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Ovarian follicular populations during early pregnancy in heifers
THERIOGENOLOGY
OVARIAN
POLLICULAR
POPULATIONS
DURING
EARLY
PREGNANCY
IN HEIFERS
R.A. Pierson and O.J. Ginther Department of Veterinary Science University of Wisconsin-Madison Madison, WI 53706 Received
for publication: Accepted:
ApriZ 2, 2986 September 18,
1986
ABSTRACT
Ovarian follicles >2mm were studied in 14 pregnant and 14 nonpregnant Holstein heifers by daily ultrasound examinations. There were significant differences among days, from Day 0 (day of ovulation) to Day 21, in the diameter of the largest follicle and the diameter of the second largest follicle in pregnant and nonpregnant heifers. There was an interaction of day and reproductive status (P < 0.001) for the diameter of the largest follicle. Significant differences among days were also observed in the numbers of follicles 2 to 3 mm, 4 to 6 mm, 7 to 10 mm, 11 to 13 mm, and >13 mm, and the total number of follicles ~2 mm. There was a significant main effect of reproductive status for the number of follicles 11 to 13 mm. An interaction of day and reproductive status was observed for the number of follicles >13 mm, but not for any of the other diameter categories. The effect of reproductive status for number of follicles 11 to 13 mm and the interactions for the number of follicles >13 mm and the diameter of the largest follicle seemed due to the selective growth and ovulation of the follicle destined to ovulate in nonpregnant heifers. The differences in ovarian follicular populations between pregnant and nonpregnant heifers were attributed solely to the presence of a physiological mechanism for the selection of an ovulatory follicle in nonpregnant heifers. There were no significant differences among days for any follicular endpoint during Days 22 to 60 in the pregnant heifers.
Key words:
ovary, follicles,
pregnancy,
bovine
Supported by the College of Agricultural and Life Sciences, University of WisconsinMadison and USDA Grant 84-CRSR-2-2451. The authors wish to thank Mary J. Lindstrom, Department of Statistics and Peter Crump, Department of Computing and Biometry. Appreciation is expressed to Marcia Campbell, Peggy Hoge, Susan Krebs, Lisa Kulick, and Diane Stuber for technical assistance and to the Upjohn Company, Kalamazoo, MI for Lutalyse.
NOVEMBER
1986 VOL.
26 NO. 5
649
THERIOGENOLOGY
INTRODUCTION
Studies on the changing morphology of ovaries in cattle have been limited to examination of slaughterhouse specimens or to transrectal palpation (l-5). No information was found in the literature which compared follicular populations during early pregnancy with the comparable period of the interovulatory interval in cycling heifers. There have been only limited slaughterhouse studies on follicle numbers in which ovaries were sliced and follicles counted on selected, known days of pregnancy (earliest day of examination, Day 19; 3) or in which the stage of pregnancy was estimated by the crown-rump length of the fetus (4). A preliminary study in our laboratory was done to assess the use of ultrasonic imaging for measuring and of the ovaries counting follicles in cattle (6). The three principal components (follicles, corpora lutea, and stroma) were distinguishable, and ultrasonography was judged to be a suitable approach to the study of ovarian changes in cattle and for detection of follicles as small as 2 mm. Diagnostic ultrasonography has been used to study ovarian follicular populations during the estrous cycle in mares (7), including a study in which follicular profiles of pregnant and nonpregnant mares were compared (6). The purpose of the present experiment was to characterize the changes in the ovarian follicular populations during the first 60 days of pregnancy in heifers. Special attention was given to the first 21 days; follicular profiles were compared between pregnant and nonpregnant heifers for a period equivalent to the length of an interovulatory interval in nonpregnant heifers. MATERIALS
AND METHODS
Nulliparous Holstein heifers between 1.5 and 2.0 years of age and weighing 300 to 450 kg were used. The heifers were randomly put into a bred group (n = 19) or a nonbred group (n = 21). Ten of the 19 heifers assigned to the bred group were artificially inseminated approximately 12 hours after first observation of standing estrus if a regressing corpus luteum and a follicle >14 mm were detected by The nine remaining heifers were given prostaglandin F2, ultrasonography (6). (Lutalyse, Upjohn Company, Kalamazoo, MI) when an apparently mature corpus The nine heifers were artificially luteum was detected ultrasonically (6). inseminated 72 hours later if a follicle >14 mm and a regressing corpus luteum were present. A real-time B-mode diagnostic ultrasound instrument (Equisonics 210, Equisonics Inc., Roselle, IL) equipped with a linear-array, 5 MHz transducer designed for intrarectal placement was used for the examinations. Fecal material was removed from the rectum and the transducer was inserted. The transducer was moved along the dorsal surface of the reproductive tract for orientation and then moved laterally to examine the ovaries. Follicles >2 mm were counted. The antra of follicles larger than 10 mm were measured withintegral electronic calipers at the interface of the follicular wall and the follicular fluid. The diameters of follicles smaller than 10 mm were estimated by comparison with the centimeter scale displayed at the side of the ultrasound image. This was done to minimize errors due to loss of identity of individual small follicles during freezing of the image. The day of ovulation (Day 0) was determined by the disappearance of a large (>I1 mm) follicle as described (6). The examinations were made without knowledge of the
650
NOVEMBER
1986 VOL. 26 NO. 5
THERIOGENOLOGY
number of days post ovulation or of the previous day’s results. The reproductive tract was not directly manipulated before or during the ultrasound examination. Examination of the uterus for the presence of a conceptus was done after the ovarian examinations were made. The first day of detection of a discrete nonechogenic structure within the uterine lumen was recorded as the first day of detection of a presumptive embryonic vesicle. A confirmed pregnancy was defined as subsequent progressive elongation of the nonechogenic area within the uterus and the eventual detection of an embryo proper as described (9). The heifers were examined daily from at least three days prior to an ovulation until Day 60 of pregnancy or until at least three days after the nonpregnant heifers returned to estrus and ovulated. Fourteen of the heifers in the bred group maintained an embryo until at least Day 60, so data for the first 14 heifers to complete an interovulatory interval in the nonbred group were compared Four of the bred heifers lost the conceptus prior to the to the pregnant group. detection of an embryo proper and were considered as a separate group. Results specific to the conceptuses are reported elsewhere (9,lO). For descriptive purposes, data for Days -3 to -1 before the first ovulation were added to the data for both pregnant and cycling heifers, and Days +l to +3 after the subsequent ovulation were added to the data for the interovulatory interval of nonpregnant heifers. Follicular patterns were studied by grouping the follicles into diameter ranges of 2 to 3 mm, 4 to 6 mm, 7 to 10 mm, 11 to 13 mm, and >13 mm. Diameters of the largest follicle and second largest follicle were also determined. Data for the nonbred heifers were normalized to the mean length of the interovulatory interval (20.7 ?r 0.4 days, mean + SEM). For heifers with intervals longer than 21 days, the appropriate number of days was randomly deleted; for heifers with intervals shorter than 21 days, data for the appropriate number of days was randomly inserted by averaging the data from the day before and the day after the insert. A separate randomization was done for each follicular diameter category. Statistical analyses were performed using the Statistical Analysis System @AS) by general linear models univariate analyses for repeated measures (11). Specific analyses were performed to determine the effect of day and reproductive status from Days 0 to 21 for the pregnant and nonpregnant heifers. The effect of day was also examined from Days 22 to 60 for the pregnant heifers. Detailed analyses of the day effects during the interovulatory interval in nonpregnant heifers are part of another experiment. The main effects were tested using F tests with degrees of freedom modified by the Greenhouse-Geyser epsilon (11). Days of significant difference due to reproductive status were determined by using univariate t tests for each day. The interactions between day and reproductive status for Days 0 to 21 were examined.
RESULTS
The mean diameters of the largest and second largest follicles are depicted for the interovulatory interval for nonpregnant heifers and up to Day 30 for pregnant heifers (Figure 1). Additionally, profiles are shown for the diameters of the largest and second largest follicles for pregnant heifers from Days 31 to 60 (Figure 2). There were significant differences among days for the diameters of the largest and second largest follicles from Days 0 to 21 in pregnant and nonpregnant heifers. The interaction of day and reproductive status was significant (P < 0.0001) for the diameter of the largest follicle. The interaction appeared to be due to the growth
NOVEMBER 1986 VOL. 26 NO. 5
651
THERIOGENOLOGY
111(1111)~11~(~1”~~“‘~““~““~
o
5
10
15
20
25
30
Ow Figure 1. The mean diameter C-+SEMI of the largest and the second largest follicles until Day 30 for pregnant heifers (n = 14) and the interovulatory interval for nonpregnant heifers (n = 14). Analysis for day and reproductive status was done from Days 0 to 21 (vertical dotted lines). Mean diameters were significantly different among days for each endpoint. There was a significant effect of reproductive status and a day by reproductive status interaction for the diameter of the largest follicle.
and ovulation of the ovulatory follicle. There was no effect of day from Days 22 to 60 for the diameter of the largest follicle or the diameter of the second largest follicle in the pregnant heifers. The mean diameter of the largest follicle on Day -1 was 16.4 k 0.4 mm for pregnant heifers and 15.9 + 0.7 mm for nonpregnant heifers. Both pregnant and nonpregnant heifers developed a large (>13 mm) follicle by Day 8 (13.3 + 0.7 mm and 14.4 +- 0.6 mm, respectively). There were differences between pregnant and nonpregnant heifers on Days 18 to 21 (P < 0.01). The Day 21 diameter of the largest follicle was 16.1 f 0.6 mm and 12.7 f 0.5 mm for nonpregnant and pregnant heifers, respectively. The Day -1 diameter of the second largest follicle was 7.6 k 0.6 mm for nonpregnant heifers and 7.8 f 0.6 mm for pregnant heifers. The Day 8 diameter for the second largest follicle of pregnant and nonpregnant heifers was 5.5 + 0.5 mm and 5.7 + 0.3 mm, respectively.
652
NOVEMBER 1986 VOL. 26 NO. 5
THERIOGENOLOGY
second largest follicle
5 1111~1111
35
ll1l~lll1
40
45
Ill1
50
Ill1
55
60
Day Figure 2. Mean diameters (k SEM) of the largest and second largest follicles for Days 31 to 60 for pregnant heifers (n = 14). There were no differences among Days 22 to 60.
Data for the 2 to 3 mm, 4 to 6 mm, and 7 to 10 mm diameter categories are depicted from Day -3 to Day 30 and from Days 31 to 60 (Figure 3). The number of follicles 11 to 13 mm, >13 mm and ~2 mm are depicted for the same time periods (Figure 4). Data for the interovulatory interval of nonpregnant heifers are included for comparison. The mean number of follicles 11 to 13 mm increased significantly from 0.1 f 0.2 on Day 1 to 1.0 f 0.2 by Day 18, and it was 0.7 f 0.3 on Day 60. The number of follicles >13 mm was 6.2 f 0.1 by Day 21 and 0.2 + 0.2 on Day 60.
The main effect of day was significant (P < 0.0001) in each of the 2 to 3 mm, 4 to 6 mm, 7 to 10 mm, 11 to 13 mm, and >13 mm follicular diameter categories from Days 0 to 21 when averaged over pregnant and nonpregnant heifers. There was a day by reproductive status interaction (P < 0.001) for the number of follicles >13 mm, but not for any of the other diameter categories. The interaction appeared to be due to growth and ovulation of the ovulatory follicle in the nonpregnant heifers (Figure 4). There was no main effect of day for any follicular endpoint from Days 22 to 60 for the pregnant heifers. There was a main effect of day (P < 0.0001) for the total number of follicles ~2 mm from Days 0 to 21 (pregnant and nonpregnant heifers), but there was no day effect for Days 22 to 60 (pregnant heifers).
NOVEMBER
1986 VOL. 26 NO. 5
653
I-HERIOGENOLOGY
lb
l-5 Day
2b
25
30
Figure 3. Mean numbers (2 SEM) of follicles 2 to 3 mm, 4 to 6 mm, and 7 to 10 mm for the interovulatory interval for nonpregnant heifers (n = 14) and until Day 60 for pregnant heifers (n = 14). Analyses for day and reproductive status were done from
654
NOVEMBER 1986 VOL. 26 NO. 5
THERIOGENOLOGY
25
Pregnant Nonpregnant ---
15
2-3 mm
4-6 mm
7-10 0-r
,
3151111 do III’
4’5 1”’
5b “‘1
mm 5’5““I 60
Day Days 0 to 21 (vertical
dotted
lines).
for Qach endpoint from Days 0 to 21. endpoint from Days 22 to 60.
There WQPQ significant differences among days There WQrQno differences among days for any
NOVEMBER 1986 VOL. 26 NO. 5
655
THERIOGENOLOGY
L
t
1 I
: :
I
,
!’f
;rr7rrrrTT
~13 mm
T
T-rT
Total22 51,
II,,,;,
,,,,,,,,,,,,
5
10
15
,,I,
20
mm 111,
25
30
Day Figure 4. Mean numbers (-+ SEM) of follicles 11 to 13 mm, ‘13 mm, and >2 mm for the interovulatory interval for nonpregnant heifers (n = 14) and until tky 60 for pregnant heifers (n = 14). Analyses for day and reproductive status were done from Days 0 to 21 (vertical dotted lines). There were significant differences among days
656
NOVEMBER
1986 VOL.
26 NO. 5
THERIOGENOLOGY
1
.5
_L
_L
1
1
I 11-13
mm
*13
mm
Total
r2
mm
Day for each endpoint from Days 0 to 21. There was a significant effect of reproductive status for the number of follicles 11 to 13 mm, and there was a significant day by reproductive status interaction for the number of follicles >13 mm. There were no differences among days for any endpoint from Days 22 to 60.
NOVEMBER
1986 VOL.
26 NO. 5
657
THERIOGENOLOGY
The mean length of the interovulatory interval in the four heifers that lost the embryonic vesicle was not significantly different (9) from that of the nonbred heifers differences for any (20.0 + 0.7, n=4; 20.7 + 0.4, n=14). There were no significant follicular endpoint between heifers that lost the conceptus prior to the detection of an embryo proper and nonbred heifers. DISCUSSlON
Data on the number of follicles ~2 mm and the diameters of the largest and second largest follicles were consistent with reports of follicular dynamics in heifers during the estrous cycle (l-6, 12). The mean profiles of diameters of the largest follicles in pregnant and nonpregnant heifers were characterized by the growth of a follicle grew to large follicle (~13 mm) by approximately Day 6. The preovulatory ovulatory size approximately between Days 17 (12 mm) and 20 (16 mm) in the cycling heifers. In the pregnant heifers, the mean diameter of the largest follicle remained constant at approximately 12 mm from Days 15 to 60. The mean diameter of the second largest follicle also remained constant from approximately Days 20 to 60 in the pregnant heifers. The day of the first significant difference in diameter of the largest follicle between pregnant and nonpregnant heifers was Day 18 (three days before ovulation). This agrees with the report that the largest follicle, as determined by marking with India Ink three days prior to estrus, was the follicle which ovulated, whereas the largest follicles marked before that time did not ovulate (2). In a preliminary study in mares (8), the ovulatory follicle of nonpregnant mares surpassed the diameter of the largest follicle in pregnant mares at approximately Day 15; that is, approximately 7 days prior to ovulation. Apparently, the interval from the time the ovulatory follicle becomes the largest follicle to the time of ovulation is much shorter in cattle than in horses. There was a significant day effect in all follicular diameter categories and for the total number of follicles ~2 mm in both pregnant and nonpregnant heifers from Days 0 to 21. The number of follicles 2 to 3 mm, 4 to 8 mm, 7 to 10 mm, and >2 mm in pregnant heifers was remarkably similar to that of the nonpregnant heifers throughout the period associated with the interovulatory interval. The effect of reproductive status in the number of follicles 11 to 13 mm was apparently related to the differences due to reproductive status on Days 18 to 20 for the number of follicles >13 mm. The significant interaction between day and reproductive status in the number of follicles >13 mm was apparently due to the selective growth of the preovulatory follicle beginning on approximately Day 18 in nonpregnant heifers. The mean number of follicles 11 to 13 mm remained at approximately one follicle, while the mean number of follicles >13 mm remained less than 0.5 from approximately Days 17 to 60 in pregnant heifers, which was consistent with a slaughterhouse study (3) that reported low numbers of large follicles on Days 19, 29, and 60. A characteristic of all follicular diameter categories, the diameters of the largest and second largest follicles, as well as the total number of follicles >2 mm, was that the mean numbers of follicles and the mean diameters of the largest and second largest follicles remained relatively constant over approximately Days 17 to 60. In conclusion, the differences in follicular populations between pregnant and nonpregnant heifers from Days 0 to 21 occurred only in the numbers of follicles 11 to 13 mm, >13 mm, and the diameter of the largest follicle. The interactions apparently became manifest approximately 3 days prior to ovulation in the nonpregnant heifers. After Day 21, there were no detectable waves of follicular
NOVEMBER
1986 VOL. 26 NO. 5
THERIOGENOLOGY
activity for any follicular endpoint. Therefore, the differences apparently were related to selective growth and ovulation of the preovulatory follicle in nonpregnant heifers. The differences in ovarian follicular populations between pregnant and nonpregnant heifers were attributed solely to the presence of a physiological mechanism for the selection of an ovulatory follicle in nonpregnant heifers.
REFERENCES 1.
Rajakoski, E. The ovarian follicular system in sexually cyclical, and left-right special reference to seasonal, Endocrinologica, Suppl. 52:1-68 (1960).
2.
Dufour, 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).
3.
Choudary, J.B., Gier, H.T. and Marion, G.B. Cyclic changes follicles. J. Anim. Sci. E:468-471 (1968).
4.
Rexroad, C.E. and Casida, L.E. Ovarian follicular development in cows, sows and ewes in different stages of pregnancy as affected by number of corpora lutea in the same ovary. 3. Anim. Sei. g:1096-1097 (1975).
5.
Matton, P., Adelakoun, V., Couture, Y. and Dufour, J. Growth and replacement of bovine ovarian follicles during the estrous cycle. J. Anim. Sci. g:813-819 (1981).
6.
Pierson, R.A. Theriogenology
7.
Pierson, mare.
8.
Ultrasonic Imaging and Reproductive Ginther, O.J. Equiservices, Cross Plains, WI, 1986 pp. 133-154.
9.
Curran, S., Pierson, R.A. and Ginther, O.J. Ultrasonic appearance of the bovine conceptus on days 10 to 20. J. Am. Vet. Med. Assoc. (in press, 1986).
10.
Curran, S., Pierson, R.A. and Ginther, O.J. Ultrasonic appearance of the bovine conceptus on days 20 to 60. J. Am. Vet. Med. Assoc. (in press, 1986)
11.
SAS User’s Guide: Statistics, 1985 pp. 956.
12.
Staigmiller, (1982).
and Ginther, O.J. 2&495-504 (1984).
Ultrasonography
mature heifers variations.
of
in bovine vesicular
the
bovine
R.A. and Ginther, O.J. Folliculogenesis during the estrous Anim. Reprod. Sci. (submitted 1986).
NOVEMBER
R.B.
Version 5 Edition.
Folliculogenesis
in the bovine.
1986 VOL. 26 NO. 5
Events
Cary, NC.
with Acta
cycle of the
in the
SAS Institute
Theriogenology
ovary.
Mare
Inc.
17: 43-52
659
OVARIAN
POLLICULAR
POPULATIONS
DURING
EARLY
PREGNANCY
IN HEIFERS
R.A. Pierson and O.J. Ginther Department of Veterinary Science University of Wisconsin-Madison Madison, WI 53706 Received
for publication: Accepted:
ApriZ 2, 2986 September 18,
1986
ABSTRACT
Ovarian follicles >2mm were studied in 14 pregnant and 14 nonpregnant Holstein heifers by daily ultrasound examinations. There were significant differences among days, from Day 0 (day of ovulation) to Day 21, in the diameter of the largest follicle and the diameter of the second largest follicle in pregnant and nonpregnant heifers. There was an interaction of day and reproductive status (P < 0.001) for the diameter of the largest follicle. Significant differences among days were also observed in the numbers of follicles 2 to 3 mm, 4 to 6 mm, 7 to 10 mm, 11 to 13 mm, and >13 mm, and the total number of follicles ~2 mm. There was a significant main effect of reproductive status for the number of follicles 11 to 13 mm. An interaction of day and reproductive status was observed for the number of follicles >13 mm, but not for any of the other diameter categories. The effect of reproductive status for number of follicles 11 to 13 mm and the interactions for the number of follicles >13 mm and the diameter of the largest follicle seemed due to the selective growth and ovulation of the follicle destined to ovulate in nonpregnant heifers. The differences in ovarian follicular populations between pregnant and nonpregnant heifers were attributed solely to the presence of a physiological mechanism for the selection of an ovulatory follicle in nonpregnant heifers. There were no significant differences among days for any follicular endpoint during Days 22 to 60 in the pregnant heifers.
Key words:
ovary, follicles,
pregnancy,
bovine
Supported by the College of Agricultural and Life Sciences, University of WisconsinMadison and USDA Grant 84-CRSR-2-2451. The authors wish to thank Mary J. Lindstrom, Department of Statistics and Peter Crump, Department of Computing and Biometry. Appreciation is expressed to Marcia Campbell, Peggy Hoge, Susan Krebs, Lisa Kulick, and Diane Stuber for technical assistance and to the Upjohn Company, Kalamazoo, MI for Lutalyse.
NOVEMBER
1986 VOL.
26 NO. 5
649
THERIOGENOLOGY
INTRODUCTION
Studies on the changing morphology of ovaries in cattle have been limited to examination of slaughterhouse specimens or to transrectal palpation (l-5). No information was found in the literature which compared follicular populations during early pregnancy with the comparable period of the interovulatory interval in cycling heifers. There have been only limited slaughterhouse studies on follicle numbers in which ovaries were sliced and follicles counted on selected, known days of pregnancy (earliest day of examination, Day 19; 3) or in which the stage of pregnancy was estimated by the crown-rump length of the fetus (4). A preliminary study in our laboratory was done to assess the use of ultrasonic imaging for measuring and of the ovaries counting follicles in cattle (6). The three principal components (follicles, corpora lutea, and stroma) were distinguishable, and ultrasonography was judged to be a suitable approach to the study of ovarian changes in cattle and for detection of follicles as small as 2 mm. Diagnostic ultrasonography has been used to study ovarian follicular populations during the estrous cycle in mares (7), including a study in which follicular profiles of pregnant and nonpregnant mares were compared (6). The purpose of the present experiment was to characterize the changes in the ovarian follicular populations during the first 60 days of pregnancy in heifers. Special attention was given to the first 21 days; follicular profiles were compared between pregnant and nonpregnant heifers for a period equivalent to the length of an interovulatory interval in nonpregnant heifers. MATERIALS
AND METHODS
Nulliparous Holstein heifers between 1.5 and 2.0 years of age and weighing 300 to 450 kg were used. The heifers were randomly put into a bred group (n = 19) or a nonbred group (n = 21). Ten of the 19 heifers assigned to the bred group were artificially inseminated approximately 12 hours after first observation of standing estrus if a regressing corpus luteum and a follicle >14 mm were detected by The nine remaining heifers were given prostaglandin F2, ultrasonography (6). (Lutalyse, Upjohn Company, Kalamazoo, MI) when an apparently mature corpus The nine heifers were artificially luteum was detected ultrasonically (6). inseminated 72 hours later if a follicle >14 mm and a regressing corpus luteum were present. A real-time B-mode diagnostic ultrasound instrument (Equisonics 210, Equisonics Inc., Roselle, IL) equipped with a linear-array, 5 MHz transducer designed for intrarectal placement was used for the examinations. Fecal material was removed from the rectum and the transducer was inserted. The transducer was moved along the dorsal surface of the reproductive tract for orientation and then moved laterally to examine the ovaries. Follicles >2 mm were counted. The antra of follicles larger than 10 mm were measured withintegral electronic calipers at the interface of the follicular wall and the follicular fluid. The diameters of follicles smaller than 10 mm were estimated by comparison with the centimeter scale displayed at the side of the ultrasound image. This was done to minimize errors due to loss of identity of individual small follicles during freezing of the image. The day of ovulation (Day 0) was determined by the disappearance of a large (>I1 mm) follicle as described (6). The examinations were made without knowledge of the
650
NOVEMBER
1986 VOL. 26 NO. 5
THERIOGENOLOGY
number of days post ovulation or of the previous day’s results. The reproductive tract was not directly manipulated before or during the ultrasound examination. Examination of the uterus for the presence of a conceptus was done after the ovarian examinations were made. The first day of detection of a discrete nonechogenic structure within the uterine lumen was recorded as the first day of detection of a presumptive embryonic vesicle. A confirmed pregnancy was defined as subsequent progressive elongation of the nonechogenic area within the uterus and the eventual detection of an embryo proper as described (9). The heifers were examined daily from at least three days prior to an ovulation until Day 60 of pregnancy or until at least three days after the nonpregnant heifers returned to estrus and ovulated. Fourteen of the heifers in the bred group maintained an embryo until at least Day 60, so data for the first 14 heifers to complete an interovulatory interval in the nonbred group were compared Four of the bred heifers lost the conceptus prior to the to the pregnant group. detection of an embryo proper and were considered as a separate group. Results specific to the conceptuses are reported elsewhere (9,lO). For descriptive purposes, data for Days -3 to -1 before the first ovulation were added to the data for both pregnant and cycling heifers, and Days +l to +3 after the subsequent ovulation were added to the data for the interovulatory interval of nonpregnant heifers. Follicular patterns were studied by grouping the follicles into diameter ranges of 2 to 3 mm, 4 to 6 mm, 7 to 10 mm, 11 to 13 mm, and >13 mm. Diameters of the largest follicle and second largest follicle were also determined. Data for the nonbred heifers were normalized to the mean length of the interovulatory interval (20.7 ?r 0.4 days, mean + SEM). For heifers with intervals longer than 21 days, the appropriate number of days was randomly deleted; for heifers with intervals shorter than 21 days, data for the appropriate number of days was randomly inserted by averaging the data from the day before and the day after the insert. A separate randomization was done for each follicular diameter category. Statistical analyses were performed using the Statistical Analysis System @AS) by general linear models univariate analyses for repeated measures (11). Specific analyses were performed to determine the effect of day and reproductive status from Days 0 to 21 for the pregnant and nonpregnant heifers. The effect of day was also examined from Days 22 to 60 for the pregnant heifers. Detailed analyses of the day effects during the interovulatory interval in nonpregnant heifers are part of another experiment. The main effects were tested using F tests with degrees of freedom modified by the Greenhouse-Geyser epsilon (11). Days of significant difference due to reproductive status were determined by using univariate t tests for each day. The interactions between day and reproductive status for Days 0 to 21 were examined.
RESULTS
The mean diameters of the largest and second largest follicles are depicted for the interovulatory interval for nonpregnant heifers and up to Day 30 for pregnant heifers (Figure 1). Additionally, profiles are shown for the diameters of the largest and second largest follicles for pregnant heifers from Days 31 to 60 (Figure 2). There were significant differences among days for the diameters of the largest and second largest follicles from Days 0 to 21 in pregnant and nonpregnant heifers. The interaction of day and reproductive status was significant (P < 0.0001) for the diameter of the largest follicle. The interaction appeared to be due to the growth
NOVEMBER 1986 VOL. 26 NO. 5
651
THERIOGENOLOGY
111(1111)~11~(~1”~~“‘~““~““~
o
5
10
15
20
25
30
Ow Figure 1. The mean diameter C-+SEMI of the largest and the second largest follicles until Day 30 for pregnant heifers (n = 14) and the interovulatory interval for nonpregnant heifers (n = 14). Analysis for day and reproductive status was done from Days 0 to 21 (vertical dotted lines). Mean diameters were significantly different among days for each endpoint. There was a significant effect of reproductive status and a day by reproductive status interaction for the diameter of the largest follicle.
and ovulation of the ovulatory follicle. There was no effect of day from Days 22 to 60 for the diameter of the largest follicle or the diameter of the second largest follicle in the pregnant heifers. The mean diameter of the largest follicle on Day -1 was 16.4 k 0.4 mm for pregnant heifers and 15.9 + 0.7 mm for nonpregnant heifers. Both pregnant and nonpregnant heifers developed a large (>13 mm) follicle by Day 8 (13.3 + 0.7 mm and 14.4 +- 0.6 mm, respectively). There were differences between pregnant and nonpregnant heifers on Days 18 to 21 (P < 0.01). The Day 21 diameter of the largest follicle was 16.1 f 0.6 mm and 12.7 f 0.5 mm for nonpregnant and pregnant heifers, respectively. The Day -1 diameter of the second largest follicle was 7.6 k 0.6 mm for nonpregnant heifers and 7.8 f 0.6 mm for pregnant heifers. The Day 8 diameter for the second largest follicle of pregnant and nonpregnant heifers was 5.5 + 0.5 mm and 5.7 + 0.3 mm, respectively.
652
NOVEMBER 1986 VOL. 26 NO. 5
THERIOGENOLOGY
second largest follicle
5 1111~1111
35
ll1l~lll1
40
45
Ill1
50
Ill1
55
60
Day Figure 2. Mean diameters (k SEM) of the largest and second largest follicles for Days 31 to 60 for pregnant heifers (n = 14). There were no differences among Days 22 to 60.
Data for the 2 to 3 mm, 4 to 6 mm, and 7 to 10 mm diameter categories are depicted from Day -3 to Day 30 and from Days 31 to 60 (Figure 3). The number of follicles 11 to 13 mm, >13 mm and ~2 mm are depicted for the same time periods (Figure 4). Data for the interovulatory interval of nonpregnant heifers are included for comparison. The mean number of follicles 11 to 13 mm increased significantly from 0.1 f 0.2 on Day 1 to 1.0 f 0.2 by Day 18, and it was 0.7 f 0.3 on Day 60. The number of follicles >13 mm was 6.2 f 0.1 by Day 21 and 0.2 + 0.2 on Day 60.
The main effect of day was significant (P < 0.0001) in each of the 2 to 3 mm, 4 to 6 mm, 7 to 10 mm, 11 to 13 mm, and >13 mm follicular diameter categories from Days 0 to 21 when averaged over pregnant and nonpregnant heifers. There was a day by reproductive status interaction (P < 0.001) for the number of follicles >13 mm, but not for any of the other diameter categories. The interaction appeared to be due to growth and ovulation of the ovulatory follicle in the nonpregnant heifers (Figure 4). There was no main effect of day for any follicular endpoint from Days 22 to 60 for the pregnant heifers. There was a main effect of day (P < 0.0001) for the total number of follicles ~2 mm from Days 0 to 21 (pregnant and nonpregnant heifers), but there was no day effect for Days 22 to 60 (pregnant heifers).
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I-HERIOGENOLOGY
lb
l-5 Day
2b
25
30
Figure 3. Mean numbers (2 SEM) of follicles 2 to 3 mm, 4 to 6 mm, and 7 to 10 mm for the interovulatory interval for nonpregnant heifers (n = 14) and until Day 60 for pregnant heifers (n = 14). Analyses for day and reproductive status were done from
654
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25
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There WQPQ significant differences among days There WQrQno differences among days for any
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THERIOGENOLOGY
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Day Figure 4. Mean numbers (-+ SEM) of follicles 11 to 13 mm, ‘13 mm, and >2 mm for the interovulatory interval for nonpregnant heifers (n = 14) and until tky 60 for pregnant heifers (n = 14). Analyses for day and reproductive status were done from Days 0 to 21 (vertical dotted lines). There were significant differences among days
656
NOVEMBER
1986 VOL.
26 NO. 5
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1
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Day for each endpoint from Days 0 to 21. There was a significant effect of reproductive status for the number of follicles 11 to 13 mm, and there was a significant day by reproductive status interaction for the number of follicles >13 mm. There were no differences among days for any endpoint from Days 22 to 60.
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The mean length of the interovulatory interval in the four heifers that lost the embryonic vesicle was not significantly different (9) from that of the nonbred heifers differences for any (20.0 + 0.7, n=4; 20.7 + 0.4, n=14). There were no significant follicular endpoint between heifers that lost the conceptus prior to the detection of an embryo proper and nonbred heifers. DISCUSSlON
Data on the number of follicles ~2 mm and the diameters of the largest and second largest follicles were consistent with reports of follicular dynamics in heifers during the estrous cycle (l-6, 12). The mean profiles of diameters of the largest follicles in pregnant and nonpregnant heifers were characterized by the growth of a follicle grew to large follicle (~13 mm) by approximately Day 6. The preovulatory ovulatory size approximately between Days 17 (12 mm) and 20 (16 mm) in the cycling heifers. In the pregnant heifers, the mean diameter of the largest follicle remained constant at approximately 12 mm from Days 15 to 60. The mean diameter of the second largest follicle also remained constant from approximately Days 20 to 60 in the pregnant heifers. The day of the first significant difference in diameter of the largest follicle between pregnant and nonpregnant heifers was Day 18 (three days before ovulation). This agrees with the report that the largest follicle, as determined by marking with India Ink three days prior to estrus, was the follicle which ovulated, whereas the largest follicles marked before that time did not ovulate (2). In a preliminary study in mares (8), the ovulatory follicle of nonpregnant mares surpassed the diameter of the largest follicle in pregnant mares at approximately Day 15; that is, approximately 7 days prior to ovulation. Apparently, the interval from the time the ovulatory follicle becomes the largest follicle to the time of ovulation is much shorter in cattle than in horses. There was a significant day effect in all follicular diameter categories and for the total number of follicles ~2 mm in both pregnant and nonpregnant heifers from Days 0 to 21. The number of follicles 2 to 3 mm, 4 to 8 mm, 7 to 10 mm, and >2 mm in pregnant heifers was remarkably similar to that of the nonpregnant heifers throughout the period associated with the interovulatory interval. The effect of reproductive status in the number of follicles 11 to 13 mm was apparently related to the differences due to reproductive status on Days 18 to 20 for the number of follicles >13 mm. The significant interaction between day and reproductive status in the number of follicles >13 mm was apparently due to the selective growth of the preovulatory follicle beginning on approximately Day 18 in nonpregnant heifers. The mean number of follicles 11 to 13 mm remained at approximately one follicle, while the mean number of follicles >13 mm remained less than 0.5 from approximately Days 17 to 60 in pregnant heifers, which was consistent with a slaughterhouse study (3) that reported low numbers of large follicles on Days 19, 29, and 60. A characteristic of all follicular diameter categories, the diameters of the largest and second largest follicles, as well as the total number of follicles >2 mm, was that the mean numbers of follicles and the mean diameters of the largest and second largest follicles remained relatively constant over approximately Days 17 to 60. In conclusion, the differences in follicular populations between pregnant and nonpregnant heifers from Days 0 to 21 occurred only in the numbers of follicles 11 to 13 mm, >13 mm, and the diameter of the largest follicle. The interactions apparently became manifest approximately 3 days prior to ovulation in the nonpregnant heifers. After Day 21, there were no detectable waves of follicular
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activity for any follicular endpoint. Therefore, the differences apparently were related to selective growth and ovulation of the preovulatory follicle in nonpregnant heifers. The differences in ovarian follicular populations between pregnant and nonpregnant heifers were attributed solely to the presence of a physiological mechanism for the selection of an ovulatory follicle in nonpregnant heifers.
REFERENCES 1.
Rajakoski, E. The ovarian follicular system in sexually cyclical, and left-right special reference to seasonal, Endocrinologica, Suppl. 52:1-68 (1960).
2.
Dufour, 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).
3.
Choudary, J.B., Gier, H.T. and Marion, G.B. Cyclic changes follicles. J. Anim. Sci. E:468-471 (1968).
4.
Rexroad, C.E. and Casida, L.E. Ovarian follicular development in cows, sows and ewes in different stages of pregnancy as affected by number of corpora lutea in the same ovary. 3. Anim. Sei. g:1096-1097 (1975).
5.
Matton, P., Adelakoun, V., Couture, Y. and Dufour, J. Growth and replacement of bovine ovarian follicles during the estrous cycle. J. Anim. Sci. g:813-819 (1981).
6.
Pierson, R.A. Theriogenology
7.
Pierson, mare.
8.
Ultrasonic Imaging and Reproductive Ginther, O.J. Equiservices, Cross Plains, WI, 1986 pp. 133-154.
9.
Curran, S., Pierson, R.A. and Ginther, O.J. Ultrasonic appearance of the bovine conceptus on days 10 to 20. J. Am. Vet. Med. Assoc. (in press, 1986).
10.
Curran, S., Pierson, R.A. and Ginther, O.J. Ultrasonic appearance of the bovine conceptus on days 20 to 60. J. Am. Vet. Med. Assoc. (in press, 1986)
11.
SAS User’s Guide: Statistics, 1985 pp. 956.
12.
Staigmiller, (1982).
and Ginther, O.J. 2&495-504 (1984).
Ultrasonography
mature heifers variations.
of
in bovine vesicular
the
bovine
R.A. and Ginther, O.J. Folliculogenesis during the estrous Anim. Reprod. Sci. (submitted 1986).
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R.B.
Version 5 Edition.
Folliculogenesis
in the bovine.
1986 VOL. 26 NO. 5
Events
Cary, NC.
with Acta
cycle of the
in the
SAS Institute
Theriogenology
ovary.
Mare
Inc.
17: 43-52
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