N. Am.: Large Anim. Pratt. 2:207-223 (1980).
Evaluation J. Am.
Fertility of post-partum
of the reproductive Vet. Med. Ass.
Luteal Thesis,
Function and University of
D.P. Ovarian N. J. Reprod.
cycle of the mare. Lectures Ky. pp. 20-30 (1970).
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Snyder, D.A., Turner, D.D., Miller, K.F., Garcia, M.C. and Ginther, O.J. Follicular and gonadotrophic changes during transition from ovulatory to anovulatory seasons. J. Reprod. Fertil. 27 (suppl.):95-101 (1979).
44.
Satoh, S. and Hoshi, S. A study of reproduction Jap. Sot. Vet. Sci. u:257-267 (1932).
45.
Asa, C.S., Goldfoot, D.A., Garcia, M.C and Ginther, behavior in ovariectomized and seasonally anovulatory (Eauus caballus). Horm. Behav. U:46-54 (I 980).
46.
Asa, C.S., Goldfoot, D.A., Garcia, M.C Dexamethasone suppression of sexual ovariectomized mare. Horm. Behav. H:55-64
in the mare.
J.
O.J. Sexual pony mares
and Ginther, O.J. behavior in the (1980).
39:631-653,
1993
SEASONAL VARIATION IN THE ESTROUS OF MARES IN THE SUBTROPICS K.F. Dowsett, Department
L.M. Knott, R.A. Woodward
CYCLE
and D.A.V.
Bodero
of Farm Animal Medicine and Production The University of Queensland Queensland, Australia 4072
Received for publication: Accepted:
May 22, 1992 January
6,
1993
ABSTRACT Data on the estrous cycles and sexual receptivity scores of 7 maiden Australian Stock Horse mares were used to study seasonal variation from December until the following November. Mares were grazed in paddocks containing both native and improved tropical pasture The study was conducted in South-East Queensland (27O27’ species. There were no differences between either the sexual South latitude). receptivity or the lengths of natural (Mean=7.5 days, SEM =0.4) and PGF-induced estruses (Mean = 7.6 days, SEM =0.4). Plasma estrogens were not related to differences in sexual receptivity scores between mares. The lowest incidence of estrus occurred at the time of the winter solstice (June 22) in the Southern Hemisphere. Winter estruses (Mean =9.3 days, SEM =0.8) were longer than those of summer (Mean = 6.6 days, SEM =0.5) and autumn (Mean =6.6 days, SEM =0.9). Approximately 30% of estruses were anovulatory. Most of these occurred in autumn and winter (P
mare, season, estrus, diestrus,
anestrus,
anovulatory
Acknowledgments The authors wish to thank Mr. W.L. Arthur for his technical assistance and Mr. B.E. Wilson for permitting use of the mares for this study.
Copyright 0 1993 Butterworth-Heinemann
Theriogenology
632
INTRODUCTION Conventional breeding programs must contend with problems caused by the seasonal pattern of estrous behavior in the mare, inconsistencies in the duration of estrus, silent estrus, split estrus, anovulatory estrus and persistent corpora lutea which can extend the duration of diestrus from 16 or 17 days to 3 months (1). In addition to these problems, the conventional breeding season in Australia (September 8 to December 31) is out of phase with the physiological breeding season of the mare and stallion (2, 3). The stimulus of increasing daylength on the reproductive behavior and ovarian activity of mares is well documented (4-7). Differences in latitude have been suggested to influence ovarian function in mares. Hammond (8) concluded that the natural breeding season of mares in higher latitudes occurred during the spring and summer but was extended in the subtropics and tropics and that there was a tendency to have 2 breeding seasons per year. Gonzalez and Valencia (9) found that mares in Mexico had 2 peaks of sexual receptivity (April to June and September to October). However, they presented no data on the ovarian status of the mares, and the evidence to support these findings is inconclusive (2, IO). To gain information on the reproductive pattern of mares in a subtropical environment, a study was conducted on the sexual receptivity, length of estrus and diestrus, and frequency of ovulatory and anovulatory estrus in 7 mares during spring, summer, autumn and winter in South-East Queensland (27O28’ South latitude). Total plasma estrogens were related to sexual receptivity. Progesterone was used in conjunction with behavior and follicle palpation to distinguish between phases of the estrous cycle.
MATERIALS Animals
AND METHODS
and Management
Maiden Australian Stock Horses mares (n = 71, 3 to 4 years of age and weighing 425 to 475 kg, were used to study variation in the estrous cycle from December to the following November, as part of and in preparation for an embryo transfer program at the Equine Unit, Veterinary School Farm, The University of Queensland (1 1). The mares were grazed in paddocks containing a mixture of native and improved tropical pasture species. Adequate body condition was maintained in the mares by supplementation with lucerne hay and
633
Theriogenology
molasses when pasture quality and/or quantity deteriorated (June to October). The mares were also given 1.5 kg of horse pellets while in the stocks for physical examination. Sexual Receptivity Sexual receptivity of the mares was assessed each morning while they were in the presence of a teaser stallion for a period of 2 hours. Palpation of the ovaries, uterus and cervix as well as the behavior of the mares were used to categorize them according to the following scores: 1 - approached the teaser voluntarily, raised the tail, abducted the hind limbs, everted the clitoris and urinated; 2 - did not approach the teaser voluntarily, but exhibited a submissive stance, raised the tail, everted the clitoris but did not urinate; 3 - not positively receptive nor antagonistic to the teaser but was found to be in estrus by follicle, uterine and cervical palpation; 4 - was aggressive to the teaser, flattening the ears and intermittent kicking but had a follicle when palpated; 5 - indifferent to the presence of the teaser. Effect of Prostaglandin
on Sexual Receptivity
As part of the embryo transfer program which has been described elsewhere (1 l), injections of prostaglandin F2a (PGF) were used to induce Prostaglandin injections were given following embryo transfer estrus. collection attempts at 6 to 9 days post-ovulation. This ensured that the corpus luteum was susceptible to lysis by prostaglandin. A comparison of the patterns of sexual receptivity exhibited during induced estruses and those of natural estruses was made. Estrus Estrus was defined as the time within an estrous cycle when sexual receptivity scores for most of the mares were 1, 2 or 3 and plasma progesterone values were less than 1 .O ng/ml (12). Estrus may or may not have been associated with ovulation but was always followed by diestrus, with plasma progesterone values greater than 1 .O ng/ml. Estruses for purposes of the embryo transfer program were classified as either naturally occurring or PGF-induced. An estrus was classified as anovulatory if the largest follicle did not collapse but remained the same size and had a thickened wall when estrus had concluded. The lengths of natural, induced and anovulatory estruses were compared between and within seasons. The frequency of anovulatory estrus in both natural and induced estruses was compared within seasons.
634
Theriogenology
Diestrus The length of diestrus was taken as the time between 2 consecutive estruses when plasma progesterone levels were greater than 1.0 ng/ml (12). Diestrous periods classified as normal for statistical analyses were those not interrupted by prostaglandin injections. If the length of diestrus was longer than 30 days, approximately twice the normal range reported in other studies (13-l 5), it was classified as prolonged, due to the presence of a persistent corpus luteum. Estrous Cycle Length The duration of an estrous cycle was determined by the summation of the length of natural diestrus and the subsequent estrus. There were 5 estrous cycles which commenced in autumn and concluded in winter. These were included as autumn cycles for the purpose of statistical analyses. Blood Samples Jugular blood samples were taken from the mares daily when in estrus and 3 times per week when not. The samples were collected into lithium heparin tubes and centrifuged at 3000 rpm for 10 minutes. The plasma was separated and stored at -20°C until assayed for progesterone and total estrogens. Plasma Progesterone
Assay
Plasma progesterone concentrations were estimated using the method described by Cox et al. (16). The progesterone antibody was #334/240576 and was used at a final dilution of 1:7060. The tracer, 11,2,6,7,16,1 7-3Hl progesterone had a specific activity of 112 Ci/mM and was used at a concentration of 6000 cpm/lOO ~1. The nonspecific binding of the assay was found to be 2.4%, the intra-assay variability over the range of the standard curve was 4.7% while the inter-assay variability was 13.0%. The sensitivity of the progesterone assay was 0.08 ng/ml, and the major cross reactions were with 118 hydroxyprogesterone (27.0%), corticosterone (5.8%), and 11 -deoxycorticosterone (6.9%). Total Plasma Estrogen Assay Total plasma estrogens (conjugated and unconjugated) were estimated using a modification of the method developed by Palmer and Terqui (17). The antisera was used at a dilution of 1:400000. The tracer had a specific activity of 153 Ci/mM and was used at a concentration of
Theriogenology
635
8000 cpm/500 ~1. The concentrations of total plasma estrogen were expressed as picagram equivalents of estradiol 17-S per millilitre. The nonspecific binding of the assay was 4.0%, the intra-assay variability was 3%; the inter-assay variability was 11.4% at 163 pg/ml and 18.8% at 68 pg/ml. The major cross-reactions were with estrone (1%), estradiol 17~1 (I %) and estriol (1 %I, and the sensitivity of the assay was 31 pg equivalents of estradiol 17-S per ml. Statistical
Analyses
The data contained both continuous and discrete variables. Examples of the continuous variables are length of the estrous cycle and its components. Data analyzed as discrete variables were those recorded as frequencies, e.g. the occurrence of estrous types. Continuous variables were tested using analysis of variance and subsequent t-tests or paired t-tests. The standard errors reported were calculated from the within-group variation and are independent of the “t” value, which was calculated to determine the level of significance. Discrete variables were tested using Chi-square tests of dependence. Seasonal differences in the duration of naturally occurring and induced estruses, the duration of diestrus, the duration of the estrous cycle and the duration of ovulatory and anovulatory estruses were Within seasons, the duration of naturally compared using paired t-tests. occurring and induced estruses and the duration of ovulatory and anovulatory estruses were compared using paired t-tests. The maximum total plasma estrogen concentrations and sexual receptivity of mares in both naturally occurring and induced estruses were compared by analysis of variance. The occurrence of anovulatory estrus and natural or induced estrus was analyzed both within and between seasons. The occurrence of anovulatory estrus in individual mares within seasons and the influence of induced estrus on sexual receptivity were analyzed using Chi-square tests of dependence. Generalized log-linear modelling (I 8) was used to test the effects of mare, month, and season on distributions of estruses.
RESULTS Sexual Receptivity Sexual receptivity scores of individual mares were relatively consistent during successive estrous cycles. There were no significant
636
Theriogenofogy
differences between sexual receptivity scores of either natural or induced estrus within mares (P > 0.05; Table 1).
Table 1.
Sexual receptivity induced estrus
scores
of
mares
during
natural
and
Type of estrus
Mare Natural Receptivity score
Induced
No. of estruses per mare
Receptivity score
No. of estruses per mare
1
4 2
8 2
4 2
4 1
2
3 2
8 2
3 1
6 1
3
1
11
1
5
4
1
7
3 1
1 5
5
3 1
5 2
3 1
2 3
6
3 2 1
5 2 2
3 1
3 2
7
3 1
1 6
3 1
1 4
Total Plasma Estrogen Concentrations There were no significant differences between the maximum total plasma estrogen concentrations of mares displaying overt sexual receptivity (Scores 1 and 2) or covert sexual receptivity (Scores 3 and 4) The least squares means for total plasma estrogen during estrus. concentrations for overt and covert sexual receptivity during estrus were 13.29 and 13.25 pg/lOO/.A, respectively. Total plasma estrogen concentrations of natural and induced estruses were not significantly different. The least squares means of plasma estrogen concentrations for the 2 types of estrus were 13.13 and 13.41 pg/lOO/.A, respectively.
Theriogenology
637
Plasma Progesterone
Concentrations
Plasma progesterone concentrations of less than 1 ng/ml were obtained from mares when in either estrus or anestrus. Diagnosis of whether mares were in estrus or anestrus was made by assessing sexual receptivity and by palpation of the ovaries, cervix and uterus. Mares in diestrus had plasma progesterone concentrations of 1.8 to 10 ng/ml. Seasonal
Incidence
and Length of Estrus
The total number of estruses studied was 99. Of these, 61 were naturally occurring and 38 were induced with prostaglandin. The mean length of all estruses over the year was 7.6 days (SEM =0.3). The mean lengths of natural and induced estruses were 7.5 days (SEM=0.4) and The means and standard errors for 7.6 days (SEM =0.4), respectively. the length of natural and induced estruses within mares are presented in Table 2.
Table 2.
Means and standard errors for the induced estruses within mares
Mare
length
and
Induced estrus
Natural estrus No. estruses oer mare
of natural
Length (days) Mean
SEM
No. estruses oer mare
Length (days) Mean
SEM
1
10
7.7
0.4
5
6.8
0.8
2
10
7.5
1.1
7
5.6
0.9
3
11
6.2
0.6
5
8.2
1.7
4
7
7.3
0.9
6
7.5
0.8
5
7
9.0
1.6
5
9.0
0.4
6
9
8.7
1.5
5
7.8
1.5
7
7
6.7
0.9
5
9.2
1.5
There were 5 occasions involving 3 mares when the length of an induced estrus was 3 days or less. Four of these short estruses occurred between March and May (autumn). There were 9 induced estruses Four of these involving 5 mares which lasted for 10 or more days. occurred in February and March while 5 occurred in September and October (spring). The seasonal and monthly estruses are presented in Table 3.
distributions
of natural
and
induced
Theriogenology
638
Table 3.
Season
Summer
Autumn
Winter
Spring
Seasonal and monthly and induced estruses
distributions
Month
of natural
Type of estrus Natural
Induced
December
9
0
January
5
0
February
3
7
Total
17
7
March
4
9
April
3
6
May
7
1
Total
14
16
June
7
0
July
4
0
August
5
0
Total
16
0
September
6
4
October
3
6
November
5
5
14
15
Total
The lowest incidence of estrus occurred around the time of the winter solstice (June 22) in the Southern Hemisphere. There was also a decrease in the occurrence of estrus in January, which was attributed to 3 periods of prolonged diestrus, due to the presence of persistent corpora lutea in 3 of the 7 mares. The length of natural estrus in winter (Mean = 9.3 days; SEM =0.8) was significantly longer than the length of natural estrus in summer (Mean = 6.6 days; SEM =0.5; P
7
Spring
a,b
7
6
7
Autumn
Winter
Spring
Frequencies within columns test of dependence).
7
No. of mares
distribution estrus
different
14
16
14
17
different
of natural
superscripts
1ac
9b
7bc
0a
Anovulatory estruses
paired
different
7
0
7
6
(P
15
0
0.7
0.9
1.0
SEM
(days)
Chi-square
3b
0ab
1oa
2ab
Anovulatory estruses
8.0ab
6.6a
7.6ab
estrus
16
7
Length Mean
estrus
estruses
No. of estruses
Induced
estruses
t-test).
15
0
16
7
No. of estruses
No. of mares
anovulatory
(P
7
0
7
6
No. of mares
Induced
and prostaglandin-induced
are significantly
and prostaglandin-induced
No. of estruses
Natural
with
0.7
0.8
1.2
0.5
SEM
(days)
length
are significantly
8.2ab
9.3b
7.5ab
6.6a
Mean
Length
of natural
superscripts
14
16
14
17
No. of estruses
Summer
Season
Seasonal
different
6
Winter
with
7
Autumn
Means
7
Summer
Table 5.
ab
No. of mares
for the
estrus
errors
Natural
Means and standard per mare per season
Season
Table 4.
s 0 6 9
G
2 s *.
640
Theriogenology
Anovulatory
Estrus
Of the 99 estruses observed, there were 32 in which ovulation Anovulatory estrus occurred in 17 of the 61 natural failed to occur. estruses and in 15 of the 38 induced estruses. The seasonal distribution of anovulatory estrus, whether natural or induced, is presented in Table 5. Significantly more natural anovulatory estruses occurred during autumn and winter than during summer (P
Table 6.
Mare
Occurrence of anovulatory between seasons Summer
Autumn
estrus
between
Winter
mares
and
Spring
No. of estruses
A0
No. of estruses
A0
No. of estruses
A0
No. of estruses
A0
la
3
1
4
4
4
4
4
1
2ab
5
1
5
4
3
3
4
1
3b
4
0
4
2
4
0
4
0
qb
2
0
5
0
2
1
4
0
5ab
3
0
5
3
0
0
4
1
gab
4
0
4
2
1
1
5
1
7b
3
0
3
2
2
0
4
0
A0 a.b
Occurrence of anovulatory estruses. Mares with different superscripts had significantly different occurrences of anovulatory estruses (P < 0.05; Chi-square test of dependence).
641
Theriogenology
There were no differences between the lengths of the ovulatory and anovulatory estruses within seasons (P~0.05). The length of ovulatory estrus in spring was significantly longer than that in summer (P
Table
7.
Means and standard errors for the length ovulatory estruses per mare per season
Season
of anovulatory
and
Type of estrus Ovulatory
Anovulatory No. of mares
Length
(days)
Mean
SEM
No. of mares
Length
(days)
Mean
SEM
Summer
2
4.5
0.5
7
7.1a
0.3
Autumn
6
6.2
1.2
6
6.7 ab
0.7
Winter
4
10.1
1 .o
3
7.8 ab
0.2
Spring
4
7.0
1.8
7
8.1b
0.5
0
Length
Means with different (P-CO.05; paired t-test).
superscripts
are
significantly
different
of Diestrus
There were 58 natural diestrous periods during the study. Five of these were greater than 40 days in length and were regarded as prolonged, due to the presence of a persistent corpus luteum. One diestrus lasted only 10 days, due to the occurrence of an acute but transient endometritis and for this reason was not included in the analysis. The influence of season on the mean length of diestrus per mare is presented in Table 8. Only diestrous periods of between 10 and 30 days were included in the data. The overall mean length of diestrus was 16.6 days (SEM = 0.3; n = 50). Season had no significant influence on the length of diestrus (P>O.O51. There were fewer periods of diestrus in summer than in the other seasons, due to the occurrence of persistent corpora lutea in 3 of the mares at this time. There were 5 diestrous
Theriogenofogy
642
periods which lasted from 20 to 24 days. July to October.
Table 8.
Season
Means and standard per season
No. of diestrus
Four of these occurred
from
errors of the length of diestrus per mare
No. of mares
Length (days) Mean
Summer
11
5
16.1
0.6
Autumn
15
7
16.7
0.4
Winter
13
5
16.8
0.6
Spring
13
7
15.5
0.5
The differences in the lengths of diestrus between mares were not significant when diestrous periods of 10 to 30 days were analyzed (P>O.O5). However, when the prolonged diestrous periods were included, significant differences in the mean length of diestrus between mares were obtained (P
Theriogenology
643
The mean estrous cycle length in winter was significantly greater than that in autumn (P
Table 9.
Means and standard errors for the length of normal and total diestrous periods (including prolonged ones1 in mares Classification Normal n
Mare ID
of diestrous period Total (normal plus prolonged)
Length (days) Mean
SEM
n
Length (days) Mean
SEM
la
9
16.6
1.0
10
20.aCd
4.3
2
9
17.4
1.0
9
17.4d
1.0
3
10
16.6
0.7
10
16.6d
0.7
4a
6
16.5
0.8
7
21.4Cd
5.0
5
7
17.3
0.8
7
17.3d
0.8
6
8
15.5
0.6
8
15.5d
0.6
7a,b
3
15.3
1.3
6
41 .oc
13.7
a b cd
Winter
Indicates the presence of a persistent corpus luteum during January. This mare had a persistent corpus luteum on 3 occasions and 1 short diestrus of 3 days duration. Means with different superscripts are significantly different (P < 0.05; t-test).
Anestrus
Four mares continued to cycle during winter while 3 mares (Mares 4, 5 and 6) were in anestrus for 59, 114 and 88 days, respectively. diestrus before the onset of true These mares entered a period of
Theriogenology
644
anestrus. Because of the high incidence of anovulatory estrus, anestrus was assumed to commence on the final day of the last estrus instead of on the day of the final ovulation of the season. Anestrus was assumed to terminate at the first ovulation of the following breeding season. This definition was used since only 3 of the 7 mares entered anestrus, and they failed to ovulate during the penultimate estrus of the breeding season. Two of these mares ovulated during their final estrus, while the other mare failed to ovulate during her final estrus. Progesterone concentrations of the mares were initially consistent with those of diestrous levels and then dropped to between 0.1 ng/ml and 0.3 ng/ml until the first ovulation of the next breeding season. Mares 5 and 6 failed to ovulate during their last estrus before the onset of anestrus.
Table 10.
a,b
Means and standard per mare per season
errors of the length of estrous
cycles
Season
No. of cycles
No. of mares
Summer
12
5
22.5ab
1.1
Autumn
17
7
24.2a
0.7
Winter
11
4
25.5b
1 .o
Spring
12
7
22.3ab
0.6
Means with different (P < 0.05; paired t-test).
superscripts
Cycle length (days)
are
significantly
different
Mare 5 was passive when teased until the first large follicle developed in spring. Then she displayed erratic sexual receptivity (Scores 2, 3 and 4) until the first ovulation of the breeding season. Mare 6 remained passive in the presence of the teaser until her first follicle developed in spring, when she exhibited overt sexual receptivity (Score I). Mare 4 exhibited overt sexual receptivity (Score 2) on 4 occasions that were randomly scattered throughout winter, when both ovaries were less than 4 cm in length. Following development of the first large follicle of the breeding season, she displayed positive signs of estrus (Score 1) which continued until ovulation. Anestrus terminated in the 3 mares with an ovulation within 2 weeks of each other in September. These 3 mares showed overt sexual receptivity for extended periods of 10, 11 and 18 days, respectively (spring estrus) before entering the ovulatory season. Table 1 1 presents data on the duration of the anestrous period.
Theriogenology
645
Mares 2 and 7 displayed anestrous behavior during July and August. However, Mare 2 had progesterone concentrations that were indicative of cyclical activity during winter but she did not ovulate until September. Both ovaries were at their smallest size (4 cm) in July and her peak progesterone concentration during winter was 2.8 ng/ml, which was well below that of her other diestrous concentrations (10.0 ng/ml) during the study. Another mare (Mare 7) was in prolonged diestrus from July 7 until August 20. Her plasma progesterone concentrations were elevated during this time and ranged from 1.7 ng to 10.0 ng/ml. She failed to ovulate during this period and her ovaries were only 4 cm in length. Table 12 presents progesterone and ovarian data on these mares over the winter season.
Table 11.
Duration of anestrus in 3 mares
Characteristics of winter anestrus
Mare 4
Mare 5
Mare 6
Start of anestrus
July 16
June 5
June 16
Time from last estrus to low progesterone concentration (< 1 .O ng/ml)
17 days
15 days
18 days
Appearance
Sept 6
Aug 18
Aug 9
Time from low progesterone concentration to the first estrus
31 days
81 days
60 days
Length of the first estrus
11 days
18 days
10 days
Date of the first ovulation
Sept 13
Sept 27
Sept 12
Length of anestrus
59 days
114 days
88
Table 12.
Mare
2 7
of first follicle
days
Plasma progesterone concentrations and ovarian data from 2 mares displaying anestrous behavior during July and August Progesterone secretion pattern
Peak progesterone concentration (ng/ml)
No. of ovulations
Ovarian length (cm)
Cyclical
2.8
0
4
10.0
0
4
Persistent
CL
Theriogenology
646 DISCUSSION
The results of this study show that the intensity of sexual receptivity is repeatable within individual mares, which supports the findings of McKenzie (19) and Hillman and Loy (20). Silent estrous behavior has been associated with estruses induced with prostaglandin (21-23). This was not apparent in these mares since the intensity of sexual receptivity within the mares was not affected by prostaglandin treatment. The incidence of silent estrous behavior in pony mares was reported to be 7% (1, 15, 24). Four of the 7 mares in our study frequently had silent estruses with indifferent sexual receptivity. Group teasing by a single stallion was not adequate for assessing these mares so they had to be teased individually and their ovaries palpated to detect estrus. The lack of normal signs of estrus in these mares was unrelated to prostaglandin treatment. No consistent relationship was found between total plasma estrogen concentrations of the mares and their sexual receptivity scores. These results conflict with those of Nelson et al. (23) but agree with those of Munroe et al. (251, who found no direct association between the intensity of sexual receptivity and either plasma estrogen or plasma androgen concentrations in the mare. Ganowicz (26) found no relationship between intensity of sexual receptivity and follicular development in mares. It is possible that mares with a low sexual receptivity score of 5 (indifferent) might have responded more strongly if the teasing time was extended or if they were permitted to be mounted by a teaser stallion (27). Dominance hierarchies and the social interactions at the time of puberty may be important factors for investigation when determining why some domesticated mares fail to exhibit normal sexual receptivity during estrus. A study of New Forest ponies showed that mutual grooming was part of the mating ritual between the stallions and young mares, which even though they were in estrus, often showed signs of fear. Mutual grooming did not occur between the stallions and the older mares (28). If these conditions could be simulated on equine stud farms the incidence of silent estrus could possibly be reduced. However, the risk of injury and the spread of venereal diseases by teaser stallions would have to be considered before such a practice was initiated. The mean length of naturally occurring estruses increased as the photoperiod decreased during the transition from summer to winter, which is similar to the results of other studies (1, 15, 29, 30). It is widely accepted that the length of estrus early in the season is prolonged
Theriogenology
647
(spring estrus) and shortens as the summer solstice approaches. The length of estrus in spring in our study (Mean = 8.2 days; SEM = 0.7) was greater than that in summer (Mean =6.6 days; SEM =0.5) but this difference was not significant (P>O.O5). This result was probably due to the number of mares that continued to cycle throughout the year. Mares in the other studies, except for that of Hughes et al. (I), had estruses that decreased in length as summer approached. These findings may be due to the relatively small difference in the photoperiod in the sub-tropics as compared with that at higher latitudes. Thirty-eight percent of the estruses of the mares in this study were induced by prostaglandin injections. However, there was no significant difference between the mean length of prostaglandin-induced and natural estruses. These findings are similar to those of Douglas and Ginther (31) but differ from those of Oxender et al. (32) and of Burns et al. (33), who reported that prostaglandin-induced estruses were significantly longer than natural estruses. Nelson et al. (23) on the other hand, found that induced estruses were shorter than those of control mares. The mean length of diestrus in mares in our study was 16.6 days, which is similar to that of other data (1, 34). There was no significant difference between the lengths of diestrus due to season of year. The coefficient of variation for the duration of estrus (38%) was more than twice that for the duration of diestrus (15%). These findings are similar to those of Hughes et al. (l), Back et al. (34), Ginther et al. (35) and Greenhoff and Kenney (36). Thus the relative uniformity of the length of diestrus between seasons appears to be a constant feature of the estrous cycle of mares in both hemispheres and at different latitudes. There were 5 periods of diestrus, ranging from 20 to 24 days during winter and spring, which were not regarded as prolonged. There were another 5 diestrous periods of 43 to 90 days duration which were due to the presence of persistent corpora lutea. The average duration of prolonged diestrous due to persistent corpora lutea was reported to be 2 Neely (37) and Neely et al. (38) demonstrated that months (12, 27). either a diestrous ovulation or the release of insufficient prostaglandin to cause complete luteolysis was the likely cause of persistent corpora lutea in the mare. The definition of the estrous cycle in this study included the possible occurrence of an anovulatory estrus. Even so, the mean estrous cycle length of the mares over all seasons was 23.7 days, which is similar to the composite mean of data (21.7 days) collated by Ginther (27) and the mean of his own data (22.7 days).
640
Theriogenology
A ‘relatively low occurrence of normal sexual receptivity was recorded in our study due to the prolonged diestrous periods in 3 of the mares. However, a seasonal trend was apparent in the length of estrous This cycles, with longer cycles occurring in winter than in summer. difference in estrous cycle length was due to seasonal differences in the lengths of the actual estrus, as was shown by several other workers (1, 30, 35). Hammond (8) and Gonzalez and Valencia (9) suggested that mares This was not in tropical regions had 2 breeding seasons per year. apparent in the mares in our study. They were found to be polycyclic as were mares in a study in the tropics (39). Most of the mares in our study continued to display estrus during autumn and winter but most of these estruses were anovulatory. Both studies showed a relationship between frequency of ovulation and season of year, with fewer ovulations occurring in winter. The occurrence of anovulatory estrus in the mare has been In our study there were 6 anovulatory reported to be rare (1, 15, 40). estruses involving 4 mares during the ovulatory season, while in autumn and winter, 26 of 46 estruses were anovulatory. However, this was more common in 2 of the mares. Ginther (27) stated that mares are very dependable ovulators, but that anovulatory estrus, which occurred most commonly in the transition from the ovulatory to the anovulatory season, was due to insufficient LH to stimulate final development of the preovulatory follicle. The proportion of mares in this study that continued to display estrus during winter was twice that reported by Osborne (2), Loy (41) and Saltiel et al. (39). Sharp (42) defined anestrus as the period when less than 25% of mares ovulated. The results of the present study, with only 42% of the mares not cycling and a high proportion of anovulatory estruses during winter, support Sharp’s definition of winter anestrus. However, Sharp’s (42) concept that the number of mares cycling is indicative of the number of mares ovulating was not applicable in this study, possibly due to the reduced variation in photoperiod at this latitude. Failure of the preovulatory follicle to develop and insufficient LH were found to be the reasons for ovulatory failure at the onset of anestrus in ponies (43). It has been shown that as winter approaches the ovulatory season in ponies ceases more quickly than in horses, and that while no ponies ovulate during winter, up to 25% of horses continue to do so (15, 44). It is possible that the hypothalamus of the horse is less sensitive to melatonin than that of the pony or that the pineal of the horse is less sensitive than that of the pony to changes in photoperiod.
Theriogenology
649
The low proportion of winter anestrus in the mares of our study may have been due to the relatively small changes in photoperiod which did not stimulate sufficient melatonin secretion to inhibit GnRH secretion. This may account for the number of mares which displayed estrus during winter and why there were more anovulatory estruses in autumn and winter than in spring and summer. It has been shown by Asa et al. (45) that it is common for pony mares to exhibit sexual receptivity during the anovulatory season, and, in fact, ovariectomized pony mares also display sexual receptivity. It was suggested by Asa et al. (46) that the adrenal gland, which is a source of estrogens and androgens, may be responsible for unseasonal sexual receptivity. This was confirmed by suppression of sexual receptivity in ovariectomized mares using dexamethasone.
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