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Synchronization of estrus in beef cattle
THERIOGENOLOGY
I.
SYNCHRONIZATION OF ESTRUS IN BEEF CATTLE UTILIZATION OF A NORGESTOMET IMPST AND INJECTION OF ESTRADIOL VALERATE ’
J. c. SpitzerC, W. C. Burrell, D. G. LeFever, R. W. Whitmand and J. N. Wiltbanke Departments of Physiology and Biophysics and Animal Sciences Colorado State University, Fort Collins, Colorado 80523 Received for Publication:
May 10, 1978
Abstract
Five experiments were conducted with 647 virgin heifers and 821 lactating cows to determine the effectiveness of an estrous synchronization treatment involving an ear implant containing 6 mg of 17a-acetoxy-118-methyl-l9-nor-preg-4-ene-3,20-dione (norgestomet) and an injection of varying levels (5 to 7.5 mg) of estradiol valerate (EV) at the time of implantation. One of the experiments additionally involved utilizing estradiol-178 (E2) in an attempt to reduce variation in the interval from implant removal to estrus and ovulation. In experiments involving virgin heifers, 76 to 98% of heifers in the treated groups were observed in estrus during the 5 days immediately following implant removal (synchronized period). Heifers not showing estrus during this period were generally in early (days l-4) or late (after day 17) stages of the estrous cycle on the day of treatment. Injections of .25 and .5 mg estradiol-178 near the time of implant removal failed to decrease the variation associated with the onset of estrus or time of ovulation.
(a) Published with the approval of the Director of the Colorado State University Agricultural Experiment Station as Scientific Series Paper No. 2106. (b) G. D. Searle and Co. provided all hormones used in these experiments and partial fund support. (c) Present address:
The Texas A&M University System, Bryan.
(d) Present address:
Montana State University, Bozeman.
(e) Present address:
The Texas A&X University System, Beeville.
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When heifers received the g-day implant treatment in conjunction with 5 mg EV at implantation, fertility was not significantly decreased from that noted in controls. However, fertility was significantly reduced when heifers received the g-day implant treatment with 7.5 mg EV at implantation or the implant alone for 16 days. Of the 432 lactating cows treated with the g-day implant treatment and varying levels of EV at implantation, 359 were observed in estrus within 21 (4 trials) or 17 days (1 trial) of implant removal. Eighty-five percent of the 359 cows were in estrus during the synchronized period. The percent of cows in heat during the synchronized period increased with the dose rate of EV, being greatest when 7.5 mg EV was administered in conjunction with the implant. Conception rate after a single insemination was similar in treated and control cows with the exception of a reduction in conception rate for cows receiving 7.5 mg EV. (Key words: Beef Cattle, Synchronization of Estrus, Norgestomet, Estradiol Valerate)
Introduction Utilization of progestational compounds for periods of time approximating the length of the estrous cycle has been shown to be effective in controlling estrus, but has usually resulted in lowered fertility at the synchronized estrus (1, 2, 3, 4, 5, 6, 7, 8, 9). Fertility in animals treated for 9 to 12 days with a progestational agent and receiving an injection of estrogen near start of treatment was similar to controls (9, 10, 11, 12, 13). However, synchronization was decreased compared to longer treatments. Other workers have shown that treatment dith a progestin for 9 or 10 days in conjunction with an injection of progestin and estrogen at start of treatment was effective in synchronizing estrus and fertility was not decreased (14, 15). Estradiol has been utilized in an attempt to produce a more predictable ovulation time following synchronization of estrus. Lantz and co-workers (16), reported little or no decrease in variability of ovulation time and a decrease in fertility when an estrous synchronization treatment was followed by an injection of 5 mg of estradiol benzoate. Wiltbank and others (ll), reported that ovulation time was more predictable when an estrous synchronization treatment was followed by an injection of 2 mg of estradiol178. Roche (9) reported that use of 400 mcg of estradiol benzoate at the end of a synchronization treatment was ineffective in improving synchronization. The objectives of this study were to; (1) determine the efficacy of an ear implant containing 17a-acetoxy-ll$-methyl-l%nor-preg-4ene-3,20.-dione (norgestomet), alone or in combination with an injection of estradiol valerate (EV) for the synchronization of estrus; (2) compare fertility during the synchronized period with
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fertility during unregulated estrous periods and; (3) determine the effect on ovulation time of injecting estradiol-17S (E2) at the time the norgestomet implant was removed.
Materials and Methods Preliminary Trials .--Two preliminary trials involving 120 Angus and Angus x Hereford heifers were conducted to determine the efficacy of various types of implants containing 6 mg of 17c+acetoxy-USmethyl-19-nor-preg-4-ene-3,20-dione (norgestomet) in inhibiting estrus and ovulation for a 21-day period, In addition, an evaluation was made as to implant retention and ease of implanting and removal depending on the site and method of implantation. In the first preliminary trial, heifers were divided into six groups of 20 heifers with each group containing an equal number of heifers in early (days l-5), middle (days 6-15) and late (days 16-20) stages of the estrous cycle at time of treatment. Day 1 is day of estrus. Implants were placed subcutaneously near the tip of an ear with a modified trocar similar to that used in implanting diethylstilbestrol. Implants were 3 mm in diameter and 18 mm in length, and contained 6 mg norgestomet in a hydron polymer containing 1.2% (groups 1 and 2), 4.8% (groups 3 and 4) or 19.2% (groups 5 and 6) cross-links. The hydron used was a 3-dimensional cross-linked polymer. Threedimensional forms were produced by polymerizing solutions of hydroxy-ethyl methacrylate in the presence of small amounts of a cross-linking agent. The pliability of the hydron is positively correlated with the number of cross-links. Heifers in groups 1, 3 and 5 received one implant and those in groups 2, 4 and 6 received two implants. All implants remained -in situ for 21 days. In the second preliminary trial the 120 heifers utilized in the foregoing trial were re-allotted to six equal groups after all had exhibited one normal estrous cycle. Heifers received implants containing 6 mg norgestomet in a hydron polymer with either 1.2% (groups 1 and 2), 4% (groups 3 and 4) or 4.8% (groups 5 and 6) cross-links. Heifers in groups 1, 3 and 5 were implanted near the base of the ear, while heifers in groups 2, 4 and 6 were implanted midway between the tip and base of the ear. Implants remained in situ for 21 days. -Implants from both preliminary trials were removed through a small incision made perpendicular to and across the base of the implant. Individual implants were assayed for content of norgestomet at G. D. Searle and Co. Laboratories. Implants were finely ground, extracted with methanol and the residue removed by filtration. The methanol extract was taken to dryness, brought to a standard volume in buffer and reacted with isonicotinic acid hydrazide. Norgestomet concentration was then determined calorimetrically (G. D. Searle and Co. unpublished procedure).
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Heifers were observed for estrus twice daily during both preliminary trials. Ovarian activity was assessed by examination per rectum at the time of implant removal. Experiments 1 through 5.--Treated animals in these experiments were subcutaneously implanted in the convex surface of the ear with a 6 mg norgestomet implant. These 3 mm x 18 mm cylindrical polyhydroxy hydron polymer implants had 4% three-dimensional crosslinkage. Experiment 1, Trial l.--After the 120 Angus and Angus x Hereford heifers utilized in the preliminary trials had exhibited at least one normal estrous cycle, they were alternately allotted by breed and number of days post-estrus to one of three groups. A control in situ for 9 days and (C) group, a group where the implant was left -an intramuscular injection of 5 mg of estradiol valerate (EV) administered on the day of implantation (19 + 5EV), and a group having the implant -___ in situ for 16 days with no additional treatment (116) (Table I). Ovarian activity was assessed by palpation per rectum at the time of implantation, and at the time of implant removal. Ovaries were re-examined in heifers not showing estrus by 6 days after implant removal. Following their removal, implants were analyzed for norgestomet content as previously noted. Heifers were observed for estrus at 6-hr intervals for the 5 days following implant removal and at 12-hr intervals for the remainder of the 27-day breeding season. Heifers were inseminated with frozen semen 6 to 12 hr after detected in estrus and again 18 to 24 hr after estrus. Pregnancy was determined per rectum 35 to 60 days after the end of the breeding season. Experiment 1, Trial 2.--An additional 112 cycling Angus, Hereford and Shorthorn heifers were alternately allotted by breed and days post-estrus to the three groups described in Trial 1 (Table I). A variance from Trial lwas that these heifers were inseminated only one time at approximately 12 hr after detected in estrus. All other procedures were identical to those in Trial 1. Experiment 1, Trial 3.--One hundred and nineteen cycling Angus and Hereford heifers were alternately allotted by breed and days post-estrus to a control (C) group and a group receiving the g-day implant treatment plus 5 mg of EV on the day of implantation (I9 + 5EV) (Table I). Implants from this trial and subsequent experiments described in this report were not analyzed for norgestomet content after removal. All other procedures were identical to those in Trial 1 with the exception that heifers were inseminated only one time at approximately 12 hr after detected in estrus. No attempt was made to collect information after the first insemination.
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Experiment 2, Trial 1 .--One hundred and ten cycling Charolais x Angus and Charolais x Hereford heifers were alternately allotted by breed and days post-estrus into control (C) and treated groups. Heifers in the treated group received the 6 mg norgestomet implant for 9 days in conjunction with 7.5 mg EV at time of implantation (19 + 7.5EV) (Table I). Observations for estrus were conducted at 6-hr intervals during the 5 days immediately following implant removal and at 12-hr intervals for the remainder of the 27-day breeding period. Heifers were inseminated approximately 12 hr after observed estrus. Experiment 2, Trial 2.--An additional 73 cycling Hereford heifers were alternately allotted by days post-estrus to treated or control (C) groups as outlined in Trial 1 (Table I). These heifers were observed for estrus at 12-hr intervals throughout a 21-day breeding period and inseminated approximately 12 hr after detected in estrus. Experiment 3, Trial l.--Eighty-nine Angus, Hereford, Shorthorn and Charolais x Angus suckled cows at least 60 days post-calving were alternately allotted to either a treated or control (C) group by age, breed, date of calving and occurrence or lack of occurrence of a postpartum estrous cycle. In the treated group an implant containing 6 mg of norgestomet was placed in the ear and left -in situ for 9 days and an intramuscular injection of 5 mg of EV was given on day of implantation (19 + 5EV) (Table I). Cows were observed for estrus three times daily for 5 days after implant removal and twice daily thereafter until the end of a 45-day breeding period. All cows were artificially inseminated approximately 12 hr after being detected in estrus. Cows were examined for pregnancy per rectum 40 days after the end of the breeding period. Experiment 3, Trial 2.--A group of 51 suckled Angus cows were treated as described in Trial 1 while a group of 54 suckled Angus cows served as controls. The former group of cows calved in a 45-day period while the latter group calved over a go-day period. No individual was less than 45 days post-calving when the treatment was administered. Estrous detection, breeding and pregnancy diagnostic procedures were similar to those in Trial 1. Experiment 3, Trial 3.--Forty-two 2-year old lactating Hereford cows were alternately allotted by date of calving and the presence or absence of a corpus luteum at one of two rectal examinations 2 weeks apart, into a control (C) group and a group receiving the 6 mg norgestomet implant for 9 days and an injection of 7.5 mg of EV on the day the implant was placed in the ear (I9 + 7.5EV) (Table I). Estrous detection,breeding procedure and pregnancy diagnosis were the same as in the previous trials in this experiment.
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Experiment 3, Trial 4 .--Four hundred and fifty-two suckled Hereford cows were alternately allotted by calving date to a control (C) group and a group receiving the g-day, 6 mg norgestomet implant treatment and an injection of 6 mg of EV on the day the implant was placed in the ear (19+ 6EV) (Table I). Observations for estrus were made continuously from daylight to dark for the first 5 days after implant removal and twice daily thereafter until the end of a lJ-day breeding period. Cows were inseminated with semen from Simmental bulls during the lJ-day artificial insemination (AI) period, and were naturally mated to Hereford bulls during a 43-day period which started 3 days after the AI period. Cows were palpated per rectum approximately 4 months after ending the breeding season to diagnose pregnancy. Since accurate estimates of fetal age are difficult at this stage of gestation, calving data was used to calculate which cows became pregnant during the AI period. Experiment 3, Trial 5.--One hundred and thirty-three Red Angus x Hereford and Charolais x Hereford 5-year old suckled cows were alternately allotted by breed and date of calving into a control (C) group and two treated groups. Both treated groups received the 6 mg norgestomet implant which remained -in situ for 9 days. In addition, one treated group received an injection of 6.5 mg EV on day of implantation (19 + 6.5EV) while the other treated group received an injection of 7.5 mg EV on day of implantation (19 + 7.5EV) (Table I). All cows were confined to drylot for 5 days after implant removal and observed for estrus every 6 hr. Thereafter, checks for estrus were made twice daily for the remainder of the 45-day breeding season. Pregnancy diagnosis per rectum was made 40 days after the end of the breeding season. Experiment 4 .--Fifty-four sexually mature Charolais x Angus and Charolais x Hereford heifers were allotted by breed into five groups in such a manner that each group contained approximately an equal number of heifers in early, middle or late stages of the estrous cycle at time of treatment. One group received only the g-day implant treatment and 5 mg EV (19 f 5EV). Two groups of heifers received the g-day implant treatments, 5 mg EV at time of implantation and, in addition, .25 mg of estradiol-176 (E2) at either the time of implant removal (19 + 5EV + .25E2(0)) or 6 hr after implant removal (19 + 5EV + .25E2(6)). Heifers in the remaining 2 groups were implanted for 9 days, received 5 mg EV at implantation and .5 mg E2 at implant removal (I9 + 5EV + .5E2(0)) or 6 hr after implant removal (I9 + 5EV + .5E2(6))(Table I). Detection for estrus occurred at 4-hr intervals from time of implant removal to end of estrus. Steers or young bulls were used as an aid in detecting the onset, duration and the end of estrus. Ovaries were examined per rectum at the start and end of estrus, and every 4 hr from the end of the estrus until the time when a large follicle was no longer palpable and ovulation was presumed to have occurred. A high lumbar laparotomy was performed 30 minutes to 4 hr after presumptive ovulation and ovaries were examined for an ovulation point using a peritoneal endoscope.
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Experiment 5.--Fifty-nine Charolais x Angus and Charolais x Hereford heifers were divided into a control (C) group of 30 heifers and a treated group of 29 heifers. The 54 heifers from Experiment 4 were included in this study after a rest period of several months since the previous treatment. Fifty-one of the heifers had an estrous cycle of 17 to 25 days in length immediately prior to treatment while corpora lutea were found in three heifers which had not been detected in estrus for 25 days indicating that they had cycled and not been observed in estrus. The five additional heifers had been cycling normally prior to this time. All treated heifers received the g-day implant treatment, 5 mg EV and .25 mg E2 at implant removal (19 + 5EV + .25E2) (Table I). Heifers were observed for estrus at 6-hr intervals for the 5 days after implant removal and inseminated approximately 12 hr after detected in estrus. Heifers were observed at 12-hr intervals for the remainder of the 21-day breeding period. Statistical Analysis.--Differences observed in occurrence of estrus, conception rate at first service, and pregnancy rate after 5 days of breeding and at the end of the breeding period were analyzed with Chi square procedures (17). Data from Experiment 4 on interval from implant removal to estrus or ovulation were analyzed by analysis of variance. Variability noted in Experiment 4 was tested with Bartlett's test for homogenity of variance.
Results Preliminary Trials---Thirty-six percent of implants placed in the tip of the ear were lost compared to 5% of implants placed in the middle or at the base of the ear. Implants placed at the base of the ear were difficult to locate for removal. Infection was found in ears of many cattle where implants were not located. When asepsis was maintained and implants were placed in the middle of the ear, little or no problem with implant loss or removal was encountered. It was apparent that the amount of hormone released from implants with 19.2% cross-links was not sufficient to control estrus and ovulation. Sixty-five percent of the heifers retaining at least one implant with 19.2% cross-links showed estrus while the implant was still in place. In contrast, no heifers were observed in estrus with the implant in place with 1.2% cross-linked implants and 3% of heifers receiving implants with 4.8% cross-links. The average amount of norgestomet released in 21 days from 34 implants with 1.2% cross-links in the polymer was 4.46 mg (range 3.65 to 4.87 mg) compared to 2.07 mg (range 1.02 to 3.93) in 29 implants with 4.8% cross-links in the polymer and .82 mg (range .29 to 1.69) in 30 implants with 19.2% cross-links in the polymer.
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In the second preliminary trial where heifers received implants with 1.2, 4.0 and 4.8% cross-links, 49, 12 and 3%, respectively, of these heifers showed estrus with the implant -in situ. In the S-day period following implant removal 41, 87 and 94% of the heifers receiving implants with 1.2, 4.0 and 4.8% cross-links, respectively, were in estrus. All of the 120 implanted heifers retained these implants for the Zl-day period in this preliminary trial. The average amount of hormone released from the implants was 4.74 (4.39 to 5.33); 4.24 (3.84 to 4.39); 4.04 mg (3.41 to 4.78) for implants containing 1.2, 4 and 4.8% cross-links, respectively. The implants containing either 4 or 4.8% cross-links appeared to release the appropriate amounts of norgestomet during the entire 21-day period for control of estrus and ovulation. Experiment 1 .--The average amount of norgestomet released from implants -in situ for 16 days in Trial 1 was 3.18 mg (range 2.45 to in situ 3.71), or .20 mg per day (range .15 to .23) and for implants -for 9 days was 2.36 mg (range 1.08 to 3.07), or .26 mg per day (range .12 to .34). In the second trial the average amount of hormone released from implants -in situ for 16 days was 2.73 mg (range 2.08 to 3.61 mg) or .17 mg per day, and 2.05 mg (range 1.33 to 2.93 mg) for implants in place for 9 days, or .23 mg per day. The percentages of heifers showing estrus by 120 hr after implant removal was 98 and 92% for heifers receiving the g-day treatment in Trials 1 and 2 compared to 92 and 86% for heifers receiving the implant for 16 days (Table II). In Trial 1, 30, 12, 10 and 4% more heifers were detected in estrus by 24, 48, 72 and 96 hr after implant removal in heifers receiving implants for 16 days than in the g-day treatment group. Such differences were not found in Trial 2. In Trial 3, only 83% of heifers receiving the g-day treatment were in estrus by 120 hr after implant removal (synchronized period) compared to 98% noted in Trial 1 and 92% in Trial 2. Five of the 10 heifers not showing estrus during the synchronized period were treated on day 1 or 2 of the cycle (day 1 is day of estrus), and four were treated on day 17 or later. As assessed by palpation, corpora lutea were not regressed at the time of implant removal in four of the five heifers treated on day 1 or 2 of the cycle. The length of the cycle was unaffected by treatment in those four heifers. Three of the four heifers implanted in late stages of the cycle showed estrus by 168 hr after implant removal while the other heifer showed estrus 19 days after implant removal. Percent conception at first insemination was significantly lower (Pc.05) when heifers treated for 16 days were compared to heifers in the control (C) group (30% vs 71%, Trial 1 and 35% vs 61%, Trial 2). While the percentage of heifers conceiving at firstinsemination tended to be lower in heifers receiving the g-day treatment compared to control heifers in all three trials, these differences were not significant (P>.OS). Significantly more of the heifers receiving
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the g-day treatment were pregnant during the synchronized period in Trials 1 and 2 whfle only in Trial 1 did the heifers receiving the 16-day treatment have higher pregnancy rate than heifers in the control (C) group during this period (Px.05). After 27 days of breeding no differences in pregnancy rate among groups were noted. Experiment 2.--The proportion of treated heifers showing estrus by 120 hr after implant removal was 96% in Trial 1 and 97% in Trial 2 (Table III). Conception rate after first insemination was markedly decreased for treated heifers in both trials (Pc.05). Percent conceiving were 40% for treated heifers and 64% for control heifers in Trial 1; and 21% for treated heifers and 52% for control heifers in Trial 2. Experiment 3.--The ability of the treatment to control the estrous cycle in lactating cows can only be assessed in cows which have initiated estrous cycles after calving. Therefore, the number of cows showing estrus during the first 21 days after implant removal was determined and used as the basis for determining cow cycling for all groups. Then the number of treated cows showing estrus at various intervals after implant removal was determined. The number of cows showing estrus at various intervals after implant removal was divided by the number of cows which showed estrus in the first 21 days of breeding. In this manner the proportion of cycling cows showing estrus at various intervals after implant removal could be estimated. As an example in Trial 1, 29 cows had shown estrus by 21 days after implant removal (cycling cows). Seventeen cows had shown heat by 48 hr after implant removal or 59% of the cycling cows had shown estrus. The percentage of cows exhibiting estrus the first 21 days of the breeding season varied from 73 to 86% in the control cows and from 64 to 95% in the treated cows (Table IV). While direct comparisons cannot be made among the 5 trials, increasing the dose of EV given in conjunction with the g-day norgestomet implant treatment appeared to increase the proportion of cycling cows which were detected in estrus during the 120-hr period after implant removal. In cows receiving the implant and 5 mg of EV (Trials 1 and 2) only 79 and 74% of the cycling cows had shown estrus by 120 hr after implant removal. Most of these had been in estrus by 72 hr after implant removal. The cows which did not show estrus in these two trials were in the early stages of the estrous cycle (days 1 to 5) at the time of treatment. In Trials 4 and 5, 85 and 84% of the cows receiving 6 or 6.5 mg of EV, respectively, showed estrus by 120 hr after implant removal. In treated cows receiving 7.5 mg of EV the percentage of cycling cows showing estrus by 120 hr after implant removal was 100 and 92% in Trials 3 and 5, respectively. The difference in proportion of cows showing estrus by 120 hr after implant removal was not different (P>.O5) in cows receiving the 6.5 and the 7.5 mg dose of EV in Trial 5.
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Fertility, as measured by conception to a single service, was not significantly reduced by 5, 6 or 6.5 mg of EV (Table V). The difference in conception to a single service in control cows and in treated cows receiving 7.5 mg of EV was 23% in Trial 5 (Pc.05) and 8% in Trial 3 (P>.O5). The proportion of treated cows pregnant after 5 days of breeding was increased over the proportion noted in control cows. The difference varied from 9% in Trial 1 to 39% in Trial 2. Cows in Trial 1 were on low levels of nutrition both before and after calving and the poor reproductive performance in these cows probably resulted from these deficiencies. Significantly more treated cows than control cows were pregnant after 26 days of breeding in Trials 2 and 4 (Pc.05) while this increase in pregnancy rate was not noted in Trials 1, 3 and 5 (P>.O5). The increase in pregnancy rate after 26 days of breeding in Trial 2 should not all be attributed to synchronization since cows in the treated group calved over a 45-day period while control cows calved over a go-day period in the season prior to initiation of this study. Experiment 4 .--Injection of E2 tended to reduce the average time from implant removal to onset of estrus (Table VI). Bartlett's test for homogenity failed to reveal a difference in the variance. The proportion of heifers showing estrus in a 12-hr period for a particular group varied from 80% in heifers receiving E2 6 hr after implant removal to 40% in heifers not injected with E2 at implant removal. Neither the average length of estrus nor the variability in the length of estrus were affected by the injection of E2 near time of implant removal. The proportion of heifers ovulating in a 12-hr period immediately after the first heifer in a particular group ovulated, was significantly increased by injecting E2. However, the variability and average length of time from implant removal to ovulation were not significantly decreased. The average time from start of estrus to ovulation varied from 21.0 hr to 30.2 hr for animals in the different groups (P>.O5). Experiment 5.--The percent treated heifers in estrus by 120 hr after implant removal was low (Table VII). Percent conceiving to first insemination tended to be reduced for heifers in the treated group when compared to the controls, but this difference was not significant (Ps.05).
Discussion Preliminary trials indicated that the 3 mm x 18 mm cylindrical hydron polymer ear implant was easily administered and removed. Where proper asepsis was maintained, retention rate was 100% and subsequent experiments indicated retention rates of 98% to 100%. A single 6 mg norgestomet polyhydroxy hydron polymer implant with 4% cross-linkage was shown to be effective in preventing estrus and ovulation in cattle for a 21-day period. 190
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THERIOCENOLOGY Generaliy, the results of these experiments are in agreement with the reports of others (9, 10, 11, 12, 13, 14, 15) showing that a g-day implant treatment with progestogens and an injection of EV does not result in lowered fertility. Fertility was reduced in heifers receiving the implant for 16 days and at the higher levels of EV in conjunction with the g-day implant treatment. Synchronization of estrus was far from optimum with the described treatments which gave the best levels of fertility. It was apparent in several of these trials that the percentage of animals showing estrus by 5 days after implant removal was not as high in animals treated in either the early or late stages of the cycle as it was in those animals treated in mid-cycle. Therefore, further modifications of this treatment are necessary to maintain fertility while causing better synchrony in animals in the early or late stages of the cycle at the time of treatment. Use of E2 to decrease variability in ovulation time was not beneficial. This confirms the results of Lantz and co-workers (16) but differs from those of Wiltbank and others (11).
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THERIOGENOLOGY Literature Cited 1.
Ulberg, L. C., R C. Christian and L. E. Casida. 1951. Ovarian repsonse in heifers to progesterone injections. J. Anim. Sci. 10:752.
2.
Trimberger, G. W. and W. Hansel. 1955. Conception rate and ovarian function following estrus control by progesterone injections in dairy cattle. J. Anim. Sci. 14:224.
3.
Ulberg, L. C. and C. E. Lindley. 1960. Use of progesterone and estrogen in the control of reproductive activities in beef cattle. J. Anim. Sci. 19:1132.
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Hansel, W., P. V. Malven and D. L. Black. 1961. Estrous cycle regulation in the bovine. J. Anim. Sci. 20:621.
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Zimbleman, R. G. 1963. Determination of the minimal effective dose of 6a-methyl-17a acetoxy progesterone for control of the estrual cycle of cattle. J. Anim. Sci. 22:1051.
6.
Wiltbank, J. N., D. R. Zimmerman, J. E. Ingalls and W. W. Rowden. 1965. Use of progestational compounds alone or in combination with estrogen for synchronization of estrus. J. Anim. Sci. 24:990.
7.
Wiltbank, J. N., R. P. Shumway, W. R. Parker and D. R. Zimmerman. 1967. Duration of estrus, time of ovulation and fertilization rate in beef heifers synchronized with dihydroxy progesterone acetophenide. J. Anim. Sci. 26:764.
8.
Wagner, J. F., E. L. Veenhuizen, R. P. Gregory and L. V. Tonkinson. 1968. Fertil'ty in the beef heifer foll.owing treatment with 6-chloro A&-17 acetoxy progesterone. J. Anim. Sci. 27~1627.
9.
Synchronization of estrus in heifers Roche, J. F. 1974. with implants of progesterone. J. Reprod. Fert. 41:337.
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Wiltbank, J. N. and C. W. Kasson. 1968. Synchronization of estrus in cattle with an oral progestational agent and an injection of an estrogen. J. Anim. Sci. 27~113.
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11.
Wiltbank, J. N., J. C. Sturges, D. Wideman, D. G. LeFever and L. C. Faulkner. 1971. Control of estrus and ovulation using subcutaneous implants and estrogens in beef cattle. J. Anim. Sci. 33:600.
12.
Roche, J. F. 1974. Effect of short-term progesterone treatment on estrus response and fertility in heifers. J. Reprod. Fert. 40:433.
13.
Roche, J. F. 1976. Calving rate of cows following insemination after a 12-day treatment with silastic coils impregnated with progesterone. J. Anim. Sci. 43~164.
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Wishart, D. F. and I. M. Young. 1974. Artificial insemination of progestin (SC21009) treated cattle at predetermined times. Vet. Rec. 95:503.
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Sreenan, J. M. and P. Mulvehill. 1975. The application of long- and short-term progestogen treatments for estrus cycle control in heifers. J. Reprod. Fert. 45:367.
16.
Lantz, W. B., C. W. Rasson, A. L. Slyter and D. R. Zimmerman. 1968. Synchronization of ovulation in beef heifers. J. Anim. Sci. 27:1193 (Abstr.).
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Snedecor, G. W. and W. G. Cochran. 1967. Statistical methods. The Iowa State University Press. Ames, Iowa.
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TABLE I.
EXPERIMENTAL DESIGN
Experiment EXPERIMENT 1 Trial 1
Trial 2
Group
No. of animals
I16 IV + 5EV C 116 I9 + 5EV C
Trial 3
EXPERIMENT 2 Trial 1
19 + 5EV
Trial 2
19 + 7.5EV C IV + 7.5EV
EXPERIMENT 3 Trial 1
I9 + 5EV C
Trial 2 Trial 3 Trial 4 Trial 5
19 + C 19 + C I9 + C IV + IV + C
5EV 7.5EV 6EV 7.5EV 6.5EV
Treatment received
40 40 40 36 39 37 59 60
I only for lb days I for 9 days and 5 mg EV on day of implantation No treatment I only for lb days I for 9 days and 5 ma EV on day of implantation No treatment I for 9 days and 5 mg EV on day of implantation No treatment
55 55 35 38
I for 9 days and 7.5 mg EV on day of implantation No treatment I for 9 days and 7.5 mg EV on day of implantation No treatment
45 44 51 54 21 21 226 226 44 45 44
I for 9 days No treatment I for 9 days No treatment I for 9 days No treatment I for 9 days No treatment I for 9 days I for 9 days No treatment
and 5 mg EV on day of implantation and 5 mg EV on day of implantation and 7.5 mg EV on day of implantation and 6 mg EV on day of implantation and 7.5 mg EV on day of implantation and 6.5 mg EV on day of implantation
EXPERIMENT 4 19 + 5EV IV + 5EV + .5E2(0)
11 10
19 + 5EV + .25E2(0)
11
IV + 5EV + .5E2(6)
11
IV + 5EV + .25E2(6)
11
19 + 5EV + .25E2
29
I for 9 days and 5 mg EV on day of implantation I for 9 days and 5 mg EV on day of implantation and .5 mg E2 at implant removal I for 9 days and 5 mg EV on day of implantation and .25 mg E2 at implant removal I for 9 days and 5 mg EV on day of implantation and .5 mg E2 6 hours post-implant removal I for 9 days and 5 mg EV on day of implantation and .25 mg E2 6 hours post-implant removal
EXPERIMENT 5
C
I EV E2 c
194
= = = =
30
I for 9 days and 5 mg EV on day of implantation and .25 mg E2 at time of implant removal No treatment
6 mg norgestomet implant estradiol valerate estradiol 17-8 control
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36 39 37
59 60
TRIAL 2 116 I9 + 5EV C
TRIAL 3 19 + 5EV C
12
30 33
35 5
24
44
83 79
90 78
48
71
86 85
90 80
72
80
86 90
92 88
96
83
86 92
92 98
120
Cumulative percent showing estrus (hour after implant removal) ~-_-_-~-~_~_---_-_-
bed
45 61
35b 5oc 61'
3ob 62' 71C
3ob 5oc 27b
2gb 60' 10d
5 days
61 69 78
68 80 70
27 days
Percent pregnant (days after implant removal)a _---_-_______--_
Figures within same trial and same column with no superscript in common differ significantly (PC.05).
49 59
31 36 37
37 39 38
No. heifers inseminated
Percent conceiving at first insemination
SYNCHRONIZATION OF ESTRUS AND FERTILITY WITH A 16-DAY IMPLANT TREATMENT OR A g-DAY IMPLANT TREATMENT PLUS 5 MG EV IN VIRGIN HEIFERS.
aBased on total number of heifers in each group.
40 40 40
No. of heifers
EXPERIMENT 1.
TRIAL 1 116 I9 + 5EV C
Trial
TABLE II.
4
K
5
35 38
TRIAL 2 I9 + 7.5EV C
20
8
24
97
75
48
97
87
72
97
94
96
97
96
120
Cumulative percent showing estrus (hour after implant removal) -~-___--~---~~-~~~~
bc
4ob 64'
39 35
5 days
71 76
27 days
Percent pregnant (days after implant removal)a -__-_-_---_-_---
Figures within same trial and same column with no superscript in common differ significantly (PC.05).
34 29
53 55
No. heifers inseminated
Percent conceiving at first insemination
SYNCHRONIZATION OF ESTRUS AND FERTILITY WITH 7.5 MG EV IN CONJUNCTION WITH A g-DAY IMPLANT TREATMENT IN VIRGIN HEIFERS.
aBased on total number of heifers in each group.
55 55
No. of heifers
EXPERIMENT 2.
TRIAL 1 I9 + 7.5EV C
Trial
TABLE III.
188 (83%) 165 (73%)
38 (86%) 38 (94%) 38 (86%)
226 226
45 44
aCows were only observed 17 days in Trial 4.
I9 + 6.5EV C
C
20 (95%) 17 (81%)
21 21
TRIAL 3 19 + 7.5EV C
46 (90%) 41 (76%)
51 54
TRIAL 2 19 + 5EV C
29 (64%) 32 (73%)
Cows in estrus first 21 days of breedinga
68 60
66
7
5 3
10
65
59
5
17
14
89 79
77
80
70
72
92 84
85 80
92 79
100
74
79
90
74
72
Cumulative percent showing estrus (hour after implant removal) 120 72 24 96 48
CONTROL OF ESTRUS IN SUCKLED COWS FOLLOWING A g-DAY IMF'LANTTREATMENT AND VARIOUS LEVELS OF EV.
45 44
No. of cows
EXPERIMENT 3.
TRIAL 1 19 + 5EV C
Trial
TABLE IV.
2
51 54
21 21
TRIAL 2 I9 + 5EV C
TRIAL 3 19 + 7.5EV C
44 45 44
35 (79%) 32 (71%) 38 (86%)
160 (71%) 165 (73%)
20 (95) 17 (81%)
34 (67%) 41 (76%)
23 (51%) 32 (73%)
No. cows inseminateda
43c 56d 66d
54 50
45 53
74 63
39 47
Percent conceiving at first insemination
34c 4oc gd
4gc lod
2oc lid
64 67 68
4gc 38d
57 48
7ec 52d
47 41
Percent pregnant (days after implant removal)b ---______-__ _____--___-_-_ 5 days 26 days
FERTILITY IN SUCKLED COWS FOLLOWING A g-DAY IMPLANT TREATMENT AND VARIOUS LEVELS OF EV.
cd Figures in the same trial and the same column with no superscript in cormnondiffer significantly (PC.05).
b Based on total number of cows in each group.
aCows in treated groups inseminated first 5 days after implant removal and cows in control groups inseminated first 21 days of breeding, except in Trial 4 where cows in control group were only inseminated for a lJ-day period.
TRIAL 5 I9 + 7.5EV I9 + 6.5EV C
226 226
45 44
TRIAL 1 I9 + 5EV C
TRIAL 4 I9 + REV C
No. of cows
EXPERIMENT 3.
Dose of estradiol valerate
TABLE V.
s
8
a
e"
11
11
11
19 + 5EV + .25E2(0)
19 + 5EV + .5E2(6)
19 + 5EV + .5E2(6)
39.3
43.3
34.2
38.0
58.8
28.02
31.38
20.31
24.48
25.01
59.2
62.4
64.4
62.5
86.8
23.3
21.0
25.3
28.9
25.2
17.8
17.0
15.5
11.6
14.4
6.78
5.39
6.46
4.40
5.05
Length of estrus hr ______-__mean SD
timed from first observed estrus or ovulation within each group.
11
11
9
10
10
Implant removal to estrus hr ovulation hr ___________ ____--___sD_ mean mean SD
21.0
27.2
30.2
27.5
28.0
3.88
9.76
8.27
5.83
5.35
start of estrus to ovulation hr _____--_-sD_ mean
bcdFigures in the same column with no superscript in common differ significantly (Pc.05).
%ntervals
10
I9 + 5EV + .5E2(0)
-
11
I9 + 5EV
in estrus within 120 hr
No.
80Cd
80Cd
78bC
7obc
4ob
% heifers in estrus in 12-bra interval
80C
60'
56'
63'
2ob
% heifers ovulating in 12-hra interval
OCCURRENCE OF ESTRUS AND OWLATION IN VIRGIN HEIFERS FOLLOWING A g-DAY IMPLANT TREATMENT AND 5 MG EV AT IMPLANTATION WITH AND WITHOUT ESTRADIOL-176 AT IMPLANT REMOVAL (0) OR 6 HOURS LATER (6).
No. of heifers
EXPERIMENT 4.
Treatment
TABLE VI.
g 4
$
$
6
g
zl m
24
17
29
30
I9 + 5EV + .25E2
(:
45
48 66
72 72
96 76
120
Cumulative percent showing estrus (hour after implant removal) __----_-____________-~-~-------__
30
22
No. heifers inseminated
47
36
Percent conceiving at first insemination
SYNCHRONIZATION OF ESTRUS AND FERTILITY IN VIRGIN HEIFERS FOLLOWING A g-DAY IMPLANT TREATMENT AND 5 MG EV AT IMPLANTATION WITH .25 MG ESTRADIOL-17B AT TIME OF IMPLANT REMOVAL.
No. of heifers
EXPERIMENT 5.
___I_.
Treatment
--
TABLE VII.
I.
SYNCHRONIZATION OF ESTRUS IN BEEF CATTLE UTILIZATION OF A NORGESTOMET IMPST AND INJECTION OF ESTRADIOL VALERATE ’
J. c. SpitzerC, W. C. Burrell, D. G. LeFever, R. W. Whitmand and J. N. Wiltbanke Departments of Physiology and Biophysics and Animal Sciences Colorado State University, Fort Collins, Colorado 80523 Received for Publication:
May 10, 1978
Abstract
Five experiments were conducted with 647 virgin heifers and 821 lactating cows to determine the effectiveness of an estrous synchronization treatment involving an ear implant containing 6 mg of 17a-acetoxy-118-methyl-l9-nor-preg-4-ene-3,20-dione (norgestomet) and an injection of varying levels (5 to 7.5 mg) of estradiol valerate (EV) at the time of implantation. One of the experiments additionally involved utilizing estradiol-178 (E2) in an attempt to reduce variation in the interval from implant removal to estrus and ovulation. In experiments involving virgin heifers, 76 to 98% of heifers in the treated groups were observed in estrus during the 5 days immediately following implant removal (synchronized period). Heifers not showing estrus during this period were generally in early (days l-4) or late (after day 17) stages of the estrous cycle on the day of treatment. Injections of .25 and .5 mg estradiol-178 near the time of implant removal failed to decrease the variation associated with the onset of estrus or time of ovulation.
(a) Published with the approval of the Director of the Colorado State University Agricultural Experiment Station as Scientific Series Paper No. 2106. (b) G. D. Searle and Co. provided all hormones used in these experiments and partial fund support. (c) Present address:
The Texas A&M University System, Bryan.
(d) Present address:
Montana State University, Bozeman.
(e) Present address:
The Texas A&X University System, Beeville.
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When heifers received the g-day implant treatment in conjunction with 5 mg EV at implantation, fertility was not significantly decreased from that noted in controls. However, fertility was significantly reduced when heifers received the g-day implant treatment with 7.5 mg EV at implantation or the implant alone for 16 days. Of the 432 lactating cows treated with the g-day implant treatment and varying levels of EV at implantation, 359 were observed in estrus within 21 (4 trials) or 17 days (1 trial) of implant removal. Eighty-five percent of the 359 cows were in estrus during the synchronized period. The percent of cows in heat during the synchronized period increased with the dose rate of EV, being greatest when 7.5 mg EV was administered in conjunction with the implant. Conception rate after a single insemination was similar in treated and control cows with the exception of a reduction in conception rate for cows receiving 7.5 mg EV. (Key words: Beef Cattle, Synchronization of Estrus, Norgestomet, Estradiol Valerate)
Introduction Utilization of progestational compounds for periods of time approximating the length of the estrous cycle has been shown to be effective in controlling estrus, but has usually resulted in lowered fertility at the synchronized estrus (1, 2, 3, 4, 5, 6, 7, 8, 9). Fertility in animals treated for 9 to 12 days with a progestational agent and receiving an injection of estrogen near start of treatment was similar to controls (9, 10, 11, 12, 13). However, synchronization was decreased compared to longer treatments. Other workers have shown that treatment dith a progestin for 9 or 10 days in conjunction with an injection of progestin and estrogen at start of treatment was effective in synchronizing estrus and fertility was not decreased (14, 15). Estradiol has been utilized in an attempt to produce a more predictable ovulation time following synchronization of estrus. Lantz and co-workers (16), reported little or no decrease in variability of ovulation time and a decrease in fertility when an estrous synchronization treatment was followed by an injection of 5 mg of estradiol benzoate. Wiltbank and others (ll), reported that ovulation time was more predictable when an estrous synchronization treatment was followed by an injection of 2 mg of estradiol178. Roche (9) reported that use of 400 mcg of estradiol benzoate at the end of a synchronization treatment was ineffective in improving synchronization. The objectives of this study were to; (1) determine the efficacy of an ear implant containing 17a-acetoxy-ll$-methyl-l%nor-preg-4ene-3,20.-dione (norgestomet), alone or in combination with an injection of estradiol valerate (EV) for the synchronization of estrus; (2) compare fertility during the synchronized period with
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fertility during unregulated estrous periods and; (3) determine the effect on ovulation time of injecting estradiol-17S (E2) at the time the norgestomet implant was removed.
Materials and Methods Preliminary Trials .--Two preliminary trials involving 120 Angus and Angus x Hereford heifers were conducted to determine the efficacy of various types of implants containing 6 mg of 17c+acetoxy-USmethyl-19-nor-preg-4-ene-3,20-dione (norgestomet) in inhibiting estrus and ovulation for a 21-day period, In addition, an evaluation was made as to implant retention and ease of implanting and removal depending on the site and method of implantation. In the first preliminary trial, heifers were divided into six groups of 20 heifers with each group containing an equal number of heifers in early (days l-5), middle (days 6-15) and late (days 16-20) stages of the estrous cycle at time of treatment. Day 1 is day of estrus. Implants were placed subcutaneously near the tip of an ear with a modified trocar similar to that used in implanting diethylstilbestrol. Implants were 3 mm in diameter and 18 mm in length, and contained 6 mg norgestomet in a hydron polymer containing 1.2% (groups 1 and 2), 4.8% (groups 3 and 4) or 19.2% (groups 5 and 6) cross-links. The hydron used was a 3-dimensional cross-linked polymer. Threedimensional forms were produced by polymerizing solutions of hydroxy-ethyl methacrylate in the presence of small amounts of a cross-linking agent. The pliability of the hydron is positively correlated with the number of cross-links. Heifers in groups 1, 3 and 5 received one implant and those in groups 2, 4 and 6 received two implants. All implants remained -in situ for 21 days. In the second preliminary trial the 120 heifers utilized in the foregoing trial were re-allotted to six equal groups after all had exhibited one normal estrous cycle. Heifers received implants containing 6 mg norgestomet in a hydron polymer with either 1.2% (groups 1 and 2), 4% (groups 3 and 4) or 4.8% (groups 5 and 6) cross-links. Heifers in groups 1, 3 and 5 were implanted near the base of the ear, while heifers in groups 2, 4 and 6 were implanted midway between the tip and base of the ear. Implants remained in situ for 21 days. -Implants from both preliminary trials were removed through a small incision made perpendicular to and across the base of the implant. Individual implants were assayed for content of norgestomet at G. D. Searle and Co. Laboratories. Implants were finely ground, extracted with methanol and the residue removed by filtration. The methanol extract was taken to dryness, brought to a standard volume in buffer and reacted with isonicotinic acid hydrazide. Norgestomet concentration was then determined calorimetrically (G. D. Searle and Co. unpublished procedure).
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THERIOGENOLOGY
Heifers were observed for estrus twice daily during both preliminary trials. Ovarian activity was assessed by examination per rectum at the time of implant removal. Experiments 1 through 5.--Treated animals in these experiments were subcutaneously implanted in the convex surface of the ear with a 6 mg norgestomet implant. These 3 mm x 18 mm cylindrical polyhydroxy hydron polymer implants had 4% three-dimensional crosslinkage. Experiment 1, Trial l.--After the 120 Angus and Angus x Hereford heifers utilized in the preliminary trials had exhibited at least one normal estrous cycle, they were alternately allotted by breed and number of days post-estrus to one of three groups. A control in situ for 9 days and (C) group, a group where the implant was left -an intramuscular injection of 5 mg of estradiol valerate (EV) administered on the day of implantation (19 + 5EV), and a group having the implant -___ in situ for 16 days with no additional treatment (116) (Table I). Ovarian activity was assessed by palpation per rectum at the time of implantation, and at the time of implant removal. Ovaries were re-examined in heifers not showing estrus by 6 days after implant removal. Following their removal, implants were analyzed for norgestomet content as previously noted. Heifers were observed for estrus at 6-hr intervals for the 5 days following implant removal and at 12-hr intervals for the remainder of the 27-day breeding season. Heifers were inseminated with frozen semen 6 to 12 hr after detected in estrus and again 18 to 24 hr after estrus. Pregnancy was determined per rectum 35 to 60 days after the end of the breeding season. Experiment 1, Trial 2.--An additional 112 cycling Angus, Hereford and Shorthorn heifers were alternately allotted by breed and days post-estrus to the three groups described in Trial 1 (Table I). A variance from Trial lwas that these heifers were inseminated only one time at approximately 12 hr after detected in estrus. All other procedures were identical to those in Trial 1. Experiment 1, Trial 3.--One hundred and nineteen cycling Angus and Hereford heifers were alternately allotted by breed and days post-estrus to a control (C) group and a group receiving the g-day implant treatment plus 5 mg of EV on the day of implantation (I9 + 5EV) (Table I). Implants from this trial and subsequent experiments described in this report were not analyzed for norgestomet content after removal. All other procedures were identical to those in Trial 1 with the exception that heifers were inseminated only one time at approximately 12 hr after detected in estrus. No attempt was made to collect information after the first insemination.
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Experiment 2, Trial 1 .--One hundred and ten cycling Charolais x Angus and Charolais x Hereford heifers were alternately allotted by breed and days post-estrus into control (C) and treated groups. Heifers in the treated group received the 6 mg norgestomet implant for 9 days in conjunction with 7.5 mg EV at time of implantation (19 + 7.5EV) (Table I). Observations for estrus were conducted at 6-hr intervals during the 5 days immediately following implant removal and at 12-hr intervals for the remainder of the 27-day breeding period. Heifers were inseminated approximately 12 hr after observed estrus. Experiment 2, Trial 2.--An additional 73 cycling Hereford heifers were alternately allotted by days post-estrus to treated or control (C) groups as outlined in Trial 1 (Table I). These heifers were observed for estrus at 12-hr intervals throughout a 21-day breeding period and inseminated approximately 12 hr after detected in estrus. Experiment 3, Trial l.--Eighty-nine Angus, Hereford, Shorthorn and Charolais x Angus suckled cows at least 60 days post-calving were alternately allotted to either a treated or control (C) group by age, breed, date of calving and occurrence or lack of occurrence of a postpartum estrous cycle. In the treated group an implant containing 6 mg of norgestomet was placed in the ear and left -in situ for 9 days and an intramuscular injection of 5 mg of EV was given on day of implantation (19 + 5EV) (Table I). Cows were observed for estrus three times daily for 5 days after implant removal and twice daily thereafter until the end of a 45-day breeding period. All cows were artificially inseminated approximately 12 hr after being detected in estrus. Cows were examined for pregnancy per rectum 40 days after the end of the breeding period. Experiment 3, Trial 2.--A group of 51 suckled Angus cows were treated as described in Trial 1 while a group of 54 suckled Angus cows served as controls. The former group of cows calved in a 45-day period while the latter group calved over a go-day period. No individual was less than 45 days post-calving when the treatment was administered. Estrous detection, breeding and pregnancy diagnostic procedures were similar to those in Trial 1. Experiment 3, Trial 3.--Forty-two 2-year old lactating Hereford cows were alternately allotted by date of calving and the presence or absence of a corpus luteum at one of two rectal examinations 2 weeks apart, into a control (C) group and a group receiving the 6 mg norgestomet implant for 9 days and an injection of 7.5 mg of EV on the day the implant was placed in the ear (I9 + 7.5EV) (Table I). Estrous detection,breeding procedure and pregnancy diagnosis were the same as in the previous trials in this experiment.
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Experiment 3, Trial 4 .--Four hundred and fifty-two suckled Hereford cows were alternately allotted by calving date to a control (C) group and a group receiving the g-day, 6 mg norgestomet implant treatment and an injection of 6 mg of EV on the day the implant was placed in the ear (19+ 6EV) (Table I). Observations for estrus were made continuously from daylight to dark for the first 5 days after implant removal and twice daily thereafter until the end of a lJ-day breeding period. Cows were inseminated with semen from Simmental bulls during the lJ-day artificial insemination (AI) period, and were naturally mated to Hereford bulls during a 43-day period which started 3 days after the AI period. Cows were palpated per rectum approximately 4 months after ending the breeding season to diagnose pregnancy. Since accurate estimates of fetal age are difficult at this stage of gestation, calving data was used to calculate which cows became pregnant during the AI period. Experiment 3, Trial 5.--One hundred and thirty-three Red Angus x Hereford and Charolais x Hereford 5-year old suckled cows were alternately allotted by breed and date of calving into a control (C) group and two treated groups. Both treated groups received the 6 mg norgestomet implant which remained -in situ for 9 days. In addition, one treated group received an injection of 6.5 mg EV on day of implantation (19 + 6.5EV) while the other treated group received an injection of 7.5 mg EV on day of implantation (19 + 7.5EV) (Table I). All cows were confined to drylot for 5 days after implant removal and observed for estrus every 6 hr. Thereafter, checks for estrus were made twice daily for the remainder of the 45-day breeding season. Pregnancy diagnosis per rectum was made 40 days after the end of the breeding season. Experiment 4 .--Fifty-four sexually mature Charolais x Angus and Charolais x Hereford heifers were allotted by breed into five groups in such a manner that each group contained approximately an equal number of heifers in early, middle or late stages of the estrous cycle at time of treatment. One group received only the g-day implant treatment and 5 mg EV (19 f 5EV). Two groups of heifers received the g-day implant treatments, 5 mg EV at time of implantation and, in addition, .25 mg of estradiol-176 (E2) at either the time of implant removal (19 + 5EV + .25E2(0)) or 6 hr after implant removal (19 + 5EV + .25E2(6)). Heifers in the remaining 2 groups were implanted for 9 days, received 5 mg EV at implantation and .5 mg E2 at implant removal (I9 + 5EV + .5E2(0)) or 6 hr after implant removal (I9 + 5EV + .5E2(6))(Table I). Detection for estrus occurred at 4-hr intervals from time of implant removal to end of estrus. Steers or young bulls were used as an aid in detecting the onset, duration and the end of estrus. Ovaries were examined per rectum at the start and end of estrus, and every 4 hr from the end of the estrus until the time when a large follicle was no longer palpable and ovulation was presumed to have occurred. A high lumbar laparotomy was performed 30 minutes to 4 hr after presumptive ovulation and ovaries were examined for an ovulation point using a peritoneal endoscope.
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Experiment 5.--Fifty-nine Charolais x Angus and Charolais x Hereford heifers were divided into a control (C) group of 30 heifers and a treated group of 29 heifers. The 54 heifers from Experiment 4 were included in this study after a rest period of several months since the previous treatment. Fifty-one of the heifers had an estrous cycle of 17 to 25 days in length immediately prior to treatment while corpora lutea were found in three heifers which had not been detected in estrus for 25 days indicating that they had cycled and not been observed in estrus. The five additional heifers had been cycling normally prior to this time. All treated heifers received the g-day implant treatment, 5 mg EV and .25 mg E2 at implant removal (19 + 5EV + .25E2) (Table I). Heifers were observed for estrus at 6-hr intervals for the 5 days after implant removal and inseminated approximately 12 hr after detected in estrus. Heifers were observed at 12-hr intervals for the remainder of the 21-day breeding period. Statistical Analysis.--Differences observed in occurrence of estrus, conception rate at first service, and pregnancy rate after 5 days of breeding and at the end of the breeding period were analyzed with Chi square procedures (17). Data from Experiment 4 on interval from implant removal to estrus or ovulation were analyzed by analysis of variance. Variability noted in Experiment 4 was tested with Bartlett's test for homogenity of variance.
Results Preliminary Trials---Thirty-six percent of implants placed in the tip of the ear were lost compared to 5% of implants placed in the middle or at the base of the ear. Implants placed at the base of the ear were difficult to locate for removal. Infection was found in ears of many cattle where implants were not located. When asepsis was maintained and implants were placed in the middle of the ear, little or no problem with implant loss or removal was encountered. It was apparent that the amount of hormone released from implants with 19.2% cross-links was not sufficient to control estrus and ovulation. Sixty-five percent of the heifers retaining at least one implant with 19.2% cross-links showed estrus while the implant was still in place. In contrast, no heifers were observed in estrus with the implant in place with 1.2% cross-linked implants and 3% of heifers receiving implants with 4.8% cross-links. The average amount of norgestomet released in 21 days from 34 implants with 1.2% cross-links in the polymer was 4.46 mg (range 3.65 to 4.87 mg) compared to 2.07 mg (range 1.02 to 3.93) in 29 implants with 4.8% cross-links in the polymer and .82 mg (range .29 to 1.69) in 30 implants with 19.2% cross-links in the polymer.
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THERIOCENOLOGY
In the second preliminary trial where heifers received implants with 1.2, 4.0 and 4.8% cross-links, 49, 12 and 3%, respectively, of these heifers showed estrus with the implant -in situ. In the S-day period following implant removal 41, 87 and 94% of the heifers receiving implants with 1.2, 4.0 and 4.8% cross-links, respectively, were in estrus. All of the 120 implanted heifers retained these implants for the Zl-day period in this preliminary trial. The average amount of hormone released from the implants was 4.74 (4.39 to 5.33); 4.24 (3.84 to 4.39); 4.04 mg (3.41 to 4.78) for implants containing 1.2, 4 and 4.8% cross-links, respectively. The implants containing either 4 or 4.8% cross-links appeared to release the appropriate amounts of norgestomet during the entire 21-day period for control of estrus and ovulation. Experiment 1 .--The average amount of norgestomet released from implants -in situ for 16 days in Trial 1 was 3.18 mg (range 2.45 to in situ 3.71), or .20 mg per day (range .15 to .23) and for implants -for 9 days was 2.36 mg (range 1.08 to 3.07), or .26 mg per day (range .12 to .34). In the second trial the average amount of hormone released from implants -in situ for 16 days was 2.73 mg (range 2.08 to 3.61 mg) or .17 mg per day, and 2.05 mg (range 1.33 to 2.93 mg) for implants in place for 9 days, or .23 mg per day. The percentages of heifers showing estrus by 120 hr after implant removal was 98 and 92% for heifers receiving the g-day treatment in Trials 1 and 2 compared to 92 and 86% for heifers receiving the implant for 16 days (Table II). In Trial 1, 30, 12, 10 and 4% more heifers were detected in estrus by 24, 48, 72 and 96 hr after implant removal in heifers receiving implants for 16 days than in the g-day treatment group. Such differences were not found in Trial 2. In Trial 3, only 83% of heifers receiving the g-day treatment were in estrus by 120 hr after implant removal (synchronized period) compared to 98% noted in Trial 1 and 92% in Trial 2. Five of the 10 heifers not showing estrus during the synchronized period were treated on day 1 or 2 of the cycle (day 1 is day of estrus), and four were treated on day 17 or later. As assessed by palpation, corpora lutea were not regressed at the time of implant removal in four of the five heifers treated on day 1 or 2 of the cycle. The length of the cycle was unaffected by treatment in those four heifers. Three of the four heifers implanted in late stages of the cycle showed estrus by 168 hr after implant removal while the other heifer showed estrus 19 days after implant removal. Percent conception at first insemination was significantly lower (Pc.05) when heifers treated for 16 days were compared to heifers in the control (C) group (30% vs 71%, Trial 1 and 35% vs 61%, Trial 2). While the percentage of heifers conceiving at firstinsemination tended to be lower in heifers receiving the g-day treatment compared to control heifers in all three trials, these differences were not significant (P>.OS). Significantly more of the heifers receiving
188
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THERIOGENOLOGY
the g-day treatment were pregnant during the synchronized period in Trials 1 and 2 whfle only in Trial 1 did the heifers receiving the 16-day treatment have higher pregnancy rate than heifers in the control (C) group during this period (Px.05). After 27 days of breeding no differences in pregnancy rate among groups were noted. Experiment 2.--The proportion of treated heifers showing estrus by 120 hr after implant removal was 96% in Trial 1 and 97% in Trial 2 (Table III). Conception rate after first insemination was markedly decreased for treated heifers in both trials (Pc.05). Percent conceiving were 40% for treated heifers and 64% for control heifers in Trial 1; and 21% for treated heifers and 52% for control heifers in Trial 2. Experiment 3.--The ability of the treatment to control the estrous cycle in lactating cows can only be assessed in cows which have initiated estrous cycles after calving. Therefore, the number of cows showing estrus during the first 21 days after implant removal was determined and used as the basis for determining cow cycling for all groups. Then the number of treated cows showing estrus at various intervals after implant removal was determined. The number of cows showing estrus at various intervals after implant removal was divided by the number of cows which showed estrus in the first 21 days of breeding. In this manner the proportion of cycling cows showing estrus at various intervals after implant removal could be estimated. As an example in Trial 1, 29 cows had shown estrus by 21 days after implant removal (cycling cows). Seventeen cows had shown heat by 48 hr after implant removal or 59% of the cycling cows had shown estrus. The percentage of cows exhibiting estrus the first 21 days of the breeding season varied from 73 to 86% in the control cows and from 64 to 95% in the treated cows (Table IV). While direct comparisons cannot be made among the 5 trials, increasing the dose of EV given in conjunction with the g-day norgestomet implant treatment appeared to increase the proportion of cycling cows which were detected in estrus during the 120-hr period after implant removal. In cows receiving the implant and 5 mg of EV (Trials 1 and 2) only 79 and 74% of the cycling cows had shown estrus by 120 hr after implant removal. Most of these had been in estrus by 72 hr after implant removal. The cows which did not show estrus in these two trials were in the early stages of the estrous cycle (days 1 to 5) at the time of treatment. In Trials 4 and 5, 85 and 84% of the cows receiving 6 or 6.5 mg of EV, respectively, showed estrus by 120 hr after implant removal. In treated cows receiving 7.5 mg of EV the percentage of cycling cows showing estrus by 120 hr after implant removal was 100 and 92% in Trials 3 and 5, respectively. The difference in proportion of cows showing estrus by 120 hr after implant removal was not different (P>.O5) in cows receiving the 6.5 and the 7.5 mg dose of EV in Trial 5.
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THERIOGENOLOGY
Fertility, as measured by conception to a single service, was not significantly reduced by 5, 6 or 6.5 mg of EV (Table V). The difference in conception to a single service in control cows and in treated cows receiving 7.5 mg of EV was 23% in Trial 5 (Pc.05) and 8% in Trial 3 (P>.O5). The proportion of treated cows pregnant after 5 days of breeding was increased over the proportion noted in control cows. The difference varied from 9% in Trial 1 to 39% in Trial 2. Cows in Trial 1 were on low levels of nutrition both before and after calving and the poor reproductive performance in these cows probably resulted from these deficiencies. Significantly more treated cows than control cows were pregnant after 26 days of breeding in Trials 2 and 4 (Pc.05) while this increase in pregnancy rate was not noted in Trials 1, 3 and 5 (P>.O5). The increase in pregnancy rate after 26 days of breeding in Trial 2 should not all be attributed to synchronization since cows in the treated group calved over a 45-day period while control cows calved over a go-day period in the season prior to initiation of this study. Experiment 4 .--Injection of E2 tended to reduce the average time from implant removal to onset of estrus (Table VI). Bartlett's test for homogenity failed to reveal a difference in the variance. The proportion of heifers showing estrus in a 12-hr period for a particular group varied from 80% in heifers receiving E2 6 hr after implant removal to 40% in heifers not injected with E2 at implant removal. Neither the average length of estrus nor the variability in the length of estrus were affected by the injection of E2 near time of implant removal. The proportion of heifers ovulating in a 12-hr period immediately after the first heifer in a particular group ovulated, was significantly increased by injecting E2. However, the variability and average length of time from implant removal to ovulation were not significantly decreased. The average time from start of estrus to ovulation varied from 21.0 hr to 30.2 hr for animals in the different groups (P>.O5). Experiment 5.--The percent treated heifers in estrus by 120 hr after implant removal was low (Table VII). Percent conceiving to first insemination tended to be reduced for heifers in the treated group when compared to the controls, but this difference was not significant (Ps.05).
Discussion Preliminary trials indicated that the 3 mm x 18 mm cylindrical hydron polymer ear implant was easily administered and removed. Where proper asepsis was maintained, retention rate was 100% and subsequent experiments indicated retention rates of 98% to 100%. A single 6 mg norgestomet polyhydroxy hydron polymer implant with 4% cross-linkage was shown to be effective in preventing estrus and ovulation in cattle for a 21-day period. 190
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THERIOCENOLOGY Generaliy, the results of these experiments are in agreement with the reports of others (9, 10, 11, 12, 13, 14, 15) showing that a g-day implant treatment with progestogens and an injection of EV does not result in lowered fertility. Fertility was reduced in heifers receiving the implant for 16 days and at the higher levels of EV in conjunction with the g-day implant treatment. Synchronization of estrus was far from optimum with the described treatments which gave the best levels of fertility. It was apparent in several of these trials that the percentage of animals showing estrus by 5 days after implant removal was not as high in animals treated in either the early or late stages of the cycle as it was in those animals treated in mid-cycle. Therefore, further modifications of this treatment are necessary to maintain fertility while causing better synchrony in animals in the early or late stages of the cycle at the time of treatment. Use of E2 to decrease variability in ovulation time was not beneficial. This confirms the results of Lantz and co-workers (16) but differs from those of Wiltbank and others (11).
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191
THERIOGENOLOGY Literature Cited 1.
Ulberg, L. C., R C. Christian and L. E. Casida. 1951. Ovarian repsonse in heifers to progesterone injections. J. Anim. Sci. 10:752.
2.
Trimberger, G. W. and W. Hansel. 1955. Conception rate and ovarian function following estrus control by progesterone injections in dairy cattle. J. Anim. Sci. 14:224.
3.
Ulberg, L. C. and C. E. Lindley. 1960. Use of progesterone and estrogen in the control of reproductive activities in beef cattle. J. Anim. Sci. 19:1132.
4.
Hansel, W., P. V. Malven and D. L. Black. 1961. Estrous cycle regulation in the bovine. J. Anim. Sci. 20:621.
5.
Zimbleman, R. G. 1963. Determination of the minimal effective dose of 6a-methyl-17a acetoxy progesterone for control of the estrual cycle of cattle. J. Anim. Sci. 22:1051.
6.
Wiltbank, J. N., D. R. Zimmerman, J. E. Ingalls and W. W. Rowden. 1965. Use of progestational compounds alone or in combination with estrogen for synchronization of estrus. J. Anim. Sci. 24:990.
7.
Wiltbank, J. N., R. P. Shumway, W. R. Parker and D. R. Zimmerman. 1967. Duration of estrus, time of ovulation and fertilization rate in beef heifers synchronized with dihydroxy progesterone acetophenide. J. Anim. Sci. 26:764.
8.
Wagner, J. F., E. L. Veenhuizen, R. P. Gregory and L. V. Tonkinson. 1968. Fertil'ty in the beef heifer foll.owing treatment with 6-chloro A&-17 acetoxy progesterone. J. Anim. Sci. 27~1627.
9.
Synchronization of estrus in heifers Roche, J. F. 1974. with implants of progesterone. J. Reprod. Fert. 41:337.
10.
192
Wiltbank, J. N. and C. W. Kasson. 1968. Synchronization of estrus in cattle with an oral progestational agent and an injection of an estrogen. J. Anim. Sci. 27~113.
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THERIOGENOLOGY
11.
Wiltbank, J. N., J. C. Sturges, D. Wideman, D. G. LeFever and L. C. Faulkner. 1971. Control of estrus and ovulation using subcutaneous implants and estrogens in beef cattle. J. Anim. Sci. 33:600.
12.
Roche, J. F. 1974. Effect of short-term progesterone treatment on estrus response and fertility in heifers. J. Reprod. Fert. 40:433.
13.
Roche, J. F. 1976. Calving rate of cows following insemination after a 12-day treatment with silastic coils impregnated with progesterone. J. Anim. Sci. 43~164.
14.
Wishart, D. F. and I. M. Young. 1974. Artificial insemination of progestin (SC21009) treated cattle at predetermined times. Vet. Rec. 95:503.
15.
Sreenan, J. M. and P. Mulvehill. 1975. The application of long- and short-term progestogen treatments for estrus cycle control in heifers. J. Reprod. Fert. 45:367.
16.
Lantz, W. B., C. W. Rasson, A. L. Slyter and D. R. Zimmerman. 1968. Synchronization of ovulation in beef heifers. J. Anim. Sci. 27:1193 (Abstr.).
17.
Snedecor, G. W. and W. G. Cochran. 1967. Statistical methods. The Iowa State University Press. Ames, Iowa.
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THERIOGENOLOGY
TABLE I.
EXPERIMENTAL DESIGN
Experiment EXPERIMENT 1 Trial 1
Trial 2
Group
No. of animals
I16 IV + 5EV C 116 I9 + 5EV C
Trial 3
EXPERIMENT 2 Trial 1
19 + 5EV
Trial 2
19 + 7.5EV C IV + 7.5EV
EXPERIMENT 3 Trial 1
I9 + 5EV C
Trial 2 Trial 3 Trial 4 Trial 5
19 + C 19 + C I9 + C IV + IV + C
5EV 7.5EV 6EV 7.5EV 6.5EV
Treatment received
40 40 40 36 39 37 59 60
I only for lb days I for 9 days and 5 mg EV on day of implantation No treatment I only for lb days I for 9 days and 5 ma EV on day of implantation No treatment I for 9 days and 5 mg EV on day of implantation No treatment
55 55 35 38
I for 9 days and 7.5 mg EV on day of implantation No treatment I for 9 days and 7.5 mg EV on day of implantation No treatment
45 44 51 54 21 21 226 226 44 45 44
I for 9 days No treatment I for 9 days No treatment I for 9 days No treatment I for 9 days No treatment I for 9 days I for 9 days No treatment
and 5 mg EV on day of implantation and 5 mg EV on day of implantation and 7.5 mg EV on day of implantation and 6 mg EV on day of implantation and 7.5 mg EV on day of implantation and 6.5 mg EV on day of implantation
EXPERIMENT 4 19 + 5EV IV + 5EV + .5E2(0)
11 10
19 + 5EV + .25E2(0)
11
IV + 5EV + .5E2(6)
11
IV + 5EV + .25E2(6)
11
19 + 5EV + .25E2
29
I for 9 days and 5 mg EV on day of implantation I for 9 days and 5 mg EV on day of implantation and .5 mg E2 at implant removal I for 9 days and 5 mg EV on day of implantation and .25 mg E2 at implant removal I for 9 days and 5 mg EV on day of implantation and .5 mg E2 6 hours post-implant removal I for 9 days and 5 mg EV on day of implantation and .25 mg E2 6 hours post-implant removal
EXPERIMENT 5
C
I EV E2 c
194
= = = =
30
I for 9 days and 5 mg EV on day of implantation and .25 mg E2 at time of implant removal No treatment
6 mg norgestomet implant estradiol valerate estradiol 17-8 control
AUGUST-SEPTEMBER
1978 VOL. 10 NOS. 2-3
36 39 37
59 60
TRIAL 2 116 I9 + 5EV C
TRIAL 3 19 + 5EV C
12
30 33
35 5
24
44
83 79
90 78
48
71
86 85
90 80
72
80
86 90
92 88
96
83
86 92
92 98
120
Cumulative percent showing estrus (hour after implant removal) ~-_-_-~-~_~_---_-_-
bed
45 61
35b 5oc 61'
3ob 62' 71C
3ob 5oc 27b
2gb 60' 10d
5 days
61 69 78
68 80 70
27 days
Percent pregnant (days after implant removal)a _---_-_______--_
Figures within same trial and same column with no superscript in common differ significantly (PC.05).
49 59
31 36 37
37 39 38
No. heifers inseminated
Percent conceiving at first insemination
SYNCHRONIZATION OF ESTRUS AND FERTILITY WITH A 16-DAY IMPLANT TREATMENT OR A g-DAY IMPLANT TREATMENT PLUS 5 MG EV IN VIRGIN HEIFERS.
aBased on total number of heifers in each group.
40 40 40
No. of heifers
EXPERIMENT 1.
TRIAL 1 116 I9 + 5EV C
Trial
TABLE II.
4
K
5
35 38
TRIAL 2 I9 + 7.5EV C
20
8
24
97
75
48
97
87
72
97
94
96
97
96
120
Cumulative percent showing estrus (hour after implant removal) -~-___--~---~~-~~~~
bc
4ob 64'
39 35
5 days
71 76
27 days
Percent pregnant (days after implant removal)a -__-_-_---_-_---
Figures within same trial and same column with no superscript in common differ significantly (PC.05).
34 29
53 55
No. heifers inseminated
Percent conceiving at first insemination
SYNCHRONIZATION OF ESTRUS AND FERTILITY WITH 7.5 MG EV IN CONJUNCTION WITH A g-DAY IMPLANT TREATMENT IN VIRGIN HEIFERS.
aBased on total number of heifers in each group.
55 55
No. of heifers
EXPERIMENT 2.
TRIAL 1 I9 + 7.5EV C
Trial
TABLE III.
188 (83%) 165 (73%)
38 (86%) 38 (94%) 38 (86%)
226 226
45 44
aCows were only observed 17 days in Trial 4.
I9 + 6.5EV C
C
20 (95%) 17 (81%)
21 21
TRIAL 3 19 + 7.5EV C
46 (90%) 41 (76%)
51 54
TRIAL 2 19 + 5EV C
29 (64%) 32 (73%)
Cows in estrus first 21 days of breedinga
68 60
66
7
5 3
10
65
59
5
17
14
89 79
77
80
70
72
92 84
85 80
92 79
100
74
79
90
74
72
Cumulative percent showing estrus (hour after implant removal) 120 72 24 96 48
CONTROL OF ESTRUS IN SUCKLED COWS FOLLOWING A g-DAY IMF'LANTTREATMENT AND VARIOUS LEVELS OF EV.
45 44
No. of cows
EXPERIMENT 3.
TRIAL 1 19 + 5EV C
Trial
TABLE IV.
2
51 54
21 21
TRIAL 2 I9 + 5EV C
TRIAL 3 19 + 7.5EV C
44 45 44
35 (79%) 32 (71%) 38 (86%)
160 (71%) 165 (73%)
20 (95) 17 (81%)
34 (67%) 41 (76%)
23 (51%) 32 (73%)
No. cows inseminateda
43c 56d 66d
54 50
45 53
74 63
39 47
Percent conceiving at first insemination
34c 4oc gd
4gc lod
2oc lid
64 67 68
4gc 38d
57 48
7ec 52d
47 41
Percent pregnant (days after implant removal)b ---______-__ _____--___-_-_ 5 days 26 days
FERTILITY IN SUCKLED COWS FOLLOWING A g-DAY IMPLANT TREATMENT AND VARIOUS LEVELS OF EV.
cd Figures in the same trial and the same column with no superscript in cormnondiffer significantly (PC.05).
b Based on total number of cows in each group.
aCows in treated groups inseminated first 5 days after implant removal and cows in control groups inseminated first 21 days of breeding, except in Trial 4 where cows in control group were only inseminated for a lJ-day period.
TRIAL 5 I9 + 7.5EV I9 + 6.5EV C
226 226
45 44
TRIAL 1 I9 + 5EV C
TRIAL 4 I9 + REV C
No. of cows
EXPERIMENT 3.
Dose of estradiol valerate
TABLE V.
s
8
a
e"
11
11
11
19 + 5EV + .25E2(0)
19 + 5EV + .5E2(6)
19 + 5EV + .5E2(6)
39.3
43.3
34.2
38.0
58.8
28.02
31.38
20.31
24.48
25.01
59.2
62.4
64.4
62.5
86.8
23.3
21.0
25.3
28.9
25.2
17.8
17.0
15.5
11.6
14.4
6.78
5.39
6.46
4.40
5.05
Length of estrus hr ______-__mean SD
timed from first observed estrus or ovulation within each group.
11
11
9
10
10
Implant removal to estrus hr ovulation hr ___________ ____--___sD_ mean mean SD
21.0
27.2
30.2
27.5
28.0
3.88
9.76
8.27
5.83
5.35
start of estrus to ovulation hr _____--_-sD_ mean
bcdFigures in the same column with no superscript in common differ significantly (Pc.05).
%ntervals
10
I9 + 5EV + .5E2(0)
-
11
I9 + 5EV
in estrus within 120 hr
No.
80Cd
80Cd
78bC
7obc
4ob
% heifers in estrus in 12-bra interval
80C
60'
56'
63'
2ob
% heifers ovulating in 12-hra interval
OCCURRENCE OF ESTRUS AND OWLATION IN VIRGIN HEIFERS FOLLOWING A g-DAY IMPLANT TREATMENT AND 5 MG EV AT IMPLANTATION WITH AND WITHOUT ESTRADIOL-176 AT IMPLANT REMOVAL (0) OR 6 HOURS LATER (6).
No. of heifers
EXPERIMENT 4.
Treatment
TABLE VI.
g 4
$
$
6
g
zl m
24
17
29
30
I9 + 5EV + .25E2
(:
45
48 66
72 72
96 76
120
Cumulative percent showing estrus (hour after implant removal) __----_-____________-~-~-------__
30
22
No. heifers inseminated
47
36
Percent conceiving at first insemination
SYNCHRONIZATION OF ESTRUS AND FERTILITY IN VIRGIN HEIFERS FOLLOWING A g-DAY IMPLANT TREATMENT AND 5 MG EV AT IMPLANTATION WITH .25 MG ESTRADIOL-17B AT TIME OF IMPLANT REMOVAL.
No. of heifers
EXPERIMENT 5.
___I_.
Treatment
--
TABLE VII.