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The effect of method of GnRH administration and short-term calf removal on ovarian function and reproductive performance in postpartum suckled beef cows administered PGF2α for estrous synchronization
THERIOGENOLOGY THE EFFECT OF METHOD 0~ G~RH ADMINISTRATION AND SHORT-TERM CALF REMOVAL ON OVARIAN FUNCTION AND REPRODUCTIVE PERFORMANCE IN POSTPARTUM SUCKLED BEEF COWS ADMINISTERED PGF ci FOR ESTROUS SYNCHRONIZATION(ayb) 2 T. R. Troxeltc),
G. F. Cmarik(cye), R.& and D. J. Kesler
Ott(d),
T. F. Lock(d)
Department
of Animal Science and Veterinary Clinical Medicine University of Illinois Urbana, Illinois 61801
Received
for publication: Accepted:
April 29, 1983 August 17, 1983
Abstract The first experiment was a 2 x 2 factorial experiment with calf removal (none or short-term) and method of GnRH administration (intramuscularly in saline or subcutaneously in gelatin capsules) as main The durations of the GnRH-induced LH surges were similar among effects. groups but the LH surges were delayed in the cows that received GnRH Calf removal enhanced the GnRHsubcutaneously in gelatin capsules. induced LH release for cows administered GnBH subcutaneously in a gelatin capsule but not for cows administered GnRH intramuscularly in saline. In the second experiment, 191 postpartum suckled beef cows were administered two injections of prostaglandin F2:c(PGF2u) 11 days apart. After the second PGF2a injection, the cows were assigned to a 2 x 2 factorial experiment as in Experiment 1 plus one control group. Short-term calf removal (47 h) began 28 h after the second PGF2u injection. GnRH was administered 30 h after the time of calf removal. The number of cows that ovulated following the time of the GnRH treatment, the number that had abnormal luteal phases and the first-service pregnancy rates among treatment groups within the anestrous and cyclic cows classifications were not significantly different. However, several effects were detected and are reported.
(a) PGF2" and the GnRH were generously
supplied by Dr. J. F. McAllister (The UpJohn Company) and Dr. M. Brown (CEVA Laboratories), respectively. Antisera for radioimmunoassay of LH and progesterone were generously supplied by Drs. G. D. Niswender and 0. D. Sherwood, respectively. (b) This research was conducted as part of regional research project NC-113, "Methods for Improvement of Fertility in Cows Postpartum." (c) Department of Animal Science, University of Illinois, Urbana, IL (d)61801 Veterinary Clinical Medicine, University of Illinois, Urbana, IL (e)61801 Dixon
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Springs
Agricultural
1983 VOL. 20 NO. 4
Center,
Simpson,
IL
62985
417
THERIOGENOLOGY Introduction Poor reproductive performance during the postpartum period is at least partially due to the suckling-induced suppression of ovulation (postpartum anestrous period). This postpartum anestrous period in suckled beef cows varies, but means generally range from 40 to 100 days (l-4). The postpartum anestrous period is shorter for cows milked twice a day than for suckled cows or cows milked four times a day (1,2). of pituitary During the early postpartum period, concentrations luteinizing hormone (LH) have been reported to be similar between suckled and nonsuckled cows (3,5) but suckling appears to suppress concentrations of LH in the blood plasma(6,7). There are plasma LH concentration differences between milked (7-12) and suckled cows (13) as well. It also has been reported that the pituitary regains responsiveness to exogenous gonadotropin releasing hormone (GnRH) by 7 or 8 days postpartum in milked cows (lo), while in the suckled cow the pituitary does not appear to regain responsiveness to exogenous GnRH until about two weeks postpartum (14). Suckling appears to affect the follicular maturation process as well. Even though follicles have been detected about 16 days postpartum in both suckled and milked cows (12, 15-17), the interval from the first detected ovarian follicle to the first postpartum ovulation appears to be delayed by suckling (12, 13). Furthermore, Kesler et al. (13) showed that although ovarian follicles increased in size with days postpartum, estradiol-17B concentrations in plasma did not change. Therefore, suckling appears to suppress the release of pituitary LH which may be necessary to stimulate final maturation of ovarian follicles and subsequent estradiol secretion. Without the increased estradiol secretion, the positive feedback phenomenon of estradiol on the LH surge does not appear to occur (18). Therefore, early postpartum ovarian follicles appear to become atretic rather than ovulate (13). Several treatments have been used to initiate ovarian cycles in anestrous suckled beef cows. These include early weaning (19), limited nursing (20, 21), GnRH treatment (13,22-26), and a combination of sex steroids (26-28). The luteal phase, however, following early weaning (19), limited nursing (20), and GnRH treatment (13, 23, 24) has been shown to be shorter and progesterone secretion is less than during the luteal phase of the normal estrous cycle. These short luteal phases or short estrous cycles appear to be 6 to 12 days long as compared to a normal estrous cycle length of 18 to 23 days. Because short cycles are seen about 70-80% of the time following a GnRH-induced ovulation (22-24, 29), one possible reason for short estrous cycles may be related to the profile of the GnRH-induced LH surge. Trowel et al. (26) has reported that the duration of a GnRHinduced LH surge is approximately one-half the duration of the normal preovulatory surge. The duration of the GnRH-induced LH surge can be as a carrier vehicle (26) or increased by using carboxymethylcellulose by subcutaneously implanting the GnRH in gelatin capsules (30).
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Therefore, the objectives of these experiments were to 1) determine profiles of the GnRH-induced LH released utilizing two methods of administering GnRH with or without calf removal; 2) determine if the method of GnRH administration and if short-term calf removal influence the incidence of ovulation and subsequent corpus luteum function in anestrous postpartum beef cows; and 3) determine the efficacy of these procedures in inducing fertile ovulations when used in combination with a prostaglandin F 01 estrus synchronization procedure for cyclic beef cows. 2 Materials
and Methods
Experiment 1. Twenty postpartum heef cows from the University of Illinois Dixon Springs Agricultural Center were assigned according to days postpartum to a 2 x 2 factorial experiment. Calving occurred in March and days postpartum to GnRH treatment ranged from 49 to 70 with an average of 59.2 + 1.3 days. All cows were managed together and were in good body condition. The main treatment effects were calf removal (none or short-term) and method of G&H administration (intramuscularly in saline or subcutaneously in gelatin capsules). All cows received an intramuscular injection of 25 mg PGF2" 30 h prior to GnRH treatment. Short-term calf removal consisted of a 30-h nonsuckling period beginning at the time of the PGF~N treatment. Calves that were not removed from the other ten cows were allowed to nurse Thirty hours following the PGF2cl treatment and calf removal, ad libitum. 250 pg GnRH were administered either in saline via intramuscular injection (10 cows) or in gelatin capsules via subcutaneous implantation (10 cows). All cows were bled prior to the GnRH administration (time 0) and every hour thereafter for 10 hours. Blood was collected via jugular venipuncture into evacuated tubes, placed in crushed ice immediately after collection and subsequently placed in a cold room (4 C) for 24 h. The serum was then harvested and frozen until assayed. All samples were assayed for LH concentrations. Experiment 2. One hundred and ninety-one postpartum suckled beef cows from the University of Illinois Dixon Springs Agricultural Center were used in this experiment. Calving occurred inFebruary to May and days postpartum to timed breeding ranged from 28 to 112 days with an average of 61.6 + 1.1 days. All cows were managed similarly and were wintered on either corn silage or fescue hay. Condition scores for all cows were determined by one individual on a scale of 1 (extremely thin) to 9 (excessively fat) at the time of the second PGF2a injection. Cows were assigned to five groups in a 2 x 2 factorial design plus one control group. The control group was included to determine the incidence of spontaneous ovulations during the experimental period. The main effects were method of GnRH administration (250 pg intramuscularly in saline or subcutaneously in gelatin capsules) and short-term calf removal (none or 47 h). Treatment groups were as follows: Group I was the control group (no. = 38); Group II received the GnRH intramuscularly in saline (no. = 39); Group III received GnRH subcutaneously
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in gelatin capsules (no. = 38); Group IV received GnRH subcutaneously in saline plus short-term calf removal (no. = 39) and Group V received GnRH subcutaneously in gelatin capsules plus short-term calf removal (no. = 37). In addition, all cows received two injections of PGF2e 11 days apart (25 mg/injection). The time schedule of the treatments is shown in Figure 1. Calves were removed from dams (CR) from groups Calves that were not IV and V 28 h after the second PGF2c? injection. removed (groups I, II and III) were allowed to nurse ad libitum. The GnRH was administered 58 h after the second PGF o. injection or 30 h after calf removal. All cows were then artificgally inseminated 75 h following the second PGF2e injection. Calves of the calf removal groups (IV and V) were returned to their dams following the timed breeding. Bulls were turned in with the cows 18 days after artificial insemination, and the breeding season was 100 days. Blood was collected via jugular venipuncture into evacuated tubes 10 days prior to the first PGF2s injection, immediately prior to each PGF2h injection and prior to GnRH treatment and on day 6 or 7, 11 or 12, and 15 or 16 following the time of GnRH treatment. Blood samples were placed in crushed ice immediately after collection and subsequently placed in a cold room (4 C) for 24 h. The serum was then harvested and was frozen until assayed. All samples were assayed for concentrations of progesterone. All blood samples taken prior to the time of GnRH treatment were used to determine the reproductive status of each cow (anestrous or cyclic). Cows were classified as cyclic if any pre-GnRH treatment sample had progesterone concentrations greater than 1.0 ng/ml. The blood samples taken 6 or 7, 11 or 12, and 15 or 16 days following the time of GnRH treatment were used to determine ovulation response and subsequent corpus luteum function. Cows were considered to have ovulated if progesterone concentration were greater than 0.5 ng/ml on days 6 or 7 after the time of GnRH treatment. This has been demonstrated to be indicative of an ovulation induced by GnRH treatment in anestrous postpartum suckled beef cow (31). Once cows were placed in the cyclic or anestrous categories,
Experimental
Procedure
Figure 1. The treatment and blood sampling (B) schedule for experiment 2. All cows received the two injections of PGF2ti. Additional treatments were Group I--none; Group II--GnRH in saline; Group III--GnRH in gelatin capsules; Group IV--GnRH in saline plus calf removal (CR); and Group V--GnRH in gelatin capsules plus CR.
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the concentrations of LH at the time of GnRH treatment were evaluated. If any cow had concentrations of LH greater than 15 ng/ml, the cow was eliminated from the final analysis. LH serum for Experiments 1 and 2 was quantified Assay of Hormones. by double antibody procedures reported by Niswender et al. (32), except that lz31 was used as the radioactive label. This assay has been validated in our laboratory (26). All samples were assayed in one assay for each experiment, and the intraassay coefficient of variation was 5.5% (pooled mean = 3.6 ngjml) and 4.6% (pooled mean = 3.8 ng/ml) for Experiment 1 and Experiment 2. Serum progesterone for Experiment 2 was determined by radioimmunoassay as previously described and validated in our laboratory (26, 33). All samples from Samples were assayed in duplicate in twelve assays. Intraassay and interassay coone cow were determined in one assay. efficients of variation were 6.5% and 8.6% respectively (pooled mean = Each day that unknowns were determined, two recovery aliquots 2.5 nglml). were included by adding ('H) progesterone to serum from an ovariectomized cow. Subsequently, all samples were adjusted for recovery (86.6% + 1.4). Pools of plasma from an ovariectomized cow and a diestrous Concentrations of procow were assayed in duplicate for each assay. gesterone in plasma from the ovariectomized cow and the diestrous cow averaged 0.2 20.1 ng/ml and 2.5 + 0.1 ng/ml, respectively. Duplicate estimates of hormone concentrations Data Analysis. In Experiment 1 the interval from were averaged prior to analysis. treatment to peak LH release, the peak LH release, the interval from peak LH release to baseline and the interval from baseline LH levels to baseline LH levels were analyzed by analysis of variance (34). The duration of the LH release was defined as the length of time that LH concentrations remained two standard deviations above the pretreatment concenThe GnRH-induced release of LH and the progesterone concentrations. trations of the cows with a luteal phase longer than 15 or 16 days following the time of GnRH treatment were analyzed by split-plot analysis of variance as reported by Gill and Hafs (35) due to repeated sampling of cows. For Experiment 2 the ovulation response, the first-service pregnancy rates and subsequent corpus luteum function were analyzed by a chisquare method as described by Cochran and Cox (36). Standard errors of the difference between treatment and time means were determined from the error mean squares as described by Cochran and Cox (36). Results Experiment 1. Concentrations of LH before and after GnRH treatments are shown in Figure 2. Although the main effects of the method of GnRH administration and calf removal were not significant ( P > 0.25), the change across time following GnRH treatment was signficant (P < 0.01). The durations of the G&H-induced LH surges were similar among the groups (P > O.lO), but the LH surges were delayed in the cows that received GnRH subcutaneously in a gelatin capsule as indicated in Table 1 and by the time-by-method interaction (P < 0.05). When GnRH was administered
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THERIOGENOLOGY
220.
eoo-
x-x
Soline-lM+Calf
e----e
Gelafin Capsuks-SC
x----X
Gelatin Capsules-SC+Colf
Remwd
Remow
180-
160-
='40E \ _EJ 120. 3 KIO-
80-
60.
40.
Hours AfterGnRH Treatment
of LH in serum of postpartum suckled Figure 2. Concentrations beef cows administered GnRH intramuscularly in saline or subcutaneously in gelatin capsules with or without calf removal. Values are the mean of five observations. Standard error of the difference between two treatment means for a given time is 6.1. Standard error of the difference between two time means for one treatment is 11.0.
intramuscularly in saline, cows without calf removal had a slightly In contrast, when G&H was administered higher peak LH release. subcutaneously in a gelatin capsule, calf removal enhanced the GnRHinduced LH release. This is indicated by the three-way interaction (P < 0.01). Experiment 2. Of the 191 cows, 98 (51%) were classified as cyclic and 93 (49%) were classified as anestrus. The final number of cows treated per group are reported in Tables 2 and 3. The average days postpartum for the anestrous cows and the cyclic cows did not differ among groups for both the anestrous cows (57.3 + 1.4 days) and the cyclic cows (65.5 + 1.6 days).
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Table
Main
1.
Peak LH concentrations, interval from treatment to peak, interval from peak to baseline and the duration of the LH surge in postpartum beef cows administered GnRH.
Peak LH (nalml)
Effects
Interval Treatment to Peak (h)
Interval Peak to Baselinea (h)
Durationb (h)
Method of GnRH administration: IM in saline SC in gelatin capsules
183.6 + 34.4'
2.0 _ + 0.2*
5.4 + 0.5
7.4 + 0.5
184.0 + 32.5
2.7 + 0.2*
5.7 _ + 0.3
8.4 + 0.4
161.8 + 26.9 205.8 2 37.5
2.4 + 0.2 2.3 2 0.2
5.4 + 0.5 5.7 + 0.4
7.8 + 0.5 8.0 + 0.4
Calf Removal: None Short-term
aBaseline is defined as two standard deviations bment concentrations (5.6 + 0.2 ng/ml). Interval from treatment to baseline. Sean _t standard error. P < 0.05.
greater
than pretreat-
?
The number of cows that ovulated following the time of GnRH treatment and the number of cows with abnormal luteal phases per treatment group for both the anestrous and cyclic cows are shown in Tables 2 (anestrous cows) and 3 (cyclic cows). The number of cows that ovulated after the time of GnRH treatment was similar (P > 0.10) among groups for both anestrous (mean = 85%) and cyclic cows (mean = 92%). Cows were classified as having an abnormal lureal phase if concentrations of progesterone decreased to less than 1.0 ng/ml on days 11 to 16. Seventy percent of the anestrous cows administered GnRH intramuscularly in saline had abnormal luteal phases, which is similar to previous reports (13, 23, 24). In contrast, 39% of the anestrous cows administered GnRH subcutaneously in gelatin capsules and 33% of the anestrous cows administered G&H intramuscularly in saline in combination with short-term calf removal had abnormal luteal phases. When anestrous cows were administered GnRH subcutaneously in gelatin capsules in combination with short-term calf removal, the incidence of abnormal luteal phases was 80%. The number of cows with abnormal luteal phases, however, was not significantly different (P > 0.10) among groups for both anestrous and cyclic cows. Fifty-one percent of all the anestrous cows had abnormal luteal phases, whereas only 9% of the cyclic cows had abnormal luteal phases (P < 0.01). The postpartum
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interval
to GnRH
1983 VOL. 20 NO. 4
treatment
was similar
(P >O.lO)
for
423
THERIOGENOLOGY
cows that ovulated (56.9 + 1.6 days) and cows that did not ovulate (50.0 2 3.0 days) after GnRH treatment. The postpartum interval to GnRH treatment was also similar (P >O.lO) for cows that had abnormal luteal phases (59.1 + 1.9 days) and cows that did not have abnormal luteal phases (54.4 + 2.6 days). Table 2.
Number of anestrous postpartum beef cows that ovulated after the time of GnRH treatment and the number of cows that had subsequent abnormal luteal phases after GnRH treatment and/or calf removal.
Group
GnP.H
Calf Removal
I II III IV V TOTAL
None Saling-IMa GC-SC Saling-IMa GC-SC
None None None Short-term Short-term
Number of Anestrous Cows 18 11 15 16 12 72
Number of Cows that Ovulate after the Time of GnRH Treatment 16 10 13 12 10 61
Number of Cows with Abnormal Luteal Phase
(89%) (91%) (87%) (75%) (83%) (85%)
7 7 5 4 8 31
(44%) (70%) (39%) (33%) (80%) (51%)
intramuscularly Gelatin capsule-subcutaneously
Table 3.
Groups I II III IV V TOTAL
Number of cyclic postpartum beef cows that ovulated after the time of GnRH treatment and the number of cows that had subsequent abnormal luteal phases after GnRH treatment and/or calf removal.
GnRH None Saling-IMa GC-SC Saline-IMa GC-SCn
Calf Removal None None None Short-term Short-term
Number of Cyclic Cows 15 12 11 9 12 59
Number of Cows that Ovulate after the Time of GnRH Treatment 14 12 9 8 ii 54
(93%) (100%) (82%) (89%) i9zj (92%)
Number of Cows with Abnormal Luteal Phases 0 1 2 1 1 5
(0%) (8%) (22%) (13%) (9%). (9%)
ZIntramuscularly Gelatin capsule - subcutaneously
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Progesterone concentrations in serum following the time of GnRH treatment increased (P < 0.01) across time for both anestrous and cyclic cows that had luteal phases longer than 15 or 16 days. Following the time of GnRH treatment, progesterone concentrations were higher (P < 0.01) for cyclic cows than for anestrous cows that had luteal phases longer than Since there were no differences (P >0.25) in progesterone 15 or 16 days. concentrations between the anestrous and cyclic cows that became pregnant to the timed breeding, the data for pregnant and non-pregnant cows were pooled and summarized in Figure 3.
/?c-z& Anestrus /A
cyclic
6or7
I lorl2 DAYS
15or16
Figure 3. Concentrations of progesterone in serum of anestrous and cyclic cows that ovulated following the time of GnRH treatment and had luteal phases greater than 15 or 16 days. Values are means of 30 and 49 observations. Standard error of the difference between two treatment means for a given time is 0.2. Standard error of the difference between two time means for one treatment is 0.1. The first-service pregnancy rates for both anestrous and cyclic cows are summarized in Tables 4 (anestrous COWS) and 5 (cyclic cows). The first-service pregnancy rate for the anestrous cows was low and similar among the five groups (mean = 11%). The pregnancy rate of the anestrous cows with abnormal luteal phases was 0% whereas the pregnancy rate of the anestrous cows without abnormal lureal phases was 27%. Pregnancy rate of the anestrous cows without calf removal (groups I, II and III) that ovulated after the time of GnRH treatment and did not have abnormal luteal phases was 15%. In contrast, five of the ten cows (50%) with short-term calf removal (groups IV and V) that ovulated after GnRH treatment and did not
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have abnormal luteal phases became pregnant to the timed breeding. For the cyclic cows, the first-service pregnancy rates were higher (mean = 46%; P < 0.01) than for the anestrous cows, but no significant differences (P > 0.10) were detected among the groups.
Table 4.
Group I II III IV V
The number of anestrous postpartum beef cows that became pregnant to the first-service insemination, the interval from calving to pregnancy and the number of cows not pregnant at the end of the breeding season following GnRH treatment and/or short-term calf removal.
GnRH None Salin&-IMa GC-SC Saline-IMa GC-SCb
Calf Removal None None None Shortterm Shortterm
TOTAL
Number of Cows not Pregnant at end of Breeding Season
FirstService Pregnancy Rate
Interval Calving to Pregnancy
18 II 15 16
1 0 2 4
104.6 99.6 106.3 95.2
12
1 (8%)
98.9 + 6.1
2 (17%)
72
8 (11%)
101.1 f 3.1
10 (14%)
Number of Anestrous Cows
(6%) (0%) (13%) (25%)
5 + + +
6.2 7.1 7.8 7.4
1 2 2 3
(6%) (18%) (13%) (19%)
?Intramuscularlv b Gelatin capsule - subcutaneously
Table 5.
Group I II III IV V
The number of cyclic postpartum beef cows that became pregnant to the first-service insemination, the interval from calving to pregnancy and the number of cows not pregnant at the end of the breeding season following GnRH treatment and/or short-term calf removal.
GnRH
Calf Removal
None
None
;,a;;~-"~ Saline-Ma
;,o;~ Shortterm Shortterm
GC-SCb
Number of Cyclic Cows
TOTAL
FirstService Pregnancy Rate (47%) (42%) (27%) (44%)
Interval Calving to Pregnancy
Number of cows not Pregnant at end of Breeding Season
93.8% 89.52 94.35 83.75
1 0 1 3
15 12 11 9
7 5 3 4
11.7 10.1 12.2 11.9
(7%) (0%) (9%) (33%)
12
8 (67%)
05.4-k - 9.3
0 (0%)
59
27 (46%)
89.8+ 4.9
5 (9%)
~Intramuscularly Gelatin capsule - subcutaneously
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THERIOGENOLOGY The interval from parturition to pregnancy was not different (P > 0.10) among groups for both anestrous and cyclic cows. Anestrous cows averaged 101.1 + 3.1 days and cyclic cows averaged 89.8 + 4.9 days from calving to pregnancy. The number of cows not pregnant at the end of the breeding season was also similar among groups for both anestrous and cyclic cows (Tables 4 and 5). There were significant differences in condition scores and they are summarized in Table 6. The anestrous cows had a lower condition score (4.3 + 0.1) than the cyclic cows (4.7 + 0.1; P < 0.05). In addition, the cows that conceived to the timed artificial insemination had a higher condition score than those that did not conceive to the artificial insemination (P < 0.01).
Table
6.
Condition scores for the cyclic and anestrous postpartum beef cows for when pregnancy occurred and for all cows not pregnant at the end of the breeding season.
Mean + S.E.a Reproductive
Status
Before
Timed
Breeding:
Cyclic Anestrus Pregnancy
4.7 + 0.1* 4.3 + 0.1*
by:
Timed breeding Natural breeding Not pregnant
at the end of the breeding
4.7 + 0.2x"~~ 4.3 T 0.1** season
4.2 2 0.2
$Standard error. P < 0.05. **P i 0.01.
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THERIOGENOLOGY Discussion Kesler and coworkers (30, 37) have suggested that the duration of the GnRH-induced LH surge may be related to the ovulation response and to subsequent corpus luteum function. We have since demonstrated that subcutaneous administration of 250 1~g GnRH in carboxymethylcellulose or in gelatin capsules induced an LH surge more similar in duration to a preovulatory LH surge than intramuscular administration of GnRH in saline (26). In this study, subcutaneous administration of GnRH in gelatin capsules delayed the LH surge but only prolonged the duration by about one hour. The effect of calf removal on the GnRH-induced LH release is controversial. Inskeep et al. (42) reported that short-term calf removal had no effect on the GnRH-induced LH release when 300 1.18GnRH in saline was administered intramuscularly. Troxel et al. (26), however, reported that short-term calf removal enhanced the GnRH-induced LH surge following subcutaneous administration of 250 1-lgGnRH in carboxymethylcellulose. The data from this study would appear to support both reports. Shortterm calf removal did not appear to affect the GnRH-induced LH release when GnRH was administered intramuscularly. However, when GnRH was administered in a method that has been demonstrated to prolong the GnRH-induced LH release (subcutaneously in carboxymethylcellulose or gelatin capsules), short-term calf removal enhanced the GnRH-induced LH release. The effect of short-term calf removal and the method of GnRH administration on the ovulation response of the anestrous cows was difficult to evaluate in this study since 89% of the anestrous cows not administered GnRH ovulated following the time of GnRH treatment. Short-term calf removal, however, has been previously shown to increase the ovulation response to GnRH treatment in anestrous postpartum suckled beef cows (26). Fertility of the anestrous cows was generally low. However, when anestrous cows with abnormal luteal phases, who had a pregnancy rate of O%, were removed, the first-service pregnancy rate was 27%. Therefore, at least some ova released after GnRH-induced ovulations appear to be Low pregnancy rates in anestrous cows incapable of being fertilized. duced to ovulate with GnRH treatment therefore appear to be largely due to abnormal luteal phases. It was difficult to discern the effect of method of GnRH administration on corpus luteum function of the anestrous cows for two reasons. First, the GnRH-induced LH surges were not greatly different between the two methods of administering GnRH in this study. Second, the effect of method of GnRH administration was confounded with short-term calf removal in the anestrous cows. Without short-term calf removal, there were 31% fewer abnormal luteal phases when GnRH was administered subcutaneously in gelatin capsules than when GnRH was administered intramuscularly in saline. With short-term calf removal, there were 47% more abnormal luteal phases when GnRH was administered subcutaneously in gelatin capsules than The reason for when GnRH was administered intramuscularly in saline. this difference is not clear.
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The progesterone concentrations in the anestrous cows that had luteal phases longer than 15 or 16 days were lower than in cyclic cows. The same phenomenon has been reported in postpartum cows administered indomethacin to prolong the luteal phase (43). Therefore, we have reported two types of luteal dysfunction. We called the first type "abnormal luteal phase," which were luteal phases shorter than 15 or 16 days. The other type of luteal dysfunction is suppressed progesterone secretion from corpora lutea that had a lifespan longer than 15 or 16 days. We evaluated the effect of these not detect any adverse effects.
treatments
on cyclic
cows and did
The results in the cows that received only PGF2a also merit attention. In a study using over 5,000 dairy cows, Macmillan and Day (38) reported a higher pregnancy rate in cows treated with PGF c1 than in untreated cows (69% vs 60%). A re-examination of publishe 2 reports using heifers and suckled beef cows shows that an enhanced fertility effect may have also occurred in other PGF2a studies (39-41). Roche (41) reported a calving rate of 7% in cows with inactive ovaries treated with a prostaglandin analogue. In our study, 89% of the anestrous cows administered only two injections of PGF2d ovulated and 6% of these cows became pregIn addinant to the timed insemination following the PGF2a injections. tion to PGF2a, the control cows also received considerable stimulation from handling and from being exposed to cows in estrus. Further investigation is needed to better understand the possibility of an effect on fertility by PGF2c(_ In summary, the effect of the method of GnRH treatment and shortterm calf removal on the induction of ovulation and on the incidence of abnormal luteal phases was difficult to discern. Two types of luteal dysfunctions were observed: abnormal luteal phases, which were luteal phases shorter than 15 or 16 days, and suppressed progesterone secretion by corpora lutea with lifespans longer than 15 or 16 days. At least some ova released after GnRH-induced ovulations were capable of being fertilized but pregnancy was not maintained in many cases because of abnormal luteal phases.
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3.
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4.
Short, R. E., Bellows, R. A.,Moody, E. L. and Howland, B. E. Effects of suckling and mastectomy on bovine postpartum reproduction. J. Anim. Sci. -34~70-74. (1972).
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6.
Carruthers, T. D., Resner, J. S., Convey, E. M. and Hafs, H. D. Hypothalamic GnRH and pituitary function in suckled and non-suckledI{olstein cows. J. Anim. Sci. -47(Suppl. 1): 349. (Abstr.) (1978).
per-
Short, R. E., Randel, R. D., Staigmiller, R. B. and Bellows, R. A. Factors affecting estrogen-induced LH release in the cow. Biol. Reprod. -21:683-689. (1979). 8.
Erb, R. E., Surve, A. H., Callahan, C. J., Randel, R. D. and Garverick, H. A. Reproductive steroids in the bovine. VIII. Changes postpartum. J. Anim. Sci. -33:1060-1071. (1971).
9.
Echternkamp, S. E. and Hansel, W. Concurrent changes in the bovine plasma hormone levels prior to and during the first postpartum estrous cycle. J. Anim. Sci. -37:1362-1370. (1973).
10.
Kesler, D. J., Garverick, H. A., Youngquist, R. S., Elmore, R. G. and Bierschwal, C. J. Effect of days postpartum and endogenous reproductive hormones on GnRH-induced LH release in dairy cows. J. Anim. Sci. -46:797-803. (1977).
11.
Goodale, W. S., Garverick, H. A., Kesler, D. J., Bierschwal, C. J., Elmore, R. G. andyoungquist, R. S. Transitory hormone changes J. Dairy Sci. -61:740-746. (1978). in plasma in postpartum cows.
12.
Kesler, D. J., Garverick, H. A., Bierschwal, C. J., Elmore, R. G. hormones associated with and Youngquist, R. S. Reproductive normal and abnormal changes in ovarian follicles in postpartum dairy cows. J. Dairy Sci. a:1290-1296. (1979).
430
QCTOBER
1983 VOL. 20 NO. 4
THERIOGENOLOGY
13.
Kesler, D. J., Troxel, T. R. and Hixon, D. L. partum and exogenous GnRH on reproductive changes in postpartum suckled beef cows. 13:287-296. (1980).
14.
Irvin, H. J., Garverick, H. A., Zaied, A. A., Kesler, D. J., Day, B. N. and Youngquist, R. S. LH response to GnRH in postpartum suckled beef cows. J. Anim. Sci. g:(Suppl. 1):172. (Abstr.) (1977).
15.
Callahan, C. J., Erb, R. E., Surve, A. H. and Randel, R. D. Variables influencing ovarian cycles in postpartum dairy cows. J. Anim. Sci. -33:1053-1059. (1971).
16.
Oxenreider, S. L. and Wagner, W. C. Effects of lactation and energy intake on postpartum ovarian activity in the cow. J. Anim. Sci. -33:1026-1031. (1971).
17.
Wagner, W. C. and Oxenreider, S. L. Endocrine parturition. .I. Anim. Sci. 32(Suppl.):l.
18.
Hauger, R. L., Karsh, P. J. and Foster, D. L. A new concept for control of the estrous cycle of the ewe based on the temporal relationship between luteinizing‘hormone, estradiol and progesterone in peripheral serum and evidence that progesterone inhibits tonic LH secretion. Endocrinology %:807-817. (1977).
19.
Odde,
20.
Flood, P. F., Manns, J. G., Humphrey, W. D. and Mapletoft, R. J. The first corpus luteum of the post-partum beef cows. Prog. for Sot. Study of Fertil., p. 30. (Abstr.) (1979).
21.
Randel, R. D. and Welker, G. A. calf performance. J. Anim.
22.
Britt, J. H., Kiser, T. E., Seguin, B. E., Hafs, H. D., Oxender, W. D. and Ritchie, H. D. Fertility after GnRH and PGF2" in suckling cows. J. Anim. Sci. 41:345 (Abstr.). (1975).
23.
Webb,
24.
Lishman, A. W., Allison, P. M. J., Fogwell, R. L., Butcher, R. L. and Inskeep, E. K. Follicular development and function of induced corpora lutea in underfed postpartum anestrous beef cows. J. Anim. Sci. -48~867-075. (1979).
25.
Fonesca, F. A., Britt, J. H., Kosugiyama, M. I;., Ritchie, H. D. and Dillard, E. V. Ovulation, ovarian function, and reproductive performance after treatments with GnRH in postpartum suckled beef cows. Theriogenology (1980). -13:171-181.
OCTOBER
Effect of days posthormone and ovarian Theriogenology
physiology (1971).
following
K. G., Ward, H. S., Kiracofe, G. H., McKee, R. M. and Kittok, R. J. Short estrous cycles and associated serum progesterone levels in beef cows. Theriogenology 14:105-112. (1980).
Once daily suckling effects on cowSci. -43:301 (Abstr.). (1976).
R., Lamming, G. E., Haynes, N. B., Hafs, H. D. and Manns, J. G. Response of cyclic and postpartum suckled cows to injections of synthetic LB-RH. J. Reprod. Fertil. -50:203-210. (1977).
1983 VOL. 20 NO. 4
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THERIOGENOLOGY
26.
ovuTroxel, T. R., Kesler, D. J., Noble, R. C. and Carlin, S. E. lation and reproductive hormones following steroid pretreatsuckled beef cows. ment, calf removal and GnRH in postpartum J. Anim. Sci. -51:652-659. (1980).
27.
Walters, D. L., Songster, B., Valencia, M., Burrell, W. C. and Steroids in conjunction with 48 hr calf Wiltbank, J. N. removal or early weaning in thin anestrous cows. .I. Anim. Sci. -45 (Suppl. 1):215. (1977).
28.
Smith, M. F., Burrell, W. C., Shipp, L. D., Short, L. R., Songster, Hormone treatments and use of calf W. M. and Wiltbank, J. N. J. Anim. Sci. %:1285-1294. removal in postpartum beef cows. (1979).
29.
D. S. Ovulation, Britt, J. H., Kittok, R. J. and Harrison, and endocrine response after GnRH in early postpartum Anim. Sci. -39:915-919. (1974).
30.
Effect Kesler, D. J. and Vincent, D. L. tion on luteinizing hormone release hormone. J. Dairy Sci. =:2121-2125.
31.
Kesler, D. J., Weston, P. G., Pimental, C. A., Troxel, T. R., Diminution of the -___ in vitro Vincent, D. L. and Hixon, J. E. response to luteinizing hormone by corpora lutea induced by gonadotropin releasing hormone treatment of postpartum suckled beef cows. J. Anim. Sci. -53~749-754. (1981).
32.
Niswender, G. D., Reichert, Jr., L. E., Midgley, Jr., A. R. and Radioimmunoassay for bovine and ovine Nalbandov, A. V. luteinizing hormone. Endocrinology %:1166-1173. (1969).
33.
Wiseman, B. S., Vincent, D. L., Thomford, P. J., Scheffrahn, N. S., Changes in porcine, ovine, Sargent, G. F. and Kesler, D. J. bovine and equine blood progesterone concentrations between collection and centrifugation. Anim. Reprod. Sci. >:157-165. (1983).
34.
Principles and Procedures Steel, R. G. D. and Torrie, J. H. Statistics. McGraw-Hill, New York (1960).
35.
Gill,
36.
Experimental Designs Cochran, W. G. and Cox, G. M. John Wiley and Sons, Inc., New York (1957).
37.
Kesler, D. J., Garverick, H. A., Youngquist, R. S., Elmore, R. r. Ovarian and endocrine responses and and Bierschwal, C. J. reproductive performance following GnRH treatment in early postpartum dairy cows. Theriogenology 9:363-369. (1978).
432
estrus cows.
of carrier and administraby gonadotropin releasing (1980).
J. L. and Hafs, H. D. Analysis of repeated animals. J. Anim. Sci. -33:331-336. (1971).
OCTOBER
of
measurements
of
(2nd Ed.).
1983VOL.20NO.4
THERIOGENOLOGY
38.
Macmillan, K. L. and Day, A. M. Prostaglandin F cx - A fertility drug in dairy cattle? Theriogenology 18.243-253. (1982). _.
39.
Hafs,
40.
Synchronization of oestrus and subsequent fertility Hearnshaw, H. in cattle, using the prostaglandin F2u analogue, ICI 80996 Aust. .I. Exper. Agric. and Anim. Husb. -10: (cloprostenol). 437-444. (1976).
41.
Roche, J. F. Fertility in cows after treatment with a prostaglandin analogue with or without progesterone. J. Reprod. Fert. (1976). -46:341-345.
42.
Inskeep, E. K., Lishman, A. W., Butcher, R. L., and Allison, S. M. J. Effect of short-term removal of the calf on release of LH, ovulation and CL function following GnRH in beef cows. J. Anim. Sci. -45(Suppl. 1):171. (Abstr.) (1971).
43.
to Troxel, T. R. and Kesler, D. J. The ability of indomethacin alter prostaglandin metabolite concentrations and to enhance the function of corpora lutea induced in postpartum suckled beef cows. J. Anim. Sci. (Submitted).
OCTOBER
H. D. and Manns, J. G. Onset of oestrus and fertility of dairy heifers and suckled beef cows treated with prostaglandin F2d. Anim. Prod. -21:13-20. (1975).
1983 VOL. 20 NO. 4
433
G. F. Cmarik(cye), R.& and D. J. Kesler
Ott(d),
T. F. Lock(d)
Department
of Animal Science and Veterinary Clinical Medicine University of Illinois Urbana, Illinois 61801
Received
for publication: Accepted:
April 29, 1983 August 17, 1983
Abstract The first experiment was a 2 x 2 factorial experiment with calf removal (none or short-term) and method of GnRH administration (intramuscularly in saline or subcutaneously in gelatin capsules) as main The durations of the GnRH-induced LH surges were similar among effects. groups but the LH surges were delayed in the cows that received GnRH Calf removal enhanced the GnRHsubcutaneously in gelatin capsules. induced LH release for cows administered GnBH subcutaneously in a gelatin capsule but not for cows administered GnRH intramuscularly in saline. In the second experiment, 191 postpartum suckled beef cows were administered two injections of prostaglandin F2:c(PGF2u) 11 days apart. After the second PGF2a injection, the cows were assigned to a 2 x 2 factorial experiment as in Experiment 1 plus one control group. Short-term calf removal (47 h) began 28 h after the second PGF2u injection. GnRH was administered 30 h after the time of calf removal. The number of cows that ovulated following the time of the GnRH treatment, the number that had abnormal luteal phases and the first-service pregnancy rates among treatment groups within the anestrous and cyclic cows classifications were not significantly different. However, several effects were detected and are reported.
(a) PGF2" and the GnRH were generously
supplied by Dr. J. F. McAllister (The UpJohn Company) and Dr. M. Brown (CEVA Laboratories), respectively. Antisera for radioimmunoassay of LH and progesterone were generously supplied by Drs. G. D. Niswender and 0. D. Sherwood, respectively. (b) This research was conducted as part of regional research project NC-113, "Methods for Improvement of Fertility in Cows Postpartum." (c) Department of Animal Science, University of Illinois, Urbana, IL (d)61801 Veterinary Clinical Medicine, University of Illinois, Urbana, IL (e)61801 Dixon
OCTOBER
Springs
Agricultural
1983 VOL. 20 NO. 4
Center,
Simpson,
IL
62985
417
THERIOGENOLOGY Introduction Poor reproductive performance during the postpartum period is at least partially due to the suckling-induced suppression of ovulation (postpartum anestrous period). This postpartum anestrous period in suckled beef cows varies, but means generally range from 40 to 100 days (l-4). The postpartum anestrous period is shorter for cows milked twice a day than for suckled cows or cows milked four times a day (1,2). of pituitary During the early postpartum period, concentrations luteinizing hormone (LH) have been reported to be similar between suckled and nonsuckled cows (3,5) but suckling appears to suppress concentrations of LH in the blood plasma(6,7). There are plasma LH concentration differences between milked (7-12) and suckled cows (13) as well. It also has been reported that the pituitary regains responsiveness to exogenous gonadotropin releasing hormone (GnRH) by 7 or 8 days postpartum in milked cows (lo), while in the suckled cow the pituitary does not appear to regain responsiveness to exogenous GnRH until about two weeks postpartum (14). Suckling appears to affect the follicular maturation process as well. Even though follicles have been detected about 16 days postpartum in both suckled and milked cows (12, 15-17), the interval from the first detected ovarian follicle to the first postpartum ovulation appears to be delayed by suckling (12, 13). Furthermore, Kesler et al. (13) showed that although ovarian follicles increased in size with days postpartum, estradiol-17B concentrations in plasma did not change. Therefore, suckling appears to suppress the release of pituitary LH which may be necessary to stimulate final maturation of ovarian follicles and subsequent estradiol secretion. Without the increased estradiol secretion, the positive feedback phenomenon of estradiol on the LH surge does not appear to occur (18). Therefore, early postpartum ovarian follicles appear to become atretic rather than ovulate (13). Several treatments have been used to initiate ovarian cycles in anestrous suckled beef cows. These include early weaning (19), limited nursing (20, 21), GnRH treatment (13,22-26), and a combination of sex steroids (26-28). The luteal phase, however, following early weaning (19), limited nursing (20), and GnRH treatment (13, 23, 24) has been shown to be shorter and progesterone secretion is less than during the luteal phase of the normal estrous cycle. These short luteal phases or short estrous cycles appear to be 6 to 12 days long as compared to a normal estrous cycle length of 18 to 23 days. Because short cycles are seen about 70-80% of the time following a GnRH-induced ovulation (22-24, 29), one possible reason for short estrous cycles may be related to the profile of the GnRH-induced LH surge. Trowel et al. (26) has reported that the duration of a GnRHinduced LH surge is approximately one-half the duration of the normal preovulatory surge. The duration of the GnRH-induced LH surge can be as a carrier vehicle (26) or increased by using carboxymethylcellulose by subcutaneously implanting the GnRH in gelatin capsules (30).
418
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1983 VOL. 20 NO. 4
THERIOGENOLOGY
Therefore, the objectives of these experiments were to 1) determine profiles of the GnRH-induced LH released utilizing two methods of administering GnRH with or without calf removal; 2) determine if the method of GnRH administration and if short-term calf removal influence the incidence of ovulation and subsequent corpus luteum function in anestrous postpartum beef cows; and 3) determine the efficacy of these procedures in inducing fertile ovulations when used in combination with a prostaglandin F 01 estrus synchronization procedure for cyclic beef cows. 2 Materials
and Methods
Experiment 1. Twenty postpartum heef cows from the University of Illinois Dixon Springs Agricultural Center were assigned according to days postpartum to a 2 x 2 factorial experiment. Calving occurred in March and days postpartum to GnRH treatment ranged from 49 to 70 with an average of 59.2 + 1.3 days. All cows were managed together and were in good body condition. The main treatment effects were calf removal (none or short-term) and method of G&H administration (intramuscularly in saline or subcutaneously in gelatin capsules). All cows received an intramuscular injection of 25 mg PGF2" 30 h prior to GnRH treatment. Short-term calf removal consisted of a 30-h nonsuckling period beginning at the time of the PGF~N treatment. Calves that were not removed from the other ten cows were allowed to nurse Thirty hours following the PGF2cl treatment and calf removal, ad libitum. 250 pg GnRH were administered either in saline via intramuscular injection (10 cows) or in gelatin capsules via subcutaneous implantation (10 cows). All cows were bled prior to the GnRH administration (time 0) and every hour thereafter for 10 hours. Blood was collected via jugular venipuncture into evacuated tubes, placed in crushed ice immediately after collection and subsequently placed in a cold room (4 C) for 24 h. The serum was then harvested and frozen until assayed. All samples were assayed for LH concentrations. Experiment 2. One hundred and ninety-one postpartum suckled beef cows from the University of Illinois Dixon Springs Agricultural Center were used in this experiment. Calving occurred inFebruary to May and days postpartum to timed breeding ranged from 28 to 112 days with an average of 61.6 + 1.1 days. All cows were managed similarly and were wintered on either corn silage or fescue hay. Condition scores for all cows were determined by one individual on a scale of 1 (extremely thin) to 9 (excessively fat) at the time of the second PGF2a injection. Cows were assigned to five groups in a 2 x 2 factorial design plus one control group. The control group was included to determine the incidence of spontaneous ovulations during the experimental period. The main effects were method of GnRH administration (250 pg intramuscularly in saline or subcutaneously in gelatin capsules) and short-term calf removal (none or 47 h). Treatment groups were as follows: Group I was the control group (no. = 38); Group II received the GnRH intramuscularly in saline (no. = 39); Group III received GnRH subcutaneously
OCTOBER
1983 VOL. 20 NO. 4
419
THERIOGENOLOGY
in gelatin capsules (no. = 38); Group IV received GnRH subcutaneously in saline plus short-term calf removal (no. = 39) and Group V received GnRH subcutaneously in gelatin capsules plus short-term calf removal (no. = 37). In addition, all cows received two injections of PGF2e 11 days apart (25 mg/injection). The time schedule of the treatments is shown in Figure 1. Calves were removed from dams (CR) from groups Calves that were not IV and V 28 h after the second PGF2c? injection. removed (groups I, II and III) were allowed to nurse ad libitum. The GnRH was administered 58 h after the second PGF o. injection or 30 h after calf removal. All cows were then artificgally inseminated 75 h following the second PGF2e injection. Calves of the calf removal groups (IV and V) were returned to their dams following the timed breeding. Bulls were turned in with the cows 18 days after artificial insemination, and the breeding season was 100 days. Blood was collected via jugular venipuncture into evacuated tubes 10 days prior to the first PGF2s injection, immediately prior to each PGF2h injection and prior to GnRH treatment and on day 6 or 7, 11 or 12, and 15 or 16 following the time of GnRH treatment. Blood samples were placed in crushed ice immediately after collection and subsequently placed in a cold room (4 C) for 24 h. The serum was then harvested and was frozen until assayed. All samples were assayed for concentrations of progesterone. All blood samples taken prior to the time of GnRH treatment were used to determine the reproductive status of each cow (anestrous or cyclic). Cows were classified as cyclic if any pre-GnRH treatment sample had progesterone concentrations greater than 1.0 ng/ml. The blood samples taken 6 or 7, 11 or 12, and 15 or 16 days following the time of GnRH treatment were used to determine ovulation response and subsequent corpus luteum function. Cows were considered to have ovulated if progesterone concentration were greater than 0.5 ng/ml on days 6 or 7 after the time of GnRH treatment. This has been demonstrated to be indicative of an ovulation induced by GnRH treatment in anestrous postpartum suckled beef cow (31). Once cows were placed in the cyclic or anestrous categories,
Experimental
Procedure
Figure 1. The treatment and blood sampling (B) schedule for experiment 2. All cows received the two injections of PGF2ti. Additional treatments were Group I--none; Group II--GnRH in saline; Group III--GnRH in gelatin capsules; Group IV--GnRH in saline plus calf removal (CR); and Group V--GnRH in gelatin capsules plus CR.
420
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1983VOL: 20N0.4
THERIOGENOLOGY
the concentrations of LH at the time of GnRH treatment were evaluated. If any cow had concentrations of LH greater than 15 ng/ml, the cow was eliminated from the final analysis. LH serum for Experiments 1 and 2 was quantified Assay of Hormones. by double antibody procedures reported by Niswender et al. (32), except that lz31 was used as the radioactive label. This assay has been validated in our laboratory (26). All samples were assayed in one assay for each experiment, and the intraassay coefficient of variation was 5.5% (pooled mean = 3.6 ngjml) and 4.6% (pooled mean = 3.8 ng/ml) for Experiment 1 and Experiment 2. Serum progesterone for Experiment 2 was determined by radioimmunoassay as previously described and validated in our laboratory (26, 33). All samples from Samples were assayed in duplicate in twelve assays. Intraassay and interassay coone cow were determined in one assay. efficients of variation were 6.5% and 8.6% respectively (pooled mean = Each day that unknowns were determined, two recovery aliquots 2.5 nglml). were included by adding ('H) progesterone to serum from an ovariectomized cow. Subsequently, all samples were adjusted for recovery (86.6% + 1.4). Pools of plasma from an ovariectomized cow and a diestrous Concentrations of procow were assayed in duplicate for each assay. gesterone in plasma from the ovariectomized cow and the diestrous cow averaged 0.2 20.1 ng/ml and 2.5 + 0.1 ng/ml, respectively. Duplicate estimates of hormone concentrations Data Analysis. In Experiment 1 the interval from were averaged prior to analysis. treatment to peak LH release, the peak LH release, the interval from peak LH release to baseline and the interval from baseline LH levels to baseline LH levels were analyzed by analysis of variance (34). The duration of the LH release was defined as the length of time that LH concentrations remained two standard deviations above the pretreatment concenThe GnRH-induced release of LH and the progesterone concentrations. trations of the cows with a luteal phase longer than 15 or 16 days following the time of GnRH treatment were analyzed by split-plot analysis of variance as reported by Gill and Hafs (35) due to repeated sampling of cows. For Experiment 2 the ovulation response, the first-service pregnancy rates and subsequent corpus luteum function were analyzed by a chisquare method as described by Cochran and Cox (36). Standard errors of the difference between treatment and time means were determined from the error mean squares as described by Cochran and Cox (36). Results Experiment 1. Concentrations of LH before and after GnRH treatments are shown in Figure 2. Although the main effects of the method of GnRH administration and calf removal were not significant ( P > 0.25), the change across time following GnRH treatment was signficant (P < 0.01). The durations of the G&H-induced LH surges were similar among the groups (P > O.lO), but the LH surges were delayed in the cows that received GnRH subcutaneously in a gelatin capsule as indicated in Table 1 and by the time-by-method interaction (P < 0.05). When GnRH was administered
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1983 VOL. 20 NO. 4
421
THERIOGENOLOGY
220.
eoo-
x-x
Soline-lM+Calf
e----e
Gelafin Capsuks-SC
x----X
Gelatin Capsules-SC+Colf
Remwd
Remow
180-
160-
='40E \ _EJ 120. 3 KIO-
80-
60.
40.
Hours AfterGnRH Treatment
of LH in serum of postpartum suckled Figure 2. Concentrations beef cows administered GnRH intramuscularly in saline or subcutaneously in gelatin capsules with or without calf removal. Values are the mean of five observations. Standard error of the difference between two treatment means for a given time is 6.1. Standard error of the difference between two time means for one treatment is 11.0.
intramuscularly in saline, cows without calf removal had a slightly In contrast, when G&H was administered higher peak LH release. subcutaneously in a gelatin capsule, calf removal enhanced the GnRHinduced LH release. This is indicated by the three-way interaction (P < 0.01). Experiment 2. Of the 191 cows, 98 (51%) were classified as cyclic and 93 (49%) were classified as anestrus. The final number of cows treated per group are reported in Tables 2 and 3. The average days postpartum for the anestrous cows and the cyclic cows did not differ among groups for both the anestrous cows (57.3 + 1.4 days) and the cyclic cows (65.5 + 1.6 days).
422
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Table
Main
1.
Peak LH concentrations, interval from treatment to peak, interval from peak to baseline and the duration of the LH surge in postpartum beef cows administered GnRH.
Peak LH (nalml)
Effects
Interval Treatment to Peak (h)
Interval Peak to Baselinea (h)
Durationb (h)
Method of GnRH administration: IM in saline SC in gelatin capsules
183.6 + 34.4'
2.0 _ + 0.2*
5.4 + 0.5
7.4 + 0.5
184.0 + 32.5
2.7 + 0.2*
5.7 _ + 0.3
8.4 + 0.4
161.8 + 26.9 205.8 2 37.5
2.4 + 0.2 2.3 2 0.2
5.4 + 0.5 5.7 + 0.4
7.8 + 0.5 8.0 + 0.4
Calf Removal: None Short-term
aBaseline is defined as two standard deviations bment concentrations (5.6 + 0.2 ng/ml). Interval from treatment to baseline. Sean _t standard error. P < 0.05.
greater
than pretreat-
?
The number of cows that ovulated following the time of GnRH treatment and the number of cows with abnormal luteal phases per treatment group for both the anestrous and cyclic cows are shown in Tables 2 (anestrous cows) and 3 (cyclic cows). The number of cows that ovulated after the time of GnRH treatment was similar (P > 0.10) among groups for both anestrous (mean = 85%) and cyclic cows (mean = 92%). Cows were classified as having an abnormal lureal phase if concentrations of progesterone decreased to less than 1.0 ng/ml on days 11 to 16. Seventy percent of the anestrous cows administered GnRH intramuscularly in saline had abnormal luteal phases, which is similar to previous reports (13, 23, 24). In contrast, 39% of the anestrous cows administered GnRH subcutaneously in gelatin capsules and 33% of the anestrous cows administered G&H intramuscularly in saline in combination with short-term calf removal had abnormal luteal phases. When anestrous cows were administered GnRH subcutaneously in gelatin capsules in combination with short-term calf removal, the incidence of abnormal luteal phases was 80%. The number of cows with abnormal luteal phases, however, was not significantly different (P > 0.10) among groups for both anestrous and cyclic cows. Fifty-one percent of all the anestrous cows had abnormal luteal phases, whereas only 9% of the cyclic cows had abnormal luteal phases (P < 0.01). The postpartum
OCTOBER
interval
to GnRH
1983 VOL. 20 NO. 4
treatment
was similar
(P >O.lO)
for
423
THERIOGENOLOGY
cows that ovulated (56.9 + 1.6 days) and cows that did not ovulate (50.0 2 3.0 days) after GnRH treatment. The postpartum interval to GnRH treatment was also similar (P >O.lO) for cows that had abnormal luteal phases (59.1 + 1.9 days) and cows that did not have abnormal luteal phases (54.4 + 2.6 days). Table 2.
Number of anestrous postpartum beef cows that ovulated after the time of GnRH treatment and the number of cows that had subsequent abnormal luteal phases after GnRH treatment and/or calf removal.
Group
GnP.H
Calf Removal
I II III IV V TOTAL
None Saling-IMa GC-SC Saling-IMa GC-SC
None None None Short-term Short-term
Number of Anestrous Cows 18 11 15 16 12 72
Number of Cows that Ovulate after the Time of GnRH Treatment 16 10 13 12 10 61
Number of Cows with Abnormal Luteal Phase
(89%) (91%) (87%) (75%) (83%) (85%)
7 7 5 4 8 31
(44%) (70%) (39%) (33%) (80%) (51%)
intramuscularly Gelatin capsule-subcutaneously
Table 3.
Groups I II III IV V TOTAL
Number of cyclic postpartum beef cows that ovulated after the time of GnRH treatment and the number of cows that had subsequent abnormal luteal phases after GnRH treatment and/or calf removal.
GnRH None Saling-IMa GC-SC Saline-IMa GC-SCn
Calf Removal None None None Short-term Short-term
Number of Cyclic Cows 15 12 11 9 12 59
Number of Cows that Ovulate after the Time of GnRH Treatment 14 12 9 8 ii 54
(93%) (100%) (82%) (89%) i9zj (92%)
Number of Cows with Abnormal Luteal Phases 0 1 2 1 1 5
(0%) (8%) (22%) (13%) (9%). (9%)
ZIntramuscularly Gelatin capsule - subcutaneously
424
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1983 VOL. 20 NO. 4
THERIOGENOLOGY
Progesterone concentrations in serum following the time of GnRH treatment increased (P < 0.01) across time for both anestrous and cyclic cows that had luteal phases longer than 15 or 16 days. Following the time of GnRH treatment, progesterone concentrations were higher (P < 0.01) for cyclic cows than for anestrous cows that had luteal phases longer than Since there were no differences (P >0.25) in progesterone 15 or 16 days. concentrations between the anestrous and cyclic cows that became pregnant to the timed breeding, the data for pregnant and non-pregnant cows were pooled and summarized in Figure 3.
/?c-z& Anestrus /A
cyclic
6or7
I lorl2 DAYS
15or16
Figure 3. Concentrations of progesterone in serum of anestrous and cyclic cows that ovulated following the time of GnRH treatment and had luteal phases greater than 15 or 16 days. Values are means of 30 and 49 observations. Standard error of the difference between two treatment means for a given time is 0.2. Standard error of the difference between two time means for one treatment is 0.1. The first-service pregnancy rates for both anestrous and cyclic cows are summarized in Tables 4 (anestrous COWS) and 5 (cyclic cows). The first-service pregnancy rate for the anestrous cows was low and similar among the five groups (mean = 11%). The pregnancy rate of the anestrous cows with abnormal luteal phases was 0% whereas the pregnancy rate of the anestrous cows without abnormal lureal phases was 27%. Pregnancy rate of the anestrous cows without calf removal (groups I, II and III) that ovulated after the time of GnRH treatment and did not have abnormal luteal phases was 15%. In contrast, five of the ten cows (50%) with short-term calf removal (groups IV and V) that ovulated after GnRH treatment and did not
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1983 VOL. 20 NO. 4
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THERIOGENOLOGY
have abnormal luteal phases became pregnant to the timed breeding. For the cyclic cows, the first-service pregnancy rates were higher (mean = 46%; P < 0.01) than for the anestrous cows, but no significant differences (P > 0.10) were detected among the groups.
Table 4.
Group I II III IV V
The number of anestrous postpartum beef cows that became pregnant to the first-service insemination, the interval from calving to pregnancy and the number of cows not pregnant at the end of the breeding season following GnRH treatment and/or short-term calf removal.
GnRH None Salin&-IMa GC-SC Saline-IMa GC-SCb
Calf Removal None None None Shortterm Shortterm
TOTAL
Number of Cows not Pregnant at end of Breeding Season
FirstService Pregnancy Rate
Interval Calving to Pregnancy
18 II 15 16
1 0 2 4
104.6 99.6 106.3 95.2
12
1 (8%)
98.9 + 6.1
2 (17%)
72
8 (11%)
101.1 f 3.1
10 (14%)
Number of Anestrous Cows
(6%) (0%) (13%) (25%)
5 + + +
6.2 7.1 7.8 7.4
1 2 2 3
(6%) (18%) (13%) (19%)
?Intramuscularlv b Gelatin capsule - subcutaneously
Table 5.
Group I II III IV V
The number of cyclic postpartum beef cows that became pregnant to the first-service insemination, the interval from calving to pregnancy and the number of cows not pregnant at the end of the breeding season following GnRH treatment and/or short-term calf removal.
GnRH
Calf Removal
None
None
;,a;;~-"~ Saline-Ma
;,o;~ Shortterm Shortterm
GC-SCb
Number of Cyclic Cows
TOTAL
FirstService Pregnancy Rate (47%) (42%) (27%) (44%)
Interval Calving to Pregnancy
Number of cows not Pregnant at end of Breeding Season
93.8% 89.52 94.35 83.75
1 0 1 3
15 12 11 9
7 5 3 4
11.7 10.1 12.2 11.9
(7%) (0%) (9%) (33%)
12
8 (67%)
05.4-k - 9.3
0 (0%)
59
27 (46%)
89.8+ 4.9
5 (9%)
~Intramuscularly Gelatin capsule - subcutaneously
425
OCTOBER
1983 VOL. 20 NO. 4
THERIOGENOLOGY The interval from parturition to pregnancy was not different (P > 0.10) among groups for both anestrous and cyclic cows. Anestrous cows averaged 101.1 + 3.1 days and cyclic cows averaged 89.8 + 4.9 days from calving to pregnancy. The number of cows not pregnant at the end of the breeding season was also similar among groups for both anestrous and cyclic cows (Tables 4 and 5). There were significant differences in condition scores and they are summarized in Table 6. The anestrous cows had a lower condition score (4.3 + 0.1) than the cyclic cows (4.7 + 0.1; P < 0.05). In addition, the cows that conceived to the timed artificial insemination had a higher condition score than those that did not conceive to the artificial insemination (P < 0.01).
Table
6.
Condition scores for the cyclic and anestrous postpartum beef cows for when pregnancy occurred and for all cows not pregnant at the end of the breeding season.
Mean + S.E.a Reproductive
Status
Before
Timed
Breeding:
Cyclic Anestrus Pregnancy
4.7 + 0.1* 4.3 + 0.1*
by:
Timed breeding Natural breeding Not pregnant
at the end of the breeding
4.7 + 0.2x"~~ 4.3 T 0.1** season
4.2 2 0.2
$Standard error. P < 0.05. **P i 0.01.
OCTOBER
1983 VOL. 20 NO. 4
427
THERIOGENOLOGY Discussion Kesler and coworkers (30, 37) have suggested that the duration of the GnRH-induced LH surge may be related to the ovulation response and to subsequent corpus luteum function. We have since demonstrated that subcutaneous administration of 250 1~g GnRH in carboxymethylcellulose or in gelatin capsules induced an LH surge more similar in duration to a preovulatory LH surge than intramuscular administration of GnRH in saline (26). In this study, subcutaneous administration of GnRH in gelatin capsules delayed the LH surge but only prolonged the duration by about one hour. The effect of calf removal on the GnRH-induced LH release is controversial. Inskeep et al. (42) reported that short-term calf removal had no effect on the GnRH-induced LH release when 300 1.18GnRH in saline was administered intramuscularly. Troxel et al. (26), however, reported that short-term calf removal enhanced the GnRH-induced LH surge following subcutaneous administration of 250 1-lgGnRH in carboxymethylcellulose. The data from this study would appear to support both reports. Shortterm calf removal did not appear to affect the GnRH-induced LH release when GnRH was administered intramuscularly. However, when GnRH was administered in a method that has been demonstrated to prolong the GnRH-induced LH release (subcutaneously in carboxymethylcellulose or gelatin capsules), short-term calf removal enhanced the GnRH-induced LH release. The effect of short-term calf removal and the method of GnRH administration on the ovulation response of the anestrous cows was difficult to evaluate in this study since 89% of the anestrous cows not administered GnRH ovulated following the time of GnRH treatment. Short-term calf removal, however, has been previously shown to increase the ovulation response to GnRH treatment in anestrous postpartum suckled beef cows (26). Fertility of the anestrous cows was generally low. However, when anestrous cows with abnormal luteal phases, who had a pregnancy rate of O%, were removed, the first-service pregnancy rate was 27%. Therefore, at least some ova released after GnRH-induced ovulations appear to be Low pregnancy rates in anestrous cows incapable of being fertilized. duced to ovulate with GnRH treatment therefore appear to be largely due to abnormal luteal phases. It was difficult to discern the effect of method of GnRH administration on corpus luteum function of the anestrous cows for two reasons. First, the GnRH-induced LH surges were not greatly different between the two methods of administering GnRH in this study. Second, the effect of method of GnRH administration was confounded with short-term calf removal in the anestrous cows. Without short-term calf removal, there were 31% fewer abnormal luteal phases when GnRH was administered subcutaneously in gelatin capsules than when GnRH was administered intramuscularly in saline. With short-term calf removal, there were 47% more abnormal luteal phases when GnRH was administered subcutaneously in gelatin capsules than The reason for when GnRH was administered intramuscularly in saline. this difference is not clear.
428
OCTOBER
1983 VOL. 20 NO. 4
THERIOGENOLOGY
The progesterone concentrations in the anestrous cows that had luteal phases longer than 15 or 16 days were lower than in cyclic cows. The same phenomenon has been reported in postpartum cows administered indomethacin to prolong the luteal phase (43). Therefore, we have reported two types of luteal dysfunction. We called the first type "abnormal luteal phase," which were luteal phases shorter than 15 or 16 days. The other type of luteal dysfunction is suppressed progesterone secretion from corpora lutea that had a lifespan longer than 15 or 16 days. We evaluated the effect of these not detect any adverse effects.
treatments
on cyclic
cows and did
The results in the cows that received only PGF2a also merit attention. In a study using over 5,000 dairy cows, Macmillan and Day (38) reported a higher pregnancy rate in cows treated with PGF c1 than in untreated cows (69% vs 60%). A re-examination of publishe 2 reports using heifers and suckled beef cows shows that an enhanced fertility effect may have also occurred in other PGF2a studies (39-41). Roche (41) reported a calving rate of 7% in cows with inactive ovaries treated with a prostaglandin analogue. In our study, 89% of the anestrous cows administered only two injections of PGF2d ovulated and 6% of these cows became pregIn addinant to the timed insemination following the PGF2a injections. tion to PGF2a, the control cows also received considerable stimulation from handling and from being exposed to cows in estrus. Further investigation is needed to better understand the possibility of an effect on fertility by PGF2c(_ In summary, the effect of the method of GnRH treatment and shortterm calf removal on the induction of ovulation and on the incidence of abnormal luteal phases was difficult to discern. Two types of luteal dysfunctions were observed: abnormal luteal phases, which were luteal phases shorter than 15 or 16 days, and suppressed progesterone secretion by corpora lutea with lifespans longer than 15 or 16 days. At least some ova released after GnRH-induced ovulations were capable of being fertilized but pregnancy was not maintained in many cases because of abnormal luteal phases.
OCTOBER
1983 VOL. 20 NO. 4
429
THERIOGENOLOGY
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OCTOBER
of
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of
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THERIOGENOLOGY
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H. D. and Manns, J. G. Onset of oestrus and fertility of dairy heifers and suckled beef cows treated with prostaglandin F2d. Anim. Prod. -21:13-20. (1975).
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