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Increased ovarian responses in the absence of a dominant follicle in superovulated cows
THERIOGENOLOGY
INCREASED OVARIAN RESPONSES IN THE ABSENCE OF A DOMINANT FOLLICLE IN SUPEROVULATED COWS M. Huhtinen,‘S
V. Rainio,2 J. Aalto,
P. Bredbacka’ and A. M&i-Tanilal
lAgricultural Research Centre, Department of Animal Breeding SF-31600 Jokioinen, Finland 2University of Kuopio, Veterinary Research Station P.O. Box 6, SF-70211 Kuopio, Finland 3East Breeding, P.O. Box 60 SF-76101 Pieks&n&i, Finland Received for publication: Accepted:
April 3, 1991 October 11, 1991
ABSTRACT Dairy cows (n = 35) were given a single dose of equine chorionic gonadotrophin (eCG or PMSG) between Days 9 and 12 after a previously synchronized e&us, and after their ovaries had been examined daily by ultrasound scanning from Day 4 or 5 to assess the presence of a dominant follicle and to monitor follicular development before the superovulatory treatment. Two different classification criteria for follicle dominance were tested: 1) a follicle entered the dominance phase when its diameter exceeded 8 mm and it stayed dominant until 3 days atter it stopped growing; 2) a follicle entered the dominance phase when it exceeded 9 mm in diameter and it stayed dominant until 4 days after it ceased to grow. Under both classifications the number of transferable embryos recovered nonsurgically on Day 6 after insemination was significantly higher in cows that did not have a dominant follicle on the day they received their PMSG injection. Under Classification 1, the total numbers of embryos and oocytes recovered, and the concentration of progesterone in the milk on Day 6, were also higher in the group of cows without a dominant follicle. The results suggest that the presence of a dominant follicle at the time of gonadotrophic stimulation decreases the superovulatory response. However, more precise criteria for determining follicular dominance are required in order to improve the predictability of embryo yield in future. Key words: follicle, dominance, superovulation, ultrasonography, cattle INTRODUCTION Realtime ultrasound examination has become the method of choice for monitoring follicular development in individual cows. Several studies (l-3) have reported marked individual variations in follicular dynamics among heifers, with as few as one, to as many as four, waves of follicular growth occurring within an oestrous cycle. Each wave is Acknowledgements The authors are grateful for the technical help in the Agricultural Research Centre at Jokioinen, to one of the referees for clarifying some of the issues in the paper and to Dr W.R. Allen for reading the manuscript. a Present address: Agricultural Research Centre, Equine Research Station, SF-32100 YpiljH, Finland.
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THERIOGENOLOGY
characterized by several small follicles that begin to enlarge with one of this group, the socalled dominant follicle, growing for a longer period and becoming bigger than the others. In the last wave of growth this dominant follicle is the one that ovulates. The presence of a dominant follicle in the ovaries inhibits the development of other follicles (4) and this suppression may also affect the superovulatory response. For example, it has been shown that the presence of a dominant follicle delays the appearance and reduces the number of follicles that are more than 7 mm in diameter during the superovulatory treatment, and this is associated with a reduction in the number of ovulations (5). There have been other reports (6,7) stating that the presence of a dominant follicle at the time of superovulatory treatment may alter the maturation process of recruitable follicles and their response to gonadotrophic stimulation. It is difficult to define a dominant follicle by morphological characteristics alone since the regressing dominant follicle remains the biggest in the ovaries for 3-4 days after it has passed its dominance phase (I). For this reason many investigators have stipulated that daily follow-up examinations of growing follicles are essential when undertaking studies involving follicular dynamics in the cow (Z-7). others have tried to diagnose the dominant follicle by a single examination and have failed to find any correlation between the stage of follicular growth at the beginning of gonadotrophic stimulation and the subsequent superovulatory response (8). In the present experiment we studied the influence of a “postulated dominant follicle” on the superovulatory response in PMSG-treated dairy cows, using daily ultrasound monitoring of follicular growth and two different criteria for dominance. MATERIALS AND METHODS Finnish Ayrshire dairy cows (n = 35), aged 3-12 years and ranging from 66 to 437 days from their last calving, were used as embryo donors in July 1990. Estrus was synchronized in the group by inserting a progesterone-releasing intravaginal device (PRID)b for 8 days combined with a single Km. injection of 0.625 mg cloprostenolc given 1 day Progesterone concentrations were measured by before removal of the PRID. radioimmunoassayd in whole milk samples recovered 2 days after PRID removal. Estrus was diagnosed when the milk progesterone level fell below 3 nmol/l. Beginning on Day 4 or 5 after this synchronized estrus, and continuing until the day after the subsequent injection of PMSG, the ovaries of each cow were monitored daily by realtime ultrasound scanning using a machine equipped with a 7.5 MHz linear array transrectal probee. All the examinations were carried out by the same person and the sizes of all follicles of > 5 mm diameter in both the ovaries were recorded. The presence of a corpus luteum was determined by ultrasound and confirmed by measuring elevated progesterone levels in the milk on the day of PMSG treatment and 2 days later. Each cow was given a single i.m. injection of 2,500 IU PMSGf between Days 9 and 12 after the previous synchronized estrus, with the actual days of the treatment being chosen so that there was an equal number of animals with or without a dominant follicle in their b PRID”; Abbott, France. c Estrumat?; Coopers, Germany. d Farmos Diagnostica, Turku, Finland. ’ Aloka Co, Tokyo, Japan. f Folligon; Intervet, Holland.
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ovaries on each of the 4 days. Luteolysis was induced by a single i.m. injection of 1.0 mg cloprostenol given 2 days after PMSG treatment. Estrus signs were recorded every 6 hours by a trained technician. All the cows were inseminated with frozen-thawed semen twice 12 hours apart (7 am and 7 pm) beginning 2-9 hours after the start of standing e&us. A single injection of 0.5 mg GnRHg was given to each cow 0 - 4 hours before the first insemination.
The uteri of the 35 PMSG-treated donor cows were flushed nonsurgically on Day 5.4 6.5 after e&us. The embryos were counted and classified as described by Donaldson (9). Those embryos which had viable cells making up > 25% of the total cell mass, and which were not severely retarded in development, were considered transferable. Since no precise morphological definitions of a dominant follicle were available, two different classifications for dominance were tested: 1) a follicle entered the dominance phase when it exceeded 8 mm in diameter and it stayed dominant until 3 days after it stopped growing; 2) a follicle entered the dominance phase when it exceeded 9 mm in diameter and it stayed dominant until 4 days after the cessation of growth. Follicles that fell outside these criteria were classified as nondominant. Linear model methods were used in the statistical analyses (10). RESULTS The synchronized estrus occurred 1.3 to 2.8 days (mean 1.9 f 0.4) after PRID removal. Whole milk progesterone concentrations ranged from 7 to 70 nmol/l (mean 39 mnol/l) on the day of PMSG treatment, and from 15 to 100 mnolll (mean 51 nmolll) on the day of prostaglandin treatment. The progesterone level increased after PMSG treatment in 29 cows, decreased in 5 cows, and remained steady in one cow. The difference between progesterone levels on the days of PMSG and prostaglandin treatment did not have any significant effect on the total number of embryos and non-fertilized oocytes recovered. The first signs of the superovulatory estrus were noted 1.8 f 0.2 days (range 1.2 - 2.2) after the prostaglandin injection. The group was large enough so that, by chance, the data structure over the dominance grouping was fairly balanced in terms of the age of the donors, the time from last calving, the interval from PRID removal to estrus, from estrus to PMSG injection, from PMSG treatment to e&us, and from estrus to uterine flushing under both dominance classifications. None of these factors had any significant effect on embryo yield. Cows that received their PMSG injection when a dominant follicle was not present in their ovaries produced significantly more good (transferable) embryos than those which received PMSG in the presence of a dominant follicle (Tables 1 and 2). This was true under both dominance classifications. The absence of a dominant follicle at the time of PMSG treatment (Classification 1) was also associated with a significantly higher milk progesterone concentration on Day 6 after the superovulatory estrus and with a significantly higher number of embryos and oocytes recovered (Table 1). When Classification 2 was applied, similar tendencies were seen, although with lesser statistical significance (Table 2). Under this classification the proportion of non-fertilized oocytes and dead embryos (i.e. total embryos + oocytes minus the transferable embryos) in g Fertagyl”; Intervet, Holland.
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the group of cows with a dominant follicle at the time of PMSG treatment was slightly, hut insignificantly, higher (P > 0. l), as was the milk progesterone concentration (P > 0.05). On the day of PMSG treatment the diameter of the largest follicle ranged from 13 to 22 mm (mean 17.5 mm) and this size did not have any effect on the total number of transferable embryos and unfertilized oocytes recovered. The two classification criteria for dominance are compared in Table 3. On each of the days between 6 and 12 after the synchronized estrus, there were some cows with a dominant follicle in their ovaries, whichever dominance criteria was used.
Table 1. Mean (&SEM) embryo and unfertilized oocyte recovery rates and whole milk progesterone concentrations on Day 6 after the superovulatory estrus in cows with a dominant follicle (Group D) and without a dominant follicle (Group ND) at the time of the superovulatory treatment under Classification 1. Group
No.of cows
Total embryos and oocytes recovered
Good embryos recovered
Whole milk progesterone conc.(nmol/l)
ND
22
10.3 f 1.3
6.6 * 1.1
158 f 21
D
13
4.1 + 1.6
2.2 * 1.4
82 + 28
(P < 0.05)
(P < 0.05)
(P<
0.01)
Table 2. Mean (*SEM) embryo and unfertilized oocyte recovery rates and whole milk progesterone concentrations on Day 6 after the superovulatory estrus in cows with a dominant follicle (Group D) and without a dominant follicle (Group ND) at the time of the superovulatory treatment under Classification 2.
Group
No. of cows
Total embryos and oocytes recovered
Good embryos recovered
Whole milk progesterone cont. (nmolll)
ND
19
9.6 f 1.4
7.1 f 1.1
161 f 23
D
16
6.0 f 1.6
2.4 f 1.2
94 + 25
(P > 0.05)
(P < 0.01)
(P > 0.05)
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THERIOGENOLOGY Table 3. Presence of a dominant follicle on Days 6 - 12 after the synchronized estrus under Classifications 1 and 2.
Day of estrous cycle: 6
7
8
9
10
11
12
35
35
35
34
27
18
8
Classification 1
loo
loo
77
68
37
33
50
Classification 2
94
100
100
79
78
28
38
Number of cows observed Percentage of cows with dominant follicle in:
DISCUSSION The results of this study demonstrated convincingly that the presence or absence of a dominant follicle in the ovaries at the time of exogenous gonadotrophic stimulation influences markedly the superovulatory response in the cow. In the absence of a dominant follicle at the time of PMSG injection the cows produced twice the number of transferable embryos compared to the animals treated with PMSG in the presence of a dominant follicle. The total number of oocytes and embryos found, and the progesterone concentrations on Day 6 after the superovulatory estrus both indicated that this difference between the two groups of cows was due to a higher number of ovulations in the animals that did not have a dominant follicle on the day of PMSG treatment. There was no decrease in fertility or embryo quality caused by the increase in ovulation rate. On the contrary, the percentages of unfertilized oocytes and degenerating embryos were both lower in the group of cows without a dominant follicle at the time of gonadotrophin therapy. Some previous studies in this area have given similar results ($7) while others have failed to show any significant effect of the presence of a dominant follicle on the ovarial response to hormone therapy (8,ll). Wilson et al. (8) and Gray et al, (11) both reported that the superovulatory response did not decrease in the presence of a dominant follicle. However, in the study by Wilson et al. the stage of dominance was assessed by only a single ultrasound examination on the day of the superovulatory treatment (8) and if the same parameters had been applied in the present experiment, the effect of a dominant follicle would not have been seen in the results. Similarly, in the study undertaken by Gray et al. (11) the superovulatory treatment was initiated when the largest (dominant ?) follicle had already been decreasing in size for 2 days. In our animals we did not observe any decrease in the diameter of the dominant follicle before the injection of PMSG was given. It merely flattened and became softer.
FEBRUARY 1992 VOL. 37 NO. 2
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THERIOGENOLOGY In this study a follicle was considered dominant if its diameter exceeded a chosen figure and if it was still in the growing phase or it had stopped growing less than 3-4 days previously. The choice of these parameters was suggested by the results of the previous studies undertaken in France ($7). In practice it would therefore seem to be advisable to monitor the ovaries of embryo donor cows from Day 5 or 6 after estrus until the day of gonadotrophic stimulation, or on at least 4 consecutive days before the start of the superovulatory treatment. Although the morphological definitions for a dominant follicle seem to work in practice, they nevertheless remain only approximations. Since there is appreciable variation in the size of individual follicles when they ovulate, there must also be variation in the size of follicles at the start of dominance activity. Therefore, a more accurate way of recognizing dominance is required. One possibility would be to define the inhibitory factor(s) secreted by the dominant follicle, or their precursors. However, since factors of this type could only be recognized after the dominance phase has started, their measurement would be unlikely to provide a way of improving the results of the superovulatory program. Estrogen profiles or other indicative hormones measured in blood or milk, may become a practical means for determining follicular status before an attempt is made to stimulate superovulation. On the other hand, some form of treatment given to inhibit the development of a dominant follicle before gonadotrophic stimulation is applied may give better results when developing new superovulatory treatment protocols in the future. REFERENCES 1. Fortune, J.E., Sirois, J. and Quirk, S.M. The growth and differentiation of ovarian follicles during the bovine estrous cycle. Theriogenology B:95-109 (1988). 2. Ginther, O.J., Kastelic, J.P. and Knopf, L. Intraovarian relationships among dominant and subordinate follicles and the corpus luteum in heifers. Theriogenology 2:787-795 (1989). 3. Savio, J.D., Keenan, L., Boland, M.P. and Roche, J.F. Pattern of growth of dominant follicles during the oestrous cycle in heifers. J.Reprod.Fertil. ~563-671 (1988). 4. Ireland, J.J. and Roche, J.F. Hypotheses regarding development of dominant follicles during a bovine estrous cycle. &I: Roche and O’Callaghan, (eds) Follicular Growth and Ovulation Rate in Farm Animals. Martinus Nijhoff Publishers, Dordrecht, 1987, pp. l18. 5. Grasso, F., Guilbault, L.A., Roy, G.L., Matton, P. and Lussier, J.G. The influence of the presence of a dominant follicle at the time of initiation of a superovulatory treatment on superovulatory responses in cattle. Theriogenology 2: 199 Abstr. (1989). 6. Rouillier, P., Matton, P., Guilbault, L., Grasso, F. and Lussier, J. Influence of a dominant follicle atresia and estradiol release by ovarian follicles during superovulation in cattle. Theriogenology 33:313 Abstr. (1990). 7. Guilbault, L.A., Grasso, F., Lussier, J.G., Rouillier, P. and Matton, P. Decreased superovulatory responses in heifers superovulated in the presence of a dominant follicle. J.Reprod.Fertil. 9_I:81-89 (1991).
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8. Wilson, J.M., Jones, A.L. and Miller, D.R. Influence of a dominant follicle on the superovulatory response. Theriogenology a:349 Abstr. (1990). 9. Donaldson, L.E. Embryo production in superovulated cows: transferable embryos correlated with total embryos. Theriogenology 21x517-524 (1984). 10. SAS/STATTM Guide for Personal Computers. SAS Institute, Inc.,Cary, NC; 1985. 11. Gray, B.W., Cartee, R.E., Stringfellow, D.A., Riddell, M.G., Riddell, K.P. and Wright, J.C. The effects of dominant follicular regression and FSH priming on the superovulatory response of cattle. Theriogenology =:207 Abstr. (1991).
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1992 VOL. 37 NO. 2
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INCREASED OVARIAN RESPONSES IN THE ABSENCE OF A DOMINANT FOLLICLE IN SUPEROVULATED COWS M. Huhtinen,‘S
V. Rainio,2 J. Aalto,
P. Bredbacka’ and A. M&i-Tanilal
lAgricultural Research Centre, Department of Animal Breeding SF-31600 Jokioinen, Finland 2University of Kuopio, Veterinary Research Station P.O. Box 6, SF-70211 Kuopio, Finland 3East Breeding, P.O. Box 60 SF-76101 Pieks&n&i, Finland Received for publication: Accepted:
April 3, 1991 October 11, 1991
ABSTRACT Dairy cows (n = 35) were given a single dose of equine chorionic gonadotrophin (eCG or PMSG) between Days 9 and 12 after a previously synchronized e&us, and after their ovaries had been examined daily by ultrasound scanning from Day 4 or 5 to assess the presence of a dominant follicle and to monitor follicular development before the superovulatory treatment. Two different classification criteria for follicle dominance were tested: 1) a follicle entered the dominance phase when its diameter exceeded 8 mm and it stayed dominant until 3 days atter it stopped growing; 2) a follicle entered the dominance phase when it exceeded 9 mm in diameter and it stayed dominant until 4 days after it ceased to grow. Under both classifications the number of transferable embryos recovered nonsurgically on Day 6 after insemination was significantly higher in cows that did not have a dominant follicle on the day they received their PMSG injection. Under Classification 1, the total numbers of embryos and oocytes recovered, and the concentration of progesterone in the milk on Day 6, were also higher in the group of cows without a dominant follicle. The results suggest that the presence of a dominant follicle at the time of gonadotrophic stimulation decreases the superovulatory response. However, more precise criteria for determining follicular dominance are required in order to improve the predictability of embryo yield in future. Key words: follicle, dominance, superovulation, ultrasonography, cattle INTRODUCTION Realtime ultrasound examination has become the method of choice for monitoring follicular development in individual cows. Several studies (l-3) have reported marked individual variations in follicular dynamics among heifers, with as few as one, to as many as four, waves of follicular growth occurring within an oestrous cycle. Each wave is Acknowledgements The authors are grateful for the technical help in the Agricultural Research Centre at Jokioinen, to one of the referees for clarifying some of the issues in the paper and to Dr W.R. Allen for reading the manuscript. a Present address: Agricultural Research Centre, Equine Research Station, SF-32100 YpiljH, Finland.
FEBRUARY 1992 VOL. 37 NO. 2
457
THERIOGENOLOGY
characterized by several small follicles that begin to enlarge with one of this group, the socalled dominant follicle, growing for a longer period and becoming bigger than the others. In the last wave of growth this dominant follicle is the one that ovulates. The presence of a dominant follicle in the ovaries inhibits the development of other follicles (4) and this suppression may also affect the superovulatory response. For example, it has been shown that the presence of a dominant follicle delays the appearance and reduces the number of follicles that are more than 7 mm in diameter during the superovulatory treatment, and this is associated with a reduction in the number of ovulations (5). There have been other reports (6,7) stating that the presence of a dominant follicle at the time of superovulatory treatment may alter the maturation process of recruitable follicles and their response to gonadotrophic stimulation. It is difficult to define a dominant follicle by morphological characteristics alone since the regressing dominant follicle remains the biggest in the ovaries for 3-4 days after it has passed its dominance phase (I). For this reason many investigators have stipulated that daily follow-up examinations of growing follicles are essential when undertaking studies involving follicular dynamics in the cow (Z-7). others have tried to diagnose the dominant follicle by a single examination and have failed to find any correlation between the stage of follicular growth at the beginning of gonadotrophic stimulation and the subsequent superovulatory response (8). In the present experiment we studied the influence of a “postulated dominant follicle” on the superovulatory response in PMSG-treated dairy cows, using daily ultrasound monitoring of follicular growth and two different criteria for dominance. MATERIALS AND METHODS Finnish Ayrshire dairy cows (n = 35), aged 3-12 years and ranging from 66 to 437 days from their last calving, were used as embryo donors in July 1990. Estrus was synchronized in the group by inserting a progesterone-releasing intravaginal device (PRID)b for 8 days combined with a single Km. injection of 0.625 mg cloprostenolc given 1 day Progesterone concentrations were measured by before removal of the PRID. radioimmunoassayd in whole milk samples recovered 2 days after PRID removal. Estrus was diagnosed when the milk progesterone level fell below 3 nmol/l. Beginning on Day 4 or 5 after this synchronized estrus, and continuing until the day after the subsequent injection of PMSG, the ovaries of each cow were monitored daily by realtime ultrasound scanning using a machine equipped with a 7.5 MHz linear array transrectal probee. All the examinations were carried out by the same person and the sizes of all follicles of > 5 mm diameter in both the ovaries were recorded. The presence of a corpus luteum was determined by ultrasound and confirmed by measuring elevated progesterone levels in the milk on the day of PMSG treatment and 2 days later. Each cow was given a single i.m. injection of 2,500 IU PMSGf between Days 9 and 12 after the previous synchronized estrus, with the actual days of the treatment being chosen so that there was an equal number of animals with or without a dominant follicle in their b PRID”; Abbott, France. c Estrumat?; Coopers, Germany. d Farmos Diagnostica, Turku, Finland. ’ Aloka Co, Tokyo, Japan. f Folligon; Intervet, Holland.
458
FEBRUARY
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THERIOGENOLOGY
ovaries on each of the 4 days. Luteolysis was induced by a single i.m. injection of 1.0 mg cloprostenol given 2 days after PMSG treatment. Estrus signs were recorded every 6 hours by a trained technician. All the cows were inseminated with frozen-thawed semen twice 12 hours apart (7 am and 7 pm) beginning 2-9 hours after the start of standing e&us. A single injection of 0.5 mg GnRHg was given to each cow 0 - 4 hours before the first insemination.
The uteri of the 35 PMSG-treated donor cows were flushed nonsurgically on Day 5.4 6.5 after e&us. The embryos were counted and classified as described by Donaldson (9). Those embryos which had viable cells making up > 25% of the total cell mass, and which were not severely retarded in development, were considered transferable. Since no precise morphological definitions of a dominant follicle were available, two different classifications for dominance were tested: 1) a follicle entered the dominance phase when it exceeded 8 mm in diameter and it stayed dominant until 3 days after it stopped growing; 2) a follicle entered the dominance phase when it exceeded 9 mm in diameter and it stayed dominant until 4 days after the cessation of growth. Follicles that fell outside these criteria were classified as nondominant. Linear model methods were used in the statistical analyses (10). RESULTS The synchronized estrus occurred 1.3 to 2.8 days (mean 1.9 f 0.4) after PRID removal. Whole milk progesterone concentrations ranged from 7 to 70 nmol/l (mean 39 mnol/l) on the day of PMSG treatment, and from 15 to 100 mnolll (mean 51 nmolll) on the day of prostaglandin treatment. The progesterone level increased after PMSG treatment in 29 cows, decreased in 5 cows, and remained steady in one cow. The difference between progesterone levels on the days of PMSG and prostaglandin treatment did not have any significant effect on the total number of embryos and non-fertilized oocytes recovered. The first signs of the superovulatory estrus were noted 1.8 f 0.2 days (range 1.2 - 2.2) after the prostaglandin injection. The group was large enough so that, by chance, the data structure over the dominance grouping was fairly balanced in terms of the age of the donors, the time from last calving, the interval from PRID removal to estrus, from estrus to PMSG injection, from PMSG treatment to e&us, and from estrus to uterine flushing under both dominance classifications. None of these factors had any significant effect on embryo yield. Cows that received their PMSG injection when a dominant follicle was not present in their ovaries produced significantly more good (transferable) embryos than those which received PMSG in the presence of a dominant follicle (Tables 1 and 2). This was true under both dominance classifications. The absence of a dominant follicle at the time of PMSG treatment (Classification 1) was also associated with a significantly higher milk progesterone concentration on Day 6 after the superovulatory estrus and with a significantly higher number of embryos and oocytes recovered (Table 1). When Classification 2 was applied, similar tendencies were seen, although with lesser statistical significance (Table 2). Under this classification the proportion of non-fertilized oocytes and dead embryos (i.e. total embryos + oocytes minus the transferable embryos) in g Fertagyl”; Intervet, Holland.
FEBRUARY
1992 VOL. 37 NO. 2
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THERIOGENOLOGY
the group of cows with a dominant follicle at the time of PMSG treatment was slightly, hut insignificantly, higher (P > 0. l), as was the milk progesterone concentration (P > 0.05). On the day of PMSG treatment the diameter of the largest follicle ranged from 13 to 22 mm (mean 17.5 mm) and this size did not have any effect on the total number of transferable embryos and unfertilized oocytes recovered. The two classification criteria for dominance are compared in Table 3. On each of the days between 6 and 12 after the synchronized estrus, there were some cows with a dominant follicle in their ovaries, whichever dominance criteria was used.
Table 1. Mean (&SEM) embryo and unfertilized oocyte recovery rates and whole milk progesterone concentrations on Day 6 after the superovulatory estrus in cows with a dominant follicle (Group D) and without a dominant follicle (Group ND) at the time of the superovulatory treatment under Classification 1. Group
No.of cows
Total embryos and oocytes recovered
Good embryos recovered
Whole milk progesterone conc.(nmol/l)
ND
22
10.3 f 1.3
6.6 * 1.1
158 f 21
D
13
4.1 + 1.6
2.2 * 1.4
82 + 28
(P < 0.05)
(P < 0.05)
(P<
0.01)
Table 2. Mean (*SEM) embryo and unfertilized oocyte recovery rates and whole milk progesterone concentrations on Day 6 after the superovulatory estrus in cows with a dominant follicle (Group D) and without a dominant follicle (Group ND) at the time of the superovulatory treatment under Classification 2.
Group
No. of cows
Total embryos and oocytes recovered
Good embryos recovered
Whole milk progesterone cont. (nmolll)
ND
19
9.6 f 1.4
7.1 f 1.1
161 f 23
D
16
6.0 f 1.6
2.4 f 1.2
94 + 25
(P > 0.05)
(P < 0.01)
(P > 0.05)
460
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1992 VOL. 37 NO. 2
THERIOGENOLOGY Table 3. Presence of a dominant follicle on Days 6 - 12 after the synchronized estrus under Classifications 1 and 2.
Day of estrous cycle: 6
7
8
9
10
11
12
35
35
35
34
27
18
8
Classification 1
loo
loo
77
68
37
33
50
Classification 2
94
100
100
79
78
28
38
Number of cows observed Percentage of cows with dominant follicle in:
DISCUSSION The results of this study demonstrated convincingly that the presence or absence of a dominant follicle in the ovaries at the time of exogenous gonadotrophic stimulation influences markedly the superovulatory response in the cow. In the absence of a dominant follicle at the time of PMSG injection the cows produced twice the number of transferable embryos compared to the animals treated with PMSG in the presence of a dominant follicle. The total number of oocytes and embryos found, and the progesterone concentrations on Day 6 after the superovulatory estrus both indicated that this difference between the two groups of cows was due to a higher number of ovulations in the animals that did not have a dominant follicle on the day of PMSG treatment. There was no decrease in fertility or embryo quality caused by the increase in ovulation rate. On the contrary, the percentages of unfertilized oocytes and degenerating embryos were both lower in the group of cows without a dominant follicle at the time of gonadotrophin therapy. Some previous studies in this area have given similar results ($7) while others have failed to show any significant effect of the presence of a dominant follicle on the ovarial response to hormone therapy (8,ll). Wilson et al. (8) and Gray et al, (11) both reported that the superovulatory response did not decrease in the presence of a dominant follicle. However, in the study by Wilson et al. the stage of dominance was assessed by only a single ultrasound examination on the day of the superovulatory treatment (8) and if the same parameters had been applied in the present experiment, the effect of a dominant follicle would not have been seen in the results. Similarly, in the study undertaken by Gray et al. (11) the superovulatory treatment was initiated when the largest (dominant ?) follicle had already been decreasing in size for 2 days. In our animals we did not observe any decrease in the diameter of the dominant follicle before the injection of PMSG was given. It merely flattened and became softer.
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THERIOGENOLOGY In this study a follicle was considered dominant if its diameter exceeded a chosen figure and if it was still in the growing phase or it had stopped growing less than 3-4 days previously. The choice of these parameters was suggested by the results of the previous studies undertaken in France ($7). In practice it would therefore seem to be advisable to monitor the ovaries of embryo donor cows from Day 5 or 6 after estrus until the day of gonadotrophic stimulation, or on at least 4 consecutive days before the start of the superovulatory treatment. Although the morphological definitions for a dominant follicle seem to work in practice, they nevertheless remain only approximations. Since there is appreciable variation in the size of individual follicles when they ovulate, there must also be variation in the size of follicles at the start of dominance activity. Therefore, a more accurate way of recognizing dominance is required. One possibility would be to define the inhibitory factor(s) secreted by the dominant follicle, or their precursors. However, since factors of this type could only be recognized after the dominance phase has started, their measurement would be unlikely to provide a way of improving the results of the superovulatory program. Estrogen profiles or other indicative hormones measured in blood or milk, may become a practical means for determining follicular status before an attempt is made to stimulate superovulation. On the other hand, some form of treatment given to inhibit the development of a dominant follicle before gonadotrophic stimulation is applied may give better results when developing new superovulatory treatment protocols in the future. REFERENCES 1. Fortune, J.E., Sirois, J. and Quirk, S.M. The growth and differentiation of ovarian follicles during the bovine estrous cycle. Theriogenology B:95-109 (1988). 2. Ginther, O.J., Kastelic, J.P. and Knopf, L. Intraovarian relationships among dominant and subordinate follicles and the corpus luteum in heifers. Theriogenology 2:787-795 (1989). 3. Savio, J.D., Keenan, L., Boland, M.P. and Roche, J.F. Pattern of growth of dominant follicles during the oestrous cycle in heifers. J.Reprod.Fertil. ~563-671 (1988). 4. Ireland, J.J. and Roche, J.F. Hypotheses regarding development of dominant follicles during a bovine estrous cycle. &I: Roche and O’Callaghan, (eds) Follicular Growth and Ovulation Rate in Farm Animals. Martinus Nijhoff Publishers, Dordrecht, 1987, pp. l18. 5. Grasso, F., Guilbault, L.A., Roy, G.L., Matton, P. and Lussier, J.G. The influence of the presence of a dominant follicle at the time of initiation of a superovulatory treatment on superovulatory responses in cattle. Theriogenology 2: 199 Abstr. (1989). 6. Rouillier, P., Matton, P., Guilbault, L., Grasso, F. and Lussier, J. Influence of a dominant follicle atresia and estradiol release by ovarian follicles during superovulation in cattle. Theriogenology 33:313 Abstr. (1990). 7. Guilbault, L.A., Grasso, F., Lussier, J.G., Rouillier, P. and Matton, P. Decreased superovulatory responses in heifers superovulated in the presence of a dominant follicle. J.Reprod.Fertil. 9_I:81-89 (1991).
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8. Wilson, J.M., Jones, A.L. and Miller, D.R. Influence of a dominant follicle on the superovulatory response. Theriogenology a:349 Abstr. (1990). 9. Donaldson, L.E. Embryo production in superovulated cows: transferable embryos correlated with total embryos. Theriogenology 21x517-524 (1984). 10. SAS/STATTM Guide for Personal Computers. SAS Institute, Inc.,Cary, NC; 1985. 11. Gray, B.W., Cartee, R.E., Stringfellow, D.A., Riddell, M.G., Riddell, K.P. and Wright, J.C. The effects of dominant follicular regression and FSH priming on the superovulatory response of cattle. Theriogenology =:207 Abstr. (1991).
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