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Timing of the onset and duration of ovulation in superovulated beef heifers
THERIOGENOLOGY
TIMING
OF THE ONSET
AND DURATION
M.C. Yadav,' 1
OF OVULATION
J.S. Walton,'
IN SUPEROVULATED
BEEF HEIFERS
and K.E. Leslie'
Depa;tment of Animal and Poultry Science Department of Clinical Studies University of Guelph, Guelph, Ontario
Received
for publication: Accepted:
December 31, 1985 August 22, 1986
ABSTRACT Thirty-two beef heifers were induced to superovulate by the administration of follicle stimulating hormone-porcine (FSH-P). All heifers received 32 mg FSH-P (total dose) which was injected twice daily in decreasing amounts for 4 d commencing on Days 8 to 10 of the estrous cycle. Cloprostenol was administered at 60 and 72 h after the first injection of FSH-P. Heifers were observed for estrus every 6 h and were slaughtered at known times between 48 to 100 h after the first cloprostenol treatment. The populations of ovulated and nonovulated follicles in the ovaries were quantified immediately after slaughter. Blood samples were taken at 2-h intervals from six heifers from 24 h after cloprostenol treatment until slaughter and the plasma was assayed for luteinizing hormone (LH) concentrations. The interval from cloprostenol injection to the onset of estrus was 41.3 % 1.25 h (n = 20). The interval from cloprostenol injection to the preovulatory peak of LH was 43.3 _C 1.69 h (n = 6). No ovulations were observed in animals slaughtered prior to 64.5 h after cloprostenol (n = 12). After 64.5 h, ovulation had commenced in all animals except in one animal slaughtered at 65.5 h. The ovulation rate varied from 4 to 50 ovulations. Approximately 80% of large follicles (> 10 mm diameter) had ovulated within 12 h of the onset of ovulation. Onset of ovulation was followed by a dramatic decrease in the number of large follicles (> 10 mm) and an increase in the number of small follicles (< 5 mm). These data indicate that ovulations in superovulated beef heifers occur over 12 h and commence approximately 24 h after the onset of estrus and 22 h after the peak of LH. Key words:
cattle,
superovulation,
timing
of ovulation,
estrus,
plasma
LH
INTRODUCTION A major estrus under optimum time animals came
problem faced by animal breeders is accurate detection of field conditions so that insemination can be performed at the for conception. In cows induced to superovulate, not all the into estrus,and a fairly large proportion of ovulations
Acknowledgements: We acknowledge the excellent technical assistance of C. Watson, C. Haworth, G. Werchola, and W. Szkotnicki. Financial assistance was provided by the Canadian Commonwealth Scholarship Plan (MCY), the Natural Sciences and Engineering Research Council of Canada (A7165), and the Ontario Ministry of Agriculture and Food.
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occurred silently (1, 2). For these reasons, and also because of the suggestion that ovulations are spread over time (3), predicting the optimum time to inseminate a superovulated cow has been difficult. It is often assumed that to obtain adequate fertility, repeated inseminations are required at approximately 12-h intervals around estrus. Some investigators (4) have even used two doses of semen at each insemination time. Few reports describe the timing of ovulation in superovulated cows. Maxwell et al. (3) reported that in cows superovulated using pregnant mare serum gonadotrophin (PMSG), 45 and 91% of ovulations were observed at 24 and 48 h after the onset of estrus, respectively. No ovulations were recorded in the first 18 h. Angel (5) used laparoscopy in cows treated with PMSG and follicle stimulating hormone (FSH) and reported that ovulations were spread over 24 h or more. Shea et al. (6) reported that in cows superovulated with PMSG, ovulations commenced as early as 12 h after estrus was first observed. Although PMSG has been widely used as superovulatory gonadotrophin, FSH-P is normally used in commercial embryo transfer units in North America. The onset and duration of ovulation after the use of this drug are not known precisely. The objective of this experiment was to determine the onset and duration of ovulation in heifers superovulated with FSH-P in relation to the timing of the LH surge so that an optimum time and number of inseminations can be suggested to the embryo transfer industry. MATERIALS
AND METHODS
Estrus was synchronized in batches of 10 beef heifers, 15 to 18 mo old, using a luteolytic dose of cloprostenol (Estrumate, ICI Pharma, Mississauga, Ontario, 500 ug). Within each batch, three to five heifers in which estrus was observed were chosen for superovulatory treatment. The remaining animals were treated with another luteolytic dose of cloprostenol 11 d after the first dose and were superovulated similarly. In this way a total of 32 heifers (26 Hereford and 6 crossbred) were programmed to be on Days 8 to 10 of the estrous cycle and induced to superovulate by the administration of FSH-P (Schering, Montreal, Canada Inc., Lot 574M82). These animals were housed in pens (five heifers in a pen, 2.5 x 6 m) and were fed 69% corn silage, 30% high-moisture corn, and 1% mineral mixture ad libitum. All heifers received identical treatment and were injected with the following doses of FSH-P at 12-h intervals: 5 mg, 5 mg; 4 mg, 4 mg; 4 mg, 4 mg; and 3 mg, 3 mg. The FSH-P was diluted with physiological saline to 5 ml and stored in 6-ml syringes at -18'C. Immediately prior to each injection, the contents of the syringe were allowed to thaw at room temperature and injected. Cloprostenol (500 ug) was administered 60 and 72 h after the first injection of FSH-P. Heifers were observed for estrus every 6 h and were slaughtered at known times between 48 and 100 h after the first prostaglandin treatment. Surviving animals were also inseminated at 60 h after the administration of cloprostenol. All animals were slaughtered at the abbatoir of the Department of Animal and Poultry Science, University of Guelph. The reproductive organs were recovered within 30 min of slaughter and the position of the regressed corpus luteum (CL) and the populations of ovulation sites and nonovulated follicles were quantified. Ovaries were photographed and the diameters of individual nonruptured follicles were measured using a stainless steel Follicles were classified as small (< caliper (CanLab, Toronto, Canada). - 5
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mm), medium
(6 to 10 mm), large (> 10 mm), and medium and large combined (> 6 mm). Blood samples (5 ml) were collected into heparinized vacutainers for the measurement of LH and progesterone from six heifers by jugular venepuncture at 2-h intervals from 24 h after cloprostenol treatment until slaughter. All blood samples were centrifuged immediately and the plasma was harvested and stored at -2O'C until assayed. Hormone
Analysis
Concentrations of LH were determined using a specific radioimmunoassay (7). NIAHDD-bLH-4 (2.4 x NIH-LH-Bl) was used as the assay standard. All the results were then corrected to SIH-LH-Bl. The sensitivity of the assay was 0.120 ng/ml. Interassay and intraassay coefficients of variation were 10.7 and 1.9% respectively. The onset of the preovulatory surge of LH was defined as the time when LH exceeded X + 2 SD, where X was the mean of 5 to 10 values preceding the onset of the surge, determined visually. The peak concentration was the maximum recorded concentration within the profile of an individual cow. Progesterone concentrations were measured in 200-~1 aliquots of plasma by an extraction and charcoal-separation radioimmunoassay (8). The sensitivity of the assay was 0.25 ng/ml, and interassay and intraassay coefficients of variation were 6.4 and 2.6% respectively. For statistical analysis the data were divided into two. One set (n = 12) included data until the onset of ovulation (prior to 64.5 h after cloprostenol) and the other set (n = 20) contained data after ovulations had commenced (after 64.5 h). In an attempt to account for variability in superovulatory response between individuals, the number of ovulations was expressed as a percentage of the number of large unovulated follicles plus the number of ovulations. Similarly, proportions of follicles of different sizes were expressed in relation to either the number of unovulated follicles or unovulated follicles including ovulations. The raw data and proportions obtained were subjected to either logarithmic (base e) or arcsine transformation to obtain homogeneous variances. After arcsine transformation the data of percentage ovulation were subjected to nonlinear regression using a computer programme analysis as outlined in the biomedical statistical analysis package (9). Linear regression analyses were performed on other data using the general linear model procedure of the Statistical Analysis System (10). RESULTS Only 62.5% of the heifers were detected in estrus during superovulation (stood to be mounted). The interval from cloprostenol injection to the onset of estrus in these animals was 41.3 k 1.25 h (n = 20). The interval from cloprostenol injection to the preovulatory peak of LH and the mean LH concentration (_+ SE) at the peak were 43.3 f 1.69 h (n = 6) and 90.2 _t 21.14 ng/ml (n = 6) respectively. Ovulations were not observed in animals slaughtered prior to 64.5 h after cloprostenol administration (Table 1). After this, ovulated follicles were present in all the animals except two (one at 64.5 h and another at 65.5 h). A comparison between ovaries within heifers indicated that neither ovulation rate nor number of unovulated follicles of different
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rHERIOGENOLOGY
Table 1.
Number of ovulations and nonovulated follicles in superovulated heifers slaughtered at different times after prostaglandin injection
Animal number
Time of slaughter after prostaglandin inj. (h)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
48.00 49.00 50.00 50.75 52.50 60.00 60.50 60.75 62.00 62.00 62.50 62.50 64.50 64.50 65.25 66.00 67.00 69.50 72.50 73.00 74.00 74.50 76.00 84.25 84.50 86.00 86.75 88.50 96.25 97.00 97.75 100.50
Follicles Follicles Follicles Follicles with > > 6- to lo-mm ovulatory Ovulations 5-m; dia. dia. lo-mm dia. stigma
0 0 0 0 0 0 0 0 0 0 0 0 0
4 0 29 3 31 4 51 24 2 19 17 15 22 18 15 37 16 50 18
2 0 8 3 1 11 1 11 27 0 6 8 11 3 2 12 2 17 6 26 7 6 3 22 5 12 22 20 7 14 29 10
15 5 47 24 8 13 8 19 12 L
28 2 8 3 1 11 6 16 20 2 0 4 4 10 4 2 41 8 9 5 14 4
18 10 10 19 5 20 13 41 20 38 31 19 8 18 7 2 17 6 5 2 1 8 3 1 2 14 6 5 2 1 1 1
; 0 0
1 8 1 1 1 2 1 5 1 0 0 0 1 3 1 0 2 0 0 0 0 0 0
sizes were significantly (P > 0.05) affected by the presence of the CL of the previous cycle on the ovary. After the initiation of ovulations, the logarithm number of ovulations increased linearly [ln (ovulation + 1) = -1.68 + (0.53 x time), P < 0.051 with time. Five, ten, and twenty ovulations occurred by approximately 66, 77 and 89 h after closprostenol injection respectively.
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Ovulatory stigma were observed on the surfaces of large follicles: the number of stigma varied from one to eight in animals slaughtered between 60 to 86 h after the treatment with cloprostenol (Table 1). The number of follicles with ovulatory stigma was not correlated either with ovulation The population of rate or the populations of small and medium follicles. large follicles was, however, positively correlated with the number of ovulatory stigma (r = 0.33, P = 0.07). Ovulation rate was highly variable. As many as 51 ovulations occurred in one individual (Table 1). The nonlinear regression analysis of transformed data when ovulations were expressed as a percentage of unovulated follicles of different sizes (medium + large and large) or as a percentage of total unovulated follicles, including ovulations, revealed a curvilinear relationship of ovulations with time. Re ression equations p < 0.05; were arcsine square root (% ovulations) = 0.96 (l-e O.H9(t), arcsine square root (% ovulations) = 1.24 (1-e-O*17(t)), P : 0.05; and arcsine square root (% ovulations) = 0.76 (1-e-O*21(t)), P < 0.05, for ovulation as a percentage of medium + large, large, and total unovulated follicles, respectively. Ovulations predicted over time were restored to an untransformed scale. A large proportion of follicles had ovulated by 85 h. After this time only a small increase in the proportion of follicles ovulating (approximately 1%) was observed. The overall shape of the ovulation curves remained the same when all three size classes of follicles were used as denominator except for differences in the plateau of proportion of ovulating follicles. This was highest when ovulations were represented as a percentage of the large follicles followed by the curves drawn from medium + large and total unovulated follicles. Considering only large follicles as potentially ovulatory follicles, approximately 80 and 88% of these follicles haa ovulated within 12 and 22 h, respectively, after the onset of ovulations (Figure 1). At the same times, 60 and 66% of medium + large follicles and 43 and 47,, g of total follicles had ovulated. During the preovulatory period (48 to 64.5 h), the number of large follicles increased significantly [In (follicle) = -0.55 + (0.06 x time), P < 0.051 and after the onset of ovulations (64.5 h) decreased significantly (follicle) = 4.98 + (-0.05 x time), P < 0.011. The number of small Iln follicles increased [In (follicle) = 0.15 + (0.03 x time), P < 0.051 after the onset of ovulations. No significant relationships between the number of other sized follicles with time could be established during either period. When follicles > 6 mm in diameter were expressed as a percentage of total unovulated follicles, their proportion decreased (P < 0.05 and P < 0.01) during ovulation (Figure 2). During this period, however, the increase in the numbers of small follicles was significant (P < 0.05). In the preovulatory period, the follicles of the middle class decreased significantly (P < 0.01). When follicles of different sizes were expressed as a percentage of unovulated plus ovulated follicles, similar changes in the population of follicles were apparent, except no change in the proportion of small follicles was observed during ovulation period (Figure 3). Plasma concentrations the time of slaughter were
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of progesterone measured in samples collected low (0.4 _ + 0.03 ng/ml) in all the animals.
1986 VOL. 26 NO. 4
at
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THERIOGENOLOGY
o45
1::::::::::::::::::m:::::~
55
64 Hours
72 after
50
55
96
104
prostaglandln
Figure 1. The proportion of large follicles ovulating in superovulated beef heifers with time after prostaglandin. Ovulations were expressed as a percentage of total large follicles (> 10 mm) including ovulations.
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2.0
1.8
1.6
64
Hours
72
after
80
88
prostaglandin
Figure 2. Relationship of numbers of follicles of different size with time when expressed as percentage of total unovulated follicles. Nonovulatory and ovulatory periods are separated by the vertical line. The proportion of medium follicles decreased before the onset of ovulation Iarcsine square root (% follicles) = 2.09 t (-0.03 x time), P < 0.011. During ovulation the proportion of medium t large tarcsine square root (% follicles) = 1.56 + (-0.01 x time), P < 0.051 and large [arcsine square root (% follicles) = 1.56 t (-0.01 x time), P < 0.011 follicles decreased significantly. An increase in the proportion of small follicles was observed [arcsine square root (8 follicles) = 0.01 t (0.01 x time), P < 0.051.
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THERIOGENOLOGY
2.0 1.8
.2
a14
4,(I
4
56
84
Hours
72
after
II8
80
__.
prostaglandin
Figure 3. Relationship of numbers of follicles of different sizes with time when expressed as percentage of total unovulated follicles and ovulations. Nonovulatory and ovulatory periods are separated by the vertical line. The proportion of medium follicles decreased before the onset of ovulations [arcsine square root ($ follicles) = 2.09 t (-0.03 x time), P < 0.051. During ovulation the proportion of medium t large [arcsine square root (% follicles) q 1.63 t (-0.01 x time), P < 0.051, large [arcsine square root (8 [arcsine follicles) = 1.51 t (-0.01 x time), P < 0.011, and all follicles square root ($ follicles) = 1.94 t (-1.01 x time), P < 0.051 decreased significantly.
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DISCUSSION The peak of the preovulatory surge of LH, which occurred 43.3 + 2.45 h after cloprostenol injection, is similar to other reports for cows superovulated using FSH-P (11-13). In contrast to a previous report which demonstrated that in superovulated animals the onset of estrus may occur considerably earlier than the LH surge, or well after the LH surge (14), estrus was first observed in this experiment close to the time of the peak of LH. The preovulatory increase in concentrations of estradiol triggers the onset of LH surge (15); an early buildup of this hormone in superovulated cows, compared with those that are not superovulated, must contribute to a more rapid onset of estrus and the associated LH surge (11, 16). After 21 h from the LH surge, ovulations had commenced in all the animals. In earlier reports (6, 17), it had been speculated that in superovulated animals ovulations occur earlier than in the normally cycling cow vith respect to onset of the estrus. However, the time of onset of ovulation after the onset of estrus and the LH surge closely coincided with the interval of 24 h reported for the normally cycling cow (18-20). In two animals that had not ovulated by 64.5 and 65.5 h after cloprostenol treatment, the LH surge may have occurred later than in those animals that had ovulated by this time. This opinion is supported by the observation that in Animal 15 (Table l), which had not yet started to ovulate by 65.25 h, the LH surge occurred 4 h later than that of Animal 14 (Table l), which had four ovulations by 64.5 h. Ovulatory stigma on the apex of one to two large follicles were observed in both the animals. It is known that stigma formation occurs about 1 h before ovulation and is a precise indicator of ovulation (20). Most likely, therefore, the majority of large follicles in the ovaries of both these animals would have ovulated within a few hours had the animals not been slaughtered. The number of ovulations per animal increased over time. Sixteen to twenty oocytes per animal, which have been said to be optimum (17), were released within 24 h of the initiation of ovulation. The ovulation rate in most of these animals, however, was slightly higher than reported by others (14, 21). In these reports, however, rectal palpation was used to evaluate the ovulatory response. This method is not precise and tends to underestimate ovulation rate (22). In other experiments, however, a similar range in ovulation rate (14 to 33 ovulations) has been reported in heifers treated with 32 mg FSH when the population of CL was also estimated directly from the ovaries at the time of slaughter (23). Assuming that follicles of > 10 mm diameter are almost certain to ovulate, as is the case in the normally cycling cow (24), 87 to 88% of ovulations would be expected to occur within a short period (22 h; Figure 1). The percentage of ovulation increased from 12% at the time of initiation to 80% within 12 h, followed by a smaller increase (about 8%) over the next 10 h. The assumption regarding the size (> 10 mm) of the ovulatory follicle is substantiated by the observation that there was a significant increase in numbers of these follicles during the preovulatory period followed by a dramatic decrease after the initiation of ovulation. The occurrence of a small proportion of ovulations after 12 h of initiation leads us to suggest that these follicles were either less mature
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THERIOGENOLOGY
or unable to respond normally to the LH surge. This finding may also indicate that the terminal stages of growth in medium-sized follicles was inhibited by other dominant, estrogen-active follicles that have now ovulated. A large dominant follicle with increased vascularity has the best opportunity to benefit from the preovulatory gonadotrophin surge (25). It appears, therefore, that once the large dominant follicles had ovulated, growth and maturation could occur in medium-sized follicles. Increased availability of FSH to these follicles following the rupture of large follicles probably stimulates synthesis of plasminogen-activator by granulosa cells and results in the production of ovulatory enzymes. This effect may be sequential due to the existence of a follicular hierarchy in superovulated animals and might explain delayed ovulations in medium-sized follicles and, therefore, a slight spread in the time of ovulation. During the estrous cycle in the cow, follicles are recruited from a nongrowing pool and commence development at privileged periods (26). Thereafter, the follicle grows continuously until either ovulation or atresia (27, 28). Although antrum formation and the population of normal antral follicles remain unchanged, the number of atretic follicles decreased when Friesian heifers were induced to superovulate (29). Superovulation either prevents follicles from becoming atretic or rescues some early atretic follicles from atresia (29). It is likely, therefore, that the number of medium to large and total unovulated follicles at the surface of ovaries stimulated with exogenous gonadotrophin are determined both by the influx from continuous follicular growth and the outflow from ovulations. Under these circumstances, the prediction of the number and duration of ovulations using the population of medium to large or total unovulated follicles will not be very precise. A reduction in the number of medium-sized follicles during the preovulatory period suggests that during this period they grew into large follicles (Figures 2 and 3). Since the population of small follicles was increasing and the population of large follicles was decreasing (Figures 2 and 3) during the ovulatory period, the concept that the large follicles inhibit growth of small follicles (28) appears to be confirmed. The absence of a relationship between the number of small follicles with time when the number of large follicles increased significantly during preovulation further supports this hypothesis (Figures 2 and 3). A reduction in the proportion of small follicles during ovulation was not detected when these follicles were represented as a percentage of total unovulated follicles including ovulations because the denominator was confounded with the increasing number of small and decreasing number of large follicles. Plasma progesterone concentrations were the same in animals that had ovulated, probably because the CL were too young to produce measurable amounts of progesterone in the peripheral circulation. Measurement of progesterone in ovarian venous plasma, however, may have indicated an increase in progesterone secretion with time. There was no relationship between ovulation rate and plasma progesterone concentration at slaughter. Given that after initiation of ovulation a large proportion (80%) of ovulations occur within 12 h, that the viable lifespan of spermatozoa is 24 to 48 h, and that capacitation of spermatozoa requires approximately 4 to 6 h (30), a single insemination of superovulated donors with semen from a
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bull of high fertility 60 h after cloprostenol injection should result in satisfactory conception rates. Recent reports suggest that insemination of donors superovulated with FSH-P either once with two straws at 12 or 24 h after the onset of estrus (31,32) or twice with one straw at 12 and 24 h after the detection of estrus (33) resulted in conception rates comparable to multiple inseminations. These results indirectly indicate a short spread of ovulations in superovulated animals and support the findings of this study. From this experiment, therefore, it can be concluded that the biological action of the LH surge requires approximately 24 h to induce ovulation in superovulated cows. Once initiated at 64.5 h after prostaglandin F2 alpha, ovulation of the majority of large follicles occurred within 12 h. Satisfactory conception rates from a single insemination around 60 h after cloprostenol injection would, therefore, be expected. Extensive field trials will, however, be required to test this hypothesis. REFERENCES 1.
Elsden, R.P., Lewis, S., Cumming, I.A. and Lawson, R.A.S. Superovulation in cow following treatment with PMSG and prostaglandin F2a. J. Reprod. Fert. 36~455-456 (1974).
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Greve, T., Henrik, C. and Hyttel, P. Endocrine profiles quality in superovulated cow. Nord. Vet. Med. %:408-421
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Dufour, J.J., Whitmore, H.L., Ginther, O.J. and Casida, L.E. Identification of the ovulating follicle by its size on different days of the estrous cycle in heifers. J. Anim. Sci. %:85-87 (1972).
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Monniaux, D., Mariana, follicular populations (1984).
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Hunter, R.H.F. Mating, sperm transport in the female genital tract, &: Physiology and Technology of and artificial insemination. Hunter, R.H.F. (ed). Academic Reproduction in Female Domestic Animals. Press, NY, 1980, pp. 105-118.
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Schiewe, M.C., Looney, C.R., Hill, K.G., Johnson, C.A. and Godke, R.A. Transferable embryo recovery rates following different artificial Theriogenology 19:147 insemination schedules in beef donor cattle. abstr. (1983).
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Schiewe, M.C., Voelkel, S.A. and Godke, R.A. Artificial insemination (AI) of superovulated beef cattle with a single insemination of frozen Theriogenology g:228 abstr. (1985). semen.
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West, G., West, C., Risely, D. and Donaldson, L. Effect of breeding regime on percent ova fertilized in superovulated cows. Theriogenology g:273 abstr. (1984).
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la vache.
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1986 VOL. 26 NO. 4
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TIMING
OF THE ONSET
AND DURATION
M.C. Yadav,' 1
OF OVULATION
J.S. Walton,'
IN SUPEROVULATED
BEEF HEIFERS
and K.E. Leslie'
Depa;tment of Animal and Poultry Science Department of Clinical Studies University of Guelph, Guelph, Ontario
Received
for publication: Accepted:
December 31, 1985 August 22, 1986
ABSTRACT Thirty-two beef heifers were induced to superovulate by the administration of follicle stimulating hormone-porcine (FSH-P). All heifers received 32 mg FSH-P (total dose) which was injected twice daily in decreasing amounts for 4 d commencing on Days 8 to 10 of the estrous cycle. Cloprostenol was administered at 60 and 72 h after the first injection of FSH-P. Heifers were observed for estrus every 6 h and were slaughtered at known times between 48 to 100 h after the first cloprostenol treatment. The populations of ovulated and nonovulated follicles in the ovaries were quantified immediately after slaughter. Blood samples were taken at 2-h intervals from six heifers from 24 h after cloprostenol treatment until slaughter and the plasma was assayed for luteinizing hormone (LH) concentrations. The interval from cloprostenol injection to the onset of estrus was 41.3 % 1.25 h (n = 20). The interval from cloprostenol injection to the preovulatory peak of LH was 43.3 _C 1.69 h (n = 6). No ovulations were observed in animals slaughtered prior to 64.5 h after cloprostenol (n = 12). After 64.5 h, ovulation had commenced in all animals except in one animal slaughtered at 65.5 h. The ovulation rate varied from 4 to 50 ovulations. Approximately 80% of large follicles (> 10 mm diameter) had ovulated within 12 h of the onset of ovulation. Onset of ovulation was followed by a dramatic decrease in the number of large follicles (> 10 mm) and an increase in the number of small follicles (< 5 mm). These data indicate that ovulations in superovulated beef heifers occur over 12 h and commence approximately 24 h after the onset of estrus and 22 h after the peak of LH. Key words:
cattle,
superovulation,
timing
of ovulation,
estrus,
plasma
LH
INTRODUCTION A major estrus under optimum time animals came
problem faced by animal breeders is accurate detection of field conditions so that insemination can be performed at the for conception. In cows induced to superovulate, not all the into estrus,and a fairly large proportion of ovulations
Acknowledgements: We acknowledge the excellent technical assistance of C. Watson, C. Haworth, G. Werchola, and W. Szkotnicki. Financial assistance was provided by the Canadian Commonwealth Scholarship Plan (MCY), the Natural Sciences and Engineering Research Council of Canada (A7165), and the Ontario Ministry of Agriculture and Food.
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occurred silently (1, 2). For these reasons, and also because of the suggestion that ovulations are spread over time (3), predicting the optimum time to inseminate a superovulated cow has been difficult. It is often assumed that to obtain adequate fertility, repeated inseminations are required at approximately 12-h intervals around estrus. Some investigators (4) have even used two doses of semen at each insemination time. Few reports describe the timing of ovulation in superovulated cows. Maxwell et al. (3) reported that in cows superovulated using pregnant mare serum gonadotrophin (PMSG), 45 and 91% of ovulations were observed at 24 and 48 h after the onset of estrus, respectively. No ovulations were recorded in the first 18 h. Angel (5) used laparoscopy in cows treated with PMSG and follicle stimulating hormone (FSH) and reported that ovulations were spread over 24 h or more. Shea et al. (6) reported that in cows superovulated with PMSG, ovulations commenced as early as 12 h after estrus was first observed. Although PMSG has been widely used as superovulatory gonadotrophin, FSH-P is normally used in commercial embryo transfer units in North America. The onset and duration of ovulation after the use of this drug are not known precisely. The objective of this experiment was to determine the onset and duration of ovulation in heifers superovulated with FSH-P in relation to the timing of the LH surge so that an optimum time and number of inseminations can be suggested to the embryo transfer industry. MATERIALS
AND METHODS
Estrus was synchronized in batches of 10 beef heifers, 15 to 18 mo old, using a luteolytic dose of cloprostenol (Estrumate, ICI Pharma, Mississauga, Ontario, 500 ug). Within each batch, three to five heifers in which estrus was observed were chosen for superovulatory treatment. The remaining animals were treated with another luteolytic dose of cloprostenol 11 d after the first dose and were superovulated similarly. In this way a total of 32 heifers (26 Hereford and 6 crossbred) were programmed to be on Days 8 to 10 of the estrous cycle and induced to superovulate by the administration of FSH-P (Schering, Montreal, Canada Inc., Lot 574M82). These animals were housed in pens (five heifers in a pen, 2.5 x 6 m) and were fed 69% corn silage, 30% high-moisture corn, and 1% mineral mixture ad libitum. All heifers received identical treatment and were injected with the following doses of FSH-P at 12-h intervals: 5 mg, 5 mg; 4 mg, 4 mg; 4 mg, 4 mg; and 3 mg, 3 mg. The FSH-P was diluted with physiological saline to 5 ml and stored in 6-ml syringes at -18'C. Immediately prior to each injection, the contents of the syringe were allowed to thaw at room temperature and injected. Cloprostenol (500 ug) was administered 60 and 72 h after the first injection of FSH-P. Heifers were observed for estrus every 6 h and were slaughtered at known times between 48 and 100 h after the first prostaglandin treatment. Surviving animals were also inseminated at 60 h after the administration of cloprostenol. All animals were slaughtered at the abbatoir of the Department of Animal and Poultry Science, University of Guelph. The reproductive organs were recovered within 30 min of slaughter and the position of the regressed corpus luteum (CL) and the populations of ovulation sites and nonovulated follicles were quantified. Ovaries were photographed and the diameters of individual nonruptured follicles were measured using a stainless steel Follicles were classified as small (< caliper (CanLab, Toronto, Canada). - 5
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mm), medium
(6 to 10 mm), large (> 10 mm), and medium and large combined (> 6 mm). Blood samples (5 ml) were collected into heparinized vacutainers for the measurement of LH and progesterone from six heifers by jugular venepuncture at 2-h intervals from 24 h after cloprostenol treatment until slaughter. All blood samples were centrifuged immediately and the plasma was harvested and stored at -2O'C until assayed. Hormone
Analysis
Concentrations of LH were determined using a specific radioimmunoassay (7). NIAHDD-bLH-4 (2.4 x NIH-LH-Bl) was used as the assay standard. All the results were then corrected to SIH-LH-Bl. The sensitivity of the assay was 0.120 ng/ml. Interassay and intraassay coefficients of variation were 10.7 and 1.9% respectively. The onset of the preovulatory surge of LH was defined as the time when LH exceeded X + 2 SD, where X was the mean of 5 to 10 values preceding the onset of the surge, determined visually. The peak concentration was the maximum recorded concentration within the profile of an individual cow. Progesterone concentrations were measured in 200-~1 aliquots of plasma by an extraction and charcoal-separation radioimmunoassay (8). The sensitivity of the assay was 0.25 ng/ml, and interassay and intraassay coefficients of variation were 6.4 and 2.6% respectively. For statistical analysis the data were divided into two. One set (n = 12) included data until the onset of ovulation (prior to 64.5 h after cloprostenol) and the other set (n = 20) contained data after ovulations had commenced (after 64.5 h). In an attempt to account for variability in superovulatory response between individuals, the number of ovulations was expressed as a percentage of the number of large unovulated follicles plus the number of ovulations. Similarly, proportions of follicles of different sizes were expressed in relation to either the number of unovulated follicles or unovulated follicles including ovulations. The raw data and proportions obtained were subjected to either logarithmic (base e) or arcsine transformation to obtain homogeneous variances. After arcsine transformation the data of percentage ovulation were subjected to nonlinear regression using a computer programme analysis as outlined in the biomedical statistical analysis package (9). Linear regression analyses were performed on other data using the general linear model procedure of the Statistical Analysis System (10). RESULTS Only 62.5% of the heifers were detected in estrus during superovulation (stood to be mounted). The interval from cloprostenol injection to the onset of estrus in these animals was 41.3 k 1.25 h (n = 20). The interval from cloprostenol injection to the preovulatory peak of LH and the mean LH concentration (_+ SE) at the peak were 43.3 f 1.69 h (n = 6) and 90.2 _t 21.14 ng/ml (n = 6) respectively. Ovulations were not observed in animals slaughtered prior to 64.5 h after cloprostenol administration (Table 1). After this, ovulated follicles were present in all the animals except two (one at 64.5 h and another at 65.5 h). A comparison between ovaries within heifers indicated that neither ovulation rate nor number of unovulated follicles of different
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Table 1.
Number of ovulations and nonovulated follicles in superovulated heifers slaughtered at different times after prostaglandin injection
Animal number
Time of slaughter after prostaglandin inj. (h)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
48.00 49.00 50.00 50.75 52.50 60.00 60.50 60.75 62.00 62.00 62.50 62.50 64.50 64.50 65.25 66.00 67.00 69.50 72.50 73.00 74.00 74.50 76.00 84.25 84.50 86.00 86.75 88.50 96.25 97.00 97.75 100.50
Follicles Follicles Follicles Follicles with > > 6- to lo-mm ovulatory Ovulations 5-m; dia. dia. lo-mm dia. stigma
0 0 0 0 0 0 0 0 0 0 0 0 0
4 0 29 3 31 4 51 24 2 19 17 15 22 18 15 37 16 50 18
2 0 8 3 1 11 1 11 27 0 6 8 11 3 2 12 2 17 6 26 7 6 3 22 5 12 22 20 7 14 29 10
15 5 47 24 8 13 8 19 12 L
28 2 8 3 1 11 6 16 20 2 0 4 4 10 4 2 41 8 9 5 14 4
18 10 10 19 5 20 13 41 20 38 31 19 8 18 7 2 17 6 5 2 1 8 3 1 2 14 6 5 2 1 1 1
; 0 0
1 8 1 1 1 2 1 5 1 0 0 0 1 3 1 0 2 0 0 0 0 0 0
sizes were significantly (P > 0.05) affected by the presence of the CL of the previous cycle on the ovary. After the initiation of ovulations, the logarithm number of ovulations increased linearly [ln (ovulation + 1) = -1.68 + (0.53 x time), P < 0.051 with time. Five, ten, and twenty ovulations occurred by approximately 66, 77 and 89 h after closprostenol injection respectively.
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Ovulatory stigma were observed on the surfaces of large follicles: the number of stigma varied from one to eight in animals slaughtered between 60 to 86 h after the treatment with cloprostenol (Table 1). The number of follicles with ovulatory stigma was not correlated either with ovulation The population of rate or the populations of small and medium follicles. large follicles was, however, positively correlated with the number of ovulatory stigma (r = 0.33, P = 0.07). Ovulation rate was highly variable. As many as 51 ovulations occurred in one individual (Table 1). The nonlinear regression analysis of transformed data when ovulations were expressed as a percentage of unovulated follicles of different sizes (medium + large and large) or as a percentage of total unovulated follicles, including ovulations, revealed a curvilinear relationship of ovulations with time. Re ression equations p < 0.05; were arcsine square root (% ovulations) = 0.96 (l-e O.H9(t), arcsine square root (% ovulations) = 1.24 (1-e-O*17(t)), P : 0.05; and arcsine square root (% ovulations) = 0.76 (1-e-O*21(t)), P < 0.05, for ovulation as a percentage of medium + large, large, and total unovulated follicles, respectively. Ovulations predicted over time were restored to an untransformed scale. A large proportion of follicles had ovulated by 85 h. After this time only a small increase in the proportion of follicles ovulating (approximately 1%) was observed. The overall shape of the ovulation curves remained the same when all three size classes of follicles were used as denominator except for differences in the plateau of proportion of ovulating follicles. This was highest when ovulations were represented as a percentage of the large follicles followed by the curves drawn from medium + large and total unovulated follicles. Considering only large follicles as potentially ovulatory follicles, approximately 80 and 88% of these follicles haa ovulated within 12 and 22 h, respectively, after the onset of ovulations (Figure 1). At the same times, 60 and 66% of medium + large follicles and 43 and 47,, g of total follicles had ovulated. During the preovulatory period (48 to 64.5 h), the number of large follicles increased significantly [In (follicle) = -0.55 + (0.06 x time), P < 0.051 and after the onset of ovulations (64.5 h) decreased significantly (follicle) = 4.98 + (-0.05 x time), P < 0.011. The number of small Iln follicles increased [In (follicle) = 0.15 + (0.03 x time), P < 0.051 after the onset of ovulations. No significant relationships between the number of other sized follicles with time could be established during either period. When follicles > 6 mm in diameter were expressed as a percentage of total unovulated follicles, their proportion decreased (P < 0.05 and P < 0.01) during ovulation (Figure 2). During this period, however, the increase in the numbers of small follicles was significant (P < 0.05). In the preovulatory period, the follicles of the middle class decreased significantly (P < 0.01). When follicles of different sizes were expressed as a percentage of unovulated plus ovulated follicles, similar changes in the population of follicles were apparent, except no change in the proportion of small follicles was observed during ovulation period (Figure 3). Plasma concentrations the time of slaughter were
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of progesterone measured in samples collected low (0.4 _ + 0.03 ng/ml) in all the animals.
1986 VOL. 26 NO. 4
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o45
1::::::::::::::::::m:::::~
55
64 Hours
72 after
50
55
96
104
prostaglandln
Figure 1. The proportion of large follicles ovulating in superovulated beef heifers with time after prostaglandin. Ovulations were expressed as a percentage of total large follicles (> 10 mm) including ovulations.
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2.0
1.8
1.6
64
Hours
72
after
80
88
prostaglandin
Figure 2. Relationship of numbers of follicles of different size with time when expressed as percentage of total unovulated follicles. Nonovulatory and ovulatory periods are separated by the vertical line. The proportion of medium follicles decreased before the onset of ovulation Iarcsine square root (% follicles) = 2.09 t (-0.03 x time), P < 0.011. During ovulation the proportion of medium t large tarcsine square root (% follicles) = 1.56 + (-0.01 x time), P < 0.051 and large [arcsine square root (% follicles) = 1.56 t (-0.01 x time), P < 0.011 follicles decreased significantly. An increase in the proportion of small follicles was observed [arcsine square root (8 follicles) = 0.01 t (0.01 x time), P < 0.051.
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2.0 1.8
.2
a14
4,(I
4
56
84
Hours
72
after
II8
80
__.
prostaglandin
Figure 3. Relationship of numbers of follicles of different sizes with time when expressed as percentage of total unovulated follicles and ovulations. Nonovulatory and ovulatory periods are separated by the vertical line. The proportion of medium follicles decreased before the onset of ovulations [arcsine square root ($ follicles) = 2.09 t (-0.03 x time), P < 0.051. During ovulation the proportion of medium t large [arcsine square root (% follicles) q 1.63 t (-0.01 x time), P < 0.051, large [arcsine square root (8 [arcsine follicles) = 1.51 t (-0.01 x time), P < 0.011, and all follicles square root ($ follicles) = 1.94 t (-1.01 x time), P < 0.051 decreased significantly.
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DISCUSSION The peak of the preovulatory surge of LH, which occurred 43.3 + 2.45 h after cloprostenol injection, is similar to other reports for cows superovulated using FSH-P (11-13). In contrast to a previous report which demonstrated that in superovulated animals the onset of estrus may occur considerably earlier than the LH surge, or well after the LH surge (14), estrus was first observed in this experiment close to the time of the peak of LH. The preovulatory increase in concentrations of estradiol triggers the onset of LH surge (15); an early buildup of this hormone in superovulated cows, compared with those that are not superovulated, must contribute to a more rapid onset of estrus and the associated LH surge (11, 16). After 21 h from the LH surge, ovulations had commenced in all the animals. In earlier reports (6, 17), it had been speculated that in superovulated animals ovulations occur earlier than in the normally cycling cow vith respect to onset of the estrus. However, the time of onset of ovulation after the onset of estrus and the LH surge closely coincided with the interval of 24 h reported for the normally cycling cow (18-20). In two animals that had not ovulated by 64.5 and 65.5 h after cloprostenol treatment, the LH surge may have occurred later than in those animals that had ovulated by this time. This opinion is supported by the observation that in Animal 15 (Table l), which had not yet started to ovulate by 65.25 h, the LH surge occurred 4 h later than that of Animal 14 (Table l), which had four ovulations by 64.5 h. Ovulatory stigma on the apex of one to two large follicles were observed in both the animals. It is known that stigma formation occurs about 1 h before ovulation and is a precise indicator of ovulation (20). Most likely, therefore, the majority of large follicles in the ovaries of both these animals would have ovulated within a few hours had the animals not been slaughtered. The number of ovulations per animal increased over time. Sixteen to twenty oocytes per animal, which have been said to be optimum (17), were released within 24 h of the initiation of ovulation. The ovulation rate in most of these animals, however, was slightly higher than reported by others (14, 21). In these reports, however, rectal palpation was used to evaluate the ovulatory response. This method is not precise and tends to underestimate ovulation rate (22). In other experiments, however, a similar range in ovulation rate (14 to 33 ovulations) has been reported in heifers treated with 32 mg FSH when the population of CL was also estimated directly from the ovaries at the time of slaughter (23). Assuming that follicles of > 10 mm diameter are almost certain to ovulate, as is the case in the normally cycling cow (24), 87 to 88% of ovulations would be expected to occur within a short period (22 h; Figure 1). The percentage of ovulation increased from 12% at the time of initiation to 80% within 12 h, followed by a smaller increase (about 8%) over the next 10 h. The assumption regarding the size (> 10 mm) of the ovulatory follicle is substantiated by the observation that there was a significant increase in numbers of these follicles during the preovulatory period followed by a dramatic decrease after the initiation of ovulation. The occurrence of a small proportion of ovulations after 12 h of initiation leads us to suggest that these follicles were either less mature
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or unable to respond normally to the LH surge. This finding may also indicate that the terminal stages of growth in medium-sized follicles was inhibited by other dominant, estrogen-active follicles that have now ovulated. A large dominant follicle with increased vascularity has the best opportunity to benefit from the preovulatory gonadotrophin surge (25). It appears, therefore, that once the large dominant follicles had ovulated, growth and maturation could occur in medium-sized follicles. Increased availability of FSH to these follicles following the rupture of large follicles probably stimulates synthesis of plasminogen-activator by granulosa cells and results in the production of ovulatory enzymes. This effect may be sequential due to the existence of a follicular hierarchy in superovulated animals and might explain delayed ovulations in medium-sized follicles and, therefore, a slight spread in the time of ovulation. During the estrous cycle in the cow, follicles are recruited from a nongrowing pool and commence development at privileged periods (26). Thereafter, the follicle grows continuously until either ovulation or atresia (27, 28). Although antrum formation and the population of normal antral follicles remain unchanged, the number of atretic follicles decreased when Friesian heifers were induced to superovulate (29). Superovulation either prevents follicles from becoming atretic or rescues some early atretic follicles from atresia (29). It is likely, therefore, that the number of medium to large and total unovulated follicles at the surface of ovaries stimulated with exogenous gonadotrophin are determined both by the influx from continuous follicular growth and the outflow from ovulations. Under these circumstances, the prediction of the number and duration of ovulations using the population of medium to large or total unovulated follicles will not be very precise. A reduction in the number of medium-sized follicles during the preovulatory period suggests that during this period they grew into large follicles (Figures 2 and 3). Since the population of small follicles was increasing and the population of large follicles was decreasing (Figures 2 and 3) during the ovulatory period, the concept that the large follicles inhibit growth of small follicles (28) appears to be confirmed. The absence of a relationship between the number of small follicles with time when the number of large follicles increased significantly during preovulation further supports this hypothesis (Figures 2 and 3). A reduction in the proportion of small follicles during ovulation was not detected when these follicles were represented as a percentage of total unovulated follicles including ovulations because the denominator was confounded with the increasing number of small and decreasing number of large follicles. Plasma progesterone concentrations were the same in animals that had ovulated, probably because the CL were too young to produce measurable amounts of progesterone in the peripheral circulation. Measurement of progesterone in ovarian venous plasma, however, may have indicated an increase in progesterone secretion with time. There was no relationship between ovulation rate and plasma progesterone concentration at slaughter. Given that after initiation of ovulation a large proportion (80%) of ovulations occur within 12 h, that the viable lifespan of spermatozoa is 24 to 48 h, and that capacitation of spermatozoa requires approximately 4 to 6 h (30), a single insemination of superovulated donors with semen from a
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bull of high fertility 60 h after cloprostenol injection should result in satisfactory conception rates. Recent reports suggest that insemination of donors superovulated with FSH-P either once with two straws at 12 or 24 h after the onset of estrus (31,32) or twice with one straw at 12 and 24 h after the detection of estrus (33) resulted in conception rates comparable to multiple inseminations. These results indirectly indicate a short spread of ovulations in superovulated animals and support the findings of this study. From this experiment, therefore, it can be concluded that the biological action of the LH surge requires approximately 24 h to induce ovulation in superovulated cows. Once initiated at 64.5 h after prostaglandin F2 alpha, ovulation of the majority of large follicles occurred within 12 h. Satisfactory conception rates from a single insemination around 60 h after cloprostenol injection would, therefore, be expected. Extensive field trials will, however, be required to test this hypothesis. REFERENCES 1.
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Schiewe, M.C., Voelkel, S.A. and Godke, R.A. Artificial insemination (AI) of superovulated beef cattle with a single insemination of frozen Theriogenology g:228 abstr. (1985). semen.
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