J. Anim. Sci. 2003. 81:1681-1692
© 2003 American Society of Animal Science
Resynchronization of estrus in cattle of unknown pregnancy status using estrogen, progesterone, or both1
J. S. Stevenson*,1,
S. K. Johnson
,
M. A. Medina-Britos*,
A. M. Richardson-Adams* and
G. C. Lamb
* Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506-0201;
and
Northwest Research and Extension Center, Kansas State University, Colby 67701-0786; and
and
North Central Research and Outreach Center, University of Minnesota, Grand Rapids 55744-3396
2 Correspondence:
phone: 785-532-1243; fax: 785-532-7059; E-mail:
jss{at}ksu.edu.
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Abstract
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Our objective was to develop treatments applied to cattle of unknown pregnancy status that would resynchronize the repeat estrus of nonpregnant females. In Exp. 1, previously inseminated dairy and beef heifers were assigned randomly to each of three treatments 13 d after AI: 1) no treatment (controls; n = 44); 2) 0.5 mg of estradiol cypionate (ECP) i.m. on d 13 and 20 at the time of insertion and removal of a used intravaginal progesterone (P4)-releasing insert (CIDR; P4 + ECP; n = 44); and 3) same as P4 + ECP without injections of ECP (P4; n = 42). The P4 + ECP (>90%) and P4 (>75%) protocols effectively synchronized repeat periods of estrus to 2 d and did not harm established pregnancies. In Exp. 2, treatments similar to those in Exp. 1 were applied to previously inseminated beef heifers (n = 439). Feeding 0.5 mg of melengestrol acetate (MGA) from d 13 to 19 after AI replaced the CIDR as a source of progestin. Of those heifers not pregnant (n = 65) after the initial AI, more than 86% were reinseminated, but conception was decreased (P < 0.05) by 28 to 39% compared with controls. In Exp. 3, previously inseminated lactating beef cows at four locations were assigned within herd to each of three treatments: 1) no treatment (control; n = 307); 2) same as in Exp. 1, but with P4 + 1 mg of estradiol benzoate on d 13 and 20 (P4 + EB; n = 153); and 3) same as in Exp. 1, P4 + ECP (n = 149). Treatments with P4 plus estrogen did not decrease conception rates in pregnant cows at any location, but increased (P < 0.05) the percentage of nonpregnant cows returning to estrus between 19 and 23 d after timed AI from 29% in controls to 86% in P4 + EB and 65% in P4 + ECP cows. Conception rates at the return estrus were not decreased when treatments occurred between d 13 and 20. In Exp. 4, lactating beef cows were assigned as in Exp. 3 to each of three treatments: 1) no treatment (controls; n = 51); 2) P4 + ECP (n = 47), as in Exp. 1; and 3) a single injection of ECP on d 13 (n = 48). Previously established pregnancies were not harmed (P = 0.70), and return rates of nonpregnant cows did not differ (P = 0.78) among treatments. In summary, in both heifers and lactating beef cows, the P4-based resynchronization treatments increased synchronized return rates when estrus detection rates were low, had no negative effects on established pregnancies, and decreased or tended to decrease conception rates at the resynchronized estrus.
Key Words: Estrogens • Estrus • Fertility • Progestogens • Synchronization
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Introduction
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Pregnancy outcome after synchronization of estrus and ovulation is unknown until cows return to estrus or after diagnosis of pregnancy. In such cases, the full extent of the advantages for synchronizing estrus is not realized. Because the estrous cycles of most nonpregnant cows are closely synchronized, resynchronization for a subsequent AI is possible. New and previously used progestin-releasing inserts or implants successfully reduce variation in returns to service after AI in previously synchronized cows and heifers (Stevenson and Mee, 1991; Van Cleeff et al., 1996; Purvis and Whittier, 1997). Reinsemination of nonpregnant females at the first eligible estrus can be facilitated by resynchronization of estrus (Van Cleeff et al., 1996) and may increase conception rates at the resynchronized estrus (Stevenson and Mee, 1991). Reinsertion of a progesterone (P4)-releasing intravaginal insert between 13 and 20 d after AI plus an injection of 1 or 2 mg of estradiol benzoate (EB) at insertion and removal of the progestin treatment increased the probability of identifying nonpregnant cows within 2 d after the second EB injection (Macmillan et al., 1999).
The only estrogen product approved in the United States for use in the bovine is estradiol cypionate (ECP). It has multiple label indications, including "to correct anestrus [absence of heat period] in the absence of follicular cysts." Administration of 0.5 or 1 mg of ECP induces a LH surge in lactating dairy cows (Pancarci et al., 2002; Stevenson et al., 2002) and dairy heifers (Lopes et al., 2000) when given 24 h after a luteolytic dose of PGF2
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The objective was to determine if treatments would: 1) reduce conception rates in previously inseminated cattle; 2) increase AI resubmission rates with subsequent normal fertility; and 3) increase cumulative pregnancy rates after two inseminations.
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Materials and Methods
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Experiment 1
Previously inseminated Holstein heifers (n = 68) at the Kansas State University Dairy Teaching and Research Center were studied between September 2000 and August 2001 in six replications, and purebred Angus, Simmental, and Hereford heifers (n = 62) at the Kansas State University Purebred Unit were studied during spring breeding seasons. Heifers were inseminated previously in another experiment (Richardson et al., 2002) and were then assigned randomly within breed to each of three treatments. On d 13 (range: 11 to 15 d) after the initial AI (Figure 1
), heifers received: 1) no treatment (controls; n = 44); 2) 0.5 mg of ECP (Pharmacia Animal Health, Kalamazoo, MI) i.m. on d 13 and 20 when used intravaginal P4-releasing inserts (CIDR; InterAg, Hamilton, New Zealand; P4 + ECP; n = 44) were inserted and removed, respectively; and 3) the same as P4 + ECP without injections of ECP (P4; n = 42). The CIDR inserts had been used once or twice previously, and when new, contained 1.38 or 1.9 g of P4. The CIDR inserts were applied randomly across the latter two treatments.
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Figure 1. Experimental protocols employed in each of four experiments. Blood samples were collected on d 13 and 20 after the initial AI when progesterone (P4)-releasing (CIDR) inserts were placed intravaginally and removed, respectively. Experiment 1 consisted of three treatments: CIDR (P4), CIDR + 0.5 mg of estradiol cypionate (ECP; P4 + ECP), and control. Experiment 2 consisted of three treatments: melengestrol acetate [MGA] was fed on d 13 to 19, MGA was fed on d 13 to 19 with 0.5 mg of ECP on d 13 and 20 (24 h after MGA withdrawal; MGA + ECP), and control. Experiment 3 consisted of three treatments: CIDR (P4), CIDR + 1 mg of estradiol benzoate (EB; P4 + EB), and CIDR + 0.5 mg of ECP (P4 + ECP). Experiment 4 consisted of three treatments: CIDR + 0.5 mg of ECP (P4 + ECP), a single injection of 0.5 mg of ECP on d 13 (not shown), and control. Females were observed for estrus and inseminated during MGA feeding (Exp. 2) through d 33 or on or after CIDR insert removal (d 20). CIDR = intravaginal controlled internal drug release insert that released P4.
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Beef heifers were observed visually for signs of estrus three to four times per day during daylight hours of expected estrus (d 18 to 26). Dairy heifers were fitted with electronic estrus-detection devices (HeatWatch, DDx Inc., Denver, CO). Once estrus was detected, inseminations were performed 8 to 14 h later by one technician for dairy heifers and three technicians for beef heifers. Pregnancy was diagnosed by transrectal ultrasonography once between 27 and 34 d after the initial AI to determine initial AI conception rate, and again between 40 and 54 d by uterine palpation or transrectal ultrasonography (Aloka 500V, Corimetrics, Wallingford, CT; 5.0-MHz transducer) to determine resynchronized AI conception rate.
Blood samples were collected from all heifers via puncture of a coccygeal vessel on d 13 and 20 after the initial AI corresponding to when CIDR inserts were inserted and removed. Progesterone was measured in blood sera using a specific validated radioimmunoassay (Skaggs et al., 1986). Inter- and intraassay coefficients of variation were 11.3 and 8.6%, respectively, for two assays.
Characteristics of estrus for dairy heifers calculated for the first postinsemination estrus (18 to 26 d) from electronic estrus-detection devices were: duration of estrus, number of standing events, duration of all standing events, and duration of individual standing events. Conception rates (expressed as the number pregnant divided by the number detected in estrus and inseminated x 100) at the initial insemination and after the resynchronized estrus, concentrations of P4 on d 20 when CIDR inserts were removed, percentages of heifers with low (<1 ng/mL) or high (≥1 ng/mL) concentrations of P4 on d 20, 26-d pregnancy rate (proportion pregnant after two inseminations), interval between inseminations (18 to 26 d), and percentage of nonpregnant heifers detected in estrus (AI resubmission rate for second service after resynchronization) were analyzed using the GLM and CATMOD procedures of SAS (SAS Inst., Inc., Cary, NC). The model consisted of treatment, location (dairy vs. beef), and their interaction. Because AI technicians and sires were unique to each location, those effects were confounded with location. Means were separated by orthogonal contrasts (control vs. both P4 treatments and P4 vs. P4 + ECP) or by LSD tests when associated with a protected F-test (P ≤ 0.05) in the ANOVA. Levene’s test for heterogeneity of variance (Milliken and Johnson, 1984) was used to analyze variability of return-to-estrus patterns after resynchronization treatments.
Experiment 2
This experiment was conducted at Losey Land and Cattle (Agra, KS) with yearling Angus crossbred heifers. Heifers were previously synchronized with a standard melengestrol acetate (MGA; Pharmacia Animal Health, Kalamazoo, MI) + PGF2 protocol; 0.5 mg of MGA fed per heifer daily for 14 d, and 25 mg of PGF2 (Lutalyse, Pharmacia Animal Health) injected 19 d after the last feeding of MGA. Feeding of MGA was included in the morning in half of the daily total mixed diet. Heifers were inseminated based on the AM–PM rule until 72 h after PGF2, at which time all heifers that had not shown estrus were inseminated. Average day of insemination was considered to be d 0 for the present study. A majority (431 of 439) was inseminated the first time from 1.5 d before to 1.5 d after d 0. The remaining eight heifers received a timed AI (TAI). Seventeen of the 439 heifers subsequently were observed in estrus 2 to 2.5 d later and were reinseminated. Immediately following the initial AI, heifers were returned to new pens based on the time they were inseminated. Eight days after the initial AI, each pen of heifers was gate cut to divide heifers into three treatments. Any heifer that returned to estrus before sorting (d 8) into resynchronization treatments was excluded from the experiment. Treatments for this experiment (Figure 1
) were: 1) no treatment (control; n = 87); 2) MGA (n = 176) fed at 0.5 mg•heifer-1•d-1 from d 13 (d 0 = mean day of previous insemination) through d 19; and 3) MGA + ECP (n = 176), which was similar to the previous treatment plus 0.5 mg (i.m.) of ECP administered on d 13 and 20 (1 d after MGA withdrawal). Heifers were observed for estrus at least twice daily from d 0 to 33 and were reinseminated according to the AM–PM rule.
Pregnancy was determined on d 33 (initial AI conception rate was the same as in Exp. 1) and 59 (resynchronized AI conception rate was the same as in Exp. 1) by transrectal ultrasonography. Data were analyzed using the mixed models procedure (SAS Inst. Inc.). Treatment and timing of the initial AI (estrus AI or TAI) were considered fixed effects and sire and technician were considered random effects.
Experiment 3
This experiment was conducted at four locations: University of Minnesota Research and Outreach Center, Grand Rapids (81 Angus cows); DarLynn Ranch, Pierz, MN (149 Angus, Hereford, and South Devon cows); Kansas State University Purebred Beef Unit, Manhattan (161 Angus, Hereford, and Simmental cows); and Thielen Ranch, Dorrance, KS (218 cows consisting of a three-way rotational cross of Angus, Hereford, and Simmental). Cows were inseminated previously in another experiment (Stevenson et al., 2003) and assigned within herd to three treatments according to days postpartum, parity (primiparous vs. multiparous), and previous synchronization treatment used before their initial TAI (Figure 1): 1) no treatment (control; n = 307); 2) a previously used CIDR insert was reinserted 13 d after TAI for 7 d and 1 mg of EB (Sigma Chemical, St. Louis, MO) in sesame oil was administered i.m. on d 13 when the CIDR was inserted and on d 20 when it was removed (P4 + EB; n = 153); and 3) same as in the previous treatment, but 0.5 mg of ECP was injected at the insertion and removal of the used CIDR (P4 + ECP; n = 149). Blood samples were collected at insertion and removal of CIDR inserts and assayed for P4 as in Exp. 1 (six assays with intra- and interassay CV of 6.4 and 7.8%, respectively). At the Minnesota locations, insertion of used CIDR inserts and first estradiol (EB or ECP) injections occurred on d 11 after TAI and CIDR removal and after second estradiol injections on d 18. The CIDR inserts (when new, these inserts contained 1.9 g of P4) used in this study had been used once previously in half of the cows to synchronize ovulation before the initial TAI.
Cows were observed for estrus two or three times daily from d 19 to 23 after TAI at Kansas locations and from d 17 to 21 at Minnesota locations. Cows in estrus were inseminated 8 to 14 h after first detected estrus. Sires and inseminators were distributed equally among treatments and were confounded within herd. Pregnancy was diagnosed 29 to 33 d after the initial AI (initial AI conception rate as in Exp. 1) and again 54 to 61 d after the initial AI or 35 to 42 d after the second AI (resynchronized AI conception rate as in Exp. 1) by transrectal ultrasonography. At three locations, embryo survival was calculated based on the presence of an embryo at pregnancy diagnosis on d 29 to 33 after the initial TAI and its presence or absence on d 54 to 61 after TAI.
All variables (described in Exp. 1) were analyzed using the GLM and CATMOD procedures of SAS using a model that consisted of resynchronization treatment, location (MN vs. KS), cycling status at time of the initial AI (based on previous blood collection [Stevenson et al., 2003]), parity (primiparous vs. multiparous), all two-way interactions with treatment, with days postpartum and BCS (1 = thin and 9 = fat; Whitman, 1975; assessed just before the initial TAI) as regression variables. Least squares means or unadjusted mean percentages were separated using LSD tests when associated with a protected F-test (P ≤ 0.05) in the ANOVA or by orthogonal contrasts (control vs. both CIDR treatments; and P4 + ECP vs. P4 + EB).
Experiment 4
This experiment was conducted at the Kansas State University Purebred Unit with 146 previously inseminated purebred Angus, Hereford, and Simmental lactating cows used in a previous experiment (Stevenson et al., 2003). Cows were inseminated (single TAI) and assigned randomly to each of three resynchronization treatments based on breed, parity (primiparous vs. multiparous), days postpartum, and previous synchronization treatment used before TAI (Figure 1): 1) no treatment (control; n = 51); 2) a previously used CIDR insert was reinserted on d 13 after TAI for a period of 7 d, and 0.5 mg of ECP was injected at its insertion and removal (P4 + ECP; n = 47); and 3) a single 0.5-mg injection of ECP on d 13 (ECP; n = 48; not illustrated in Figure 1). Blood samples were collected and assayed for P4 as in Exp. 1 (two assays with inter- and intraassay CV of 7.5 and 6.8%, respectively). The CIDR inserts (when new these inserts contained 1.38 or 1.9 g of P4) had been used once or twice previously.
Cows were observed at least twice daily for estrus after the initial TAI and were reinseminated as in Exp. 3. Pregnancy was diagnosed as described above at 35 to 36 d after the TAI (initial AI conception rate as in Exp. 1) and after the second AI (resynchronized AI conception rate as in Exp. 1) that followed resynchronization treatments. All variables (described for Exp. 1 and 3) were analyzed in the GLM and CATMOD procedures of SAS in a model that included resynchronization treatments, parity (primiparous vs. multiparous), breed, and cycling status at the initial TAI (based on previous blood collection [Stevenson et al., 2003]). All two-way interactions with treatment were included with days postpartum and BCS as regression variables. Least squares means or adjusted mean percentages were separated using LSD tests when associated with a protected F-test (P ≤ 0.05) from ANOVA or by orthogonal contrasts (control vs. P4 + ECP and ECP; and P4 + ECP vs. ECP).
All Experiments
These studies were conducted while availability of new CIDR inserts was limited, so used CIDR inserts were employed. We have conducted previous studies with new CIDR inserts under authorization of the U.S. FDA Investigational New Animal Drug 6450. The intent of the current treatments was to test the efficacy of supplying P4 to prevent premature occurrence of repeat estrus during the resynchronization treatment period rather than testing the used CIDR insert itself, which could not be done without concurrent administration of new CIDR inserts as controls. Application of previously used CIDR inserts in no way implies that we endorse their reuse.
Previously used (once or twice) CIDR inserts (containing 1.38 or 1.9 g of P4 when new) released sufficient P4 in situ to prevent estrus in dairy and beef heifers treated with used inserts for 7 d (Richardson et al., 2002). In that study, CIDR inserts were removed 24 h after an injection of PGF2. At insert removal, blood concentrations of P4 in sera of 88% of heifers were elevated (≥1 ng/mL; average range of 1.5 to 1.7 ng/mL) compared to 31% of nonCIDR-treated controls (average concentrations of 0.8 ng/mL).
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Results
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Experiment 1
Distribution of estrus after CIDR removal for nonpregnant dairy (visual observations plus electronic estrus detection system) and beef heifers (visual observation) is illustrated in Figure 2. Most of the returns to estrus for heifers in the P4 + ECP (64%) treatment were on the day after CIDR removal. The majority of returns to estrus for controls were before (25%), on the day of (25%), or 4 or more days after CIDR removal (19%). On the day following second ECP injections (d 1), more (P < 0.05) P4 + ECP heifers (64%) were in estrus than in either of the other treatments (P4 = 33%; control = 13%). The P4 + ECP treatment produced nearly as many heifers in estrus on d 1 (64%) as the P4 treatment did for 2 d (d 1 and 2; 76%). Variability of the return-estrus pattern was less (P < 0.01) in both P4 treatments than in that of the control.
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Figure 2. Distribution of repeat estrus in previously inseminated heifers relative to progesterone (P4)-releasing intravaginal (CIDR) insert removal for those treated with CIDR inserts for 7 d beginning on d 13 after AI (P4); P4 + estradiol cypionate (ECP); or controls. Pattern of return was less (P < 0.01) variable in both resynchronization treatments that employed P4 compared to controls (Exp. 1).
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Table 1 summarizes the reproductive characteristics of all heifers given resynchronization treatments. Concentrations of P4 were greater (P < 0.05) on d 13 before treatments were applied in the P4 (5.3 ± 0.3 ng/mL) and P4 + ECP heifers (5.0 ± 0.3 ng/mL) than in controls (4.2 ± 0.3 ng/mL). Based on elevated (≥1 ng/mL) concentrations of P4 on d 20 (CIDR removal), more (P < 0.05) P4-treated heifers had high P4 than controls. Concentrations of P4 for the P4 treatment tended (P = 0.09) to be greater than those of the P4 + ECP treatment and control, suggesting that prior ECP injection on d 13 may have reduced P4 secretion. Blood serum concentrations of P4 were lower (P < 0.05) in beef than in dairy heifers on d 13 (4.1 ± 0.5 vs. 5.5 ± 0.2 ng/mL) and 20 (3.4 ± 0.4 vs. 4.5 ± 0.4 ng/mL). Although conception rates varied from 47 to 60%, resynchronization treatments seemed to have no adverse effect (P > 0.30) on the proportion of heifers that conceived to the previous insemination before their application (Table 1). Of those heifers that failed to conceive after the initial AI before treatment, returns to estrus following resynchronization treatments were not different (P > 0.39) among treatments. Further, the interval between first and second AI did not differ (P > 0.61) among treatments. Although limited numbers of heifers were reinseminated, the P4 and P4 + ECP treatments tended (P = 0.13) to suppress conception at the repeat service compared with controls. The 26-d pregnancy rate for those conceiving after either the first or second insemination did not differ (P > 0.35) among treatments.
Sexual behavioral characteristics of dairy heifers whose estrus was resynchronized are summarized in Table 2. No significant differences among treatments were detected for the duration of standing estrus, total number and duration of all standing events, or duration of individual standing events (Table 2). However, the P4 + ECP treatment had the greatest number of standing events and durations of total standing time. Variability of these traits did not differ among treatments.
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Table 2. Sexual behavioral characteristics of dairy heifers whose estrus was resynchronized based on the HeatWatch system (Exp. 1)
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All nonpregnant dairy heifers in the P4 + ECP treatment were observed visually in estrus compared with less than 64% of heifers in the P4 and control heifers (Table 2). The ECP injection may have enhanced the proportion of heifers visually detected in estrus because of a tendency for more estrual activity induced by ECP. Further, because no significant differences were detected, one could conclude that the 0.5-mg dose of ECP used apparently produced a normal physiological estrus.
Experiment 2
Resynchronization treatments had no negative effect on conception rates resulting from the initial insemination (Table 3). Mean hours from second injections of ECP to estrus tended (P = 0.07) to be less for the control (54 ± 9) than for MGA (72 ± 6) or MGA + ECP (74 ± 5). Further, variance of the interval to estrus was greater (P < 0.05) for control than MGA or MGA + ECP (1,160, 819, and 912, respectively). Greater (P < 0.05) proportions of control than MGA- or MGA + ECP-treated heifers were in estrus before and during MGA feeding (Table 3). Feeding MGA seemed to delay estrus in the MGA and MGA + ECP treated heifers because 14% of heifers in each treatment were in estrus more than 25 d after the initial AI, whereas none of controls were in estrus during this period (Figure 3). Distribution of estrus after the second ECP injections showed no clear peak for the MGA or MGA + ECP treatments. However, the two MGA treatments reduced (P < 0.05) numbers of heifers returning to estrus between d 13 and 19 (last day of MGA feeding) after the initial AI. Resynchronized conception rates were greater in the control than MGA (P < 0.05) or MGA + ECP (P = 0.09) treated heifers (Table 3). These differences seem to be due to lower conception rates during the targeted resynchronization period (d 20 to 25) for MGA and MGA + ECP heifers than for control heifers (Table 3). The 25-d pregnancy rate after two inseminations did not differ among treatments (Table 3).
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Table 3. Reproductive traits of heifers after resynchronization with melengestrol acetate (MGA) or MGA plus estradiol cypionate (ECP) (Exp. 2)a
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Figure 3. Distribution of repeat estrus in previously inseminated heifers after receiving melengestrol acetate (MGA) for 7 d; MGA + estradiol cypionate (ECP); or controls (Exp. 2).
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Experiment 3
Timing of administration of resynchronization treatments affected distribution of returns to estrus between Kansas and Minnesota herds (Figure 4). Only one pregnant cow was detected in estrus as a consequence of the P4 + EB treatment, and it was eliminated from further analyses. In Minnesota herds, no incidence of return to estrus was observed before CIDR insert removal among cows in any treatment (Figure 4; lower panel). In Minnesota herds, the second injection of estrogen may not have been as effective in inducing estrus and the LH surge because subsequent estrus was distributed across more than 3 d. In either P4 + estrogen combination, return to estrus was uniformly distributed over a 3-d period.
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Figure 4. Distribution of repeat estrus in previously inseminated lactating beef cows after progesterone (P4)-releasing intravaginal (CIDR) insert removal for those treated with CIDR inserts for 7 d beginning on d 13 after AI (d 11 in MN) and removed on d 20 (d 18 in MN) with either estradiol benzoate (EB) or estradiol cypionate (ECP) injections given on d 13 and d 20 (d 11 and d 18 in MN herds); or controls (Exp. 3).
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In contrast, some Kansas control cows returned to estrus on d -1 and 0 (d 19 and 20 after TAI; Figure 4; upper panel). The P4 + EB treatment concentrated estrus on d 1 and 2, with the majority on d 1. In contrast, the P4 + ECP treatment concentrated estrus on d 2 and 3 for Kansas cows, but mainly on d 2.
More (P < 0.05) cows treated with P4 + estrogen than controls had elevated concentrations of P4 on d 18 or 20 after TAI when CIDR inserts were removed (Table 4). Concentrations of P4 in blood on d 13 before treatments were applied did not differ among control (2.7 ± 0.2 ng/mL), P4 + ECP (2.8 ± 0.2 ng/mL), and ECP cows (2.8 ± 0.2 ng/mL). Average concentrations of P4 on d 18 or 20 were only greater (P < 0.05) in the P4 + ECP treatment compared with controls, with the P4 + EB treatment being intermediate. Fewer (P < 0.001) cows (53%; n = 62) that were anestrus before the initial AI had elevated serum P4 (anestrus = 2.0 ± 2.0 ng/mL vs. cycling = 3.9 ± 2.5 ng/mL) on d 18 or 20 than cycling cows (87%; n = 526), which is reflected in average concentrations of P4. For each unit increase in BCS measured 2 d before the initial TAI, concentrations of P4 on d 18 or 20 were increased by 0.7 ± 0.2 ng/mL.
No harm occurred to pregnancies established in cows when resynchronization treatments were applied (conception rates after initial AI; Table 4). Conception rates after the initial TAI varied from 44 to 52% across treatments, with a smaller (P < 0.05) percentage of anestrous cows conceiving (33%; n = 66) than cycling cows (52%; n = 543). The total proportion of nonpregnant cows returning to estrus between d -1 and 3 after CIDR removal (d 0) was 2.2 to 2.9 times greater (P < 0.05) after both P4 + estrogen treatments than in controls (Table 4). No difference was detected between estrogen treatments, but proportions of primiparous cows (41%; n = 67) that returned to estrus were less (P < 0.05) than those of multiparous cows (56%; n = 235).
Intervals between TAI and the repeat AI were influenced by a treatment x location interaction (P < 0.05; Table 5). Because of earlier administration of the resynchronization treatments in Minnesota herds, concentrations of serum P4 on d 18 may have blocked the ability of the second injection of estradiol to induce estrus and the LH surge. Intervals to returned estrus among treatments were not different. In contrast, in Kansas herds in which resynchronization treatments were administered between d 13 and 20, the P4 + ECP treatment prolonged (P < 0.05) average interval to estrus compared with controls and P4 + EB.
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Table 5. Interval to estrus and conception rate after resynchronization of estrus at Kansas and Minnesota locations (Exp. 3)
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Conception rates after resynchronization also were affected (P < 0.05) by location (Table 5). No difference among treatments were observed for Kansas herds, but for Minnesota herds, the P4 + EB treatment tended (P = 0.07) to reduce conception rates compared with controls.
The 23-d pregnancy rates tended (P = 0.15) to be greater after both P4 + estrogen treatments, with the biggest difference between the P4 + ECP and controls (Table 4). The 23-d pregnancy rate was greater in cycling cows (62%; n = 543) than in anestrous cows (41%; n = 66). This relationship is substantiated by the fact that for each 10-d increase in days since calving at time of the initial AI, a 2.8 ± 1.1% increase (P < 0.01) in 23-d pregnancy rates was detected.
Embryo survival in pregnant cows assessed at 29 to 33 d after the initial TAI and d 54 to 61 at the second pregnancy diagnosis was affected (P < 0.05) by herd, but not by resynchronization treatment. Although survival was numerically greater in the P4 + ECP cows, it only tended (P = 0.10) to differ from control.
Experiment 4
Distribution of estrus after a single injection of ECP on d 13 and the 7-d CIDR plus injections of ECP on d 13 and 20 is illustrated in Figure 5. Nearly 67% of the P4 + ECP cows were in estrus on d 2 after removal of CIDR inserts, with the pattern of distribution of estrus after the single ECP injection appearing bimodal with peaks on d 1 and on and after d 4. More than 62% of the ECP-treated cows were in estrus on or after d 4 (≥d 24 or ≥11 d after ECP injection).
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Figure 5. Distribution of repeat estrus in previously inseminated lactating beef cows relative to progesterone (P4)-releasing intravaginal (CIDR) insert removal for those treated with CIDR inserts for 7 d beginning on d 13 after AI + estradiol cypionate (ECP) on d 13 and 20; ECP on d 13; or controls (Exp. 4).
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Treatments did not affect proportions of cows with elevated P4 or concentrations of P4 on d 20 (Table 6). Conception rates of cows exposed to these resynchronization treatments were not different, but as in Exp. 3, anestrous cows at time of initial AI had lower (P < 0.05) conception rates (20%; n = 10) than cycling cows (46%; n = 136). Rates of return to estrus between 0 and 5 d after CIDR insert removal (20 to 25 d after TAI) were unaffected by treatment. Intervals between inseminations tended (P = 0.09) to be longer in P4 + ECP- and ECP-treated cows compared to controls. Conception rates and 25-d pregnancy rates after resynchronization were not significantly affected by treatments, although not enough cows were tested in this experiment to detect differences.
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Discussion
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One purpose of these studies was to determine if the first eligible estrus after insemination could be resynchronized without interfering with established pregnancies resulting from an earlier insemination. We found no strong evidence that implementation of the resynchronization protocols was disruptive to established pregnancies in any of four experiments. This finding was true for both cows and heifers. Likewise, pregnancies were unaffected when injections of EB were administered on d 12, 13, or 14 after AI (Macmillan et al., 1999). Injections of 1 mg of EB or 0.5 mg of ECP administered at insertion and removal of a used CIDR did not compromise the ability of CL to produce P4 or to maintain pregnancies already established in our studies. Some caution is warranted in interpreting the lack of negative effects of the resynchronization protocols because sufficient observations were not included in each individual experiment to avoid committing a type-II error. However, collectively, results indicate that the treatments were safe for ongoing pregnancies.
Survivability of embryos in cows established after the initial AI in Exp. 3 were unaffected by resynchronization treatments. Interestingly, proportions of cows in each treatment that had elevated P4 on d 20 (Table 5) paralleled percentages of embryo survival (Table 4). Supplementing exogenous P4 may prevent low concentrations of P4 from occurring in the maternal circulation and may prevent embryo losses in dairy cows (Stevenson and Mee, 1991; El-Zarkouny et al., 2001) and dairy heifers (Van Cleeff et al., 1996). In dairy cows, providing supplemental P4 with a P4-releasing intravaginal device increased pregnancy rates when treatments were initiated no earlier than d 3 after AI (Robinson et al., 1989; Van Cleeff et al., 1996), but not consistently (Stevenson and Mee, 1991). Supplemental progestin during the luteal phase tended to increase conception rates (Wilmut et al., 1986) or calving rates of beef heifers (Favero et al., 1993). In Exp. 3, the CIDR was inserted between d 13 and 20 (Kansas herds) or d 11 and 18 (Minnesota herds) after the initial AI, thus covering that time interval when embryo loss is reported to be greatest (Inskeep, 2002). It can be assumed that supplemental P4 from the CIDR might prevent early embryo death when combined with estradiol, but further observations are warranted.
Our results show that administering P4 via CIDR inserts was also effective in preventing the occurrence of spontaneous estrus before its removal. The intravaginal combination treatment of EB and P4 (CIDR) induced atresia of dominant follicles regardless of their age or diameter (Burke et al., 1999). Further, they suggested that duration of P4 treatment must be sufficient to prevent occurrence of estrus until complete spontaneous luteolysis has occurred. Occurrence of estrus in control cows in Kansas herds in Exp. 3 on the day before and day of CIDR removal was consistent with occurrence of luteolysis before d 19 of the estrous cycle. In contrast, because CIDR inserts were removed on d 18 in Minnesota herds, some nonpregnant cows may have had a functional CL when inserts were removed. Second injections of EB and ECP were given on d 18 after TAI when endogenous concentrations of P4 may have been elevated sufficiently to block the LH surge. Because no estrus was observed in Minnesota control cows until 1 d after CIDR removal, duration of P4 treatment apparently was sufficient. In all experiments, only four pregnant females showed estrus after withdrawal of treatments (one P4-treated heifer, one P4 + EB-treated cow, one P4 + ECP-treated cow, and one ECP-treated cow).
Use of MGA as the progestin was not as efficacious as the CIDR for synchronizing returns to estrus. Although control heifers in Exp. 2 came into estrus consistently before and during resynchronization treatments, a few MGA-treated heifers were in estrus during MGA feeding. That resynchronization with MGA had no effect on conception rates to the initial AI agrees with previous studies (Purvis and Whittier, 1997). More MGA-treated heifers were in estrus after d 25, suggesting that embryonic loss may have occurred in these heifers. The total percentage of heifers reinseminated was similar among treatments, so perhaps MGA "spared" embryos for a short period of time before embryos were eventually lost. Alternatively, delayed returns to estrus of some heifers may have occurred because prolonged clearance of MGA and/or ECP failed to induce ovulation or caused turnover of the dominant follicle. Ensuring adequate consumption of MGA is a key to success of initial synchronization or resynchronization of estrus.
Concentrating the distribution of estrus into a short, predictable timeframe provides advantages for an AI program. Detection of estrus is both time consuming and labor intensive, especially for repeat periods of estrus after a failed AI because interval to estrus is more variable compared with noninseminated females (Van Cleeff et al., 1996). Therefore, another purpose of these studies was to determine whether the protocols employed effectively increased percentages of nonpregnant females returning to estrus. When P4 was used in the form of the CIDR in Exp. 1 (heifers) and 3 (cows), percentages of eligible nonpregnant females returning to estrus increased. Increases were not observed for cows in Exp. 4, where detection of estrus was greater and return rates were 73% in controls. Greater percentages of returns occurred when more females had elevated (>1 ng/mL) concentrations of P4 on d 20 (Exp. 1 and 3) or had greater average concentrations of P4 (Exp. 3). When MGA was fed to heifers in Exp. 2, 23 to 30% of nonpregnant heifers returned to estrus at odd intervals before, during, and well after (≥d 6) the MGA feeding period. That these early returns also were observed in controls might indicate that previously inseminated heifers were inseminated at an induced or pubertal estrus and were returning to estrus after an abbreviated pubertal luteal phase.
The distribution patterns of estrus for our cattle given a single injection of EB on d 13 (CIDR insertion) and on d 20 at CIDR removal are consistent with those observed in dairy cattle (Macmillan et al., 1999) in which 43% were in estrus on d 1 and 42% on d 2 after CIDR removal. That estrus activity in the P4 + EB treatment in Exp. 3 was concentrated and occurred earlier than with the P4 + ECP treatment is consistent with the half lives of the two forms of estradiol as well as their rates of absorption and conversion to estradiol-17ß. Plasma estradiol-17ß reached supraphysiologic concentrations 1 to 23 h after EB treatment and remained elevated for 20 to 30 h (Vynckier et al., 1990; Lammoglia et al., 1998). A pronounced increase in peak plasma estradiol-17ß does not occur after ECP injection. Rather, maximal concentrations after injection of 10 mg of ECP were observed 13 to 31 h after treatment and remain elevated for 170 h before decreasing steadily (Vynckier et al., 1990). Therefore, differences in patterns of estrus distribution may occur either because EB produces adequate concentrations of estradiol-17ß to induce follicular atresia earlier than ECP (d 13 injection) or because of prolonged concentration of estradiol-17ß after ECP injection (d 20 injection). In contrast, in lactating dairy cows given either EB or ECP on d 13 after TAI concurrent with insertion of a used CIDR, we were unable to turn over a high percentage of dominant follicles (El-Zarkouny et al., 2002). Asynchronous emergence or delay of a new follicular wave may occur because of prolonged elevated concentrations of estradiol-17ß (Bo et al., 1995). The difference in dosage in our study between EB (1 mg) and ECP (0.5 mg) also may have influenced emergence of a new follicular wave.
The first estrogen injection on d 13 was hypothesized to initiate a new follicular wave so that timing of a new dominant follicle corresponded to withdrawal of P4 when CIDR inserts were removed. The P4 released via a new CIDR likewise can turnover dominant follicles and initiate a new follicular wave (Kang et al., 1999). Further, exogenous estradiol is normally luteolytic when administered early in the estrous cycle (Wiltbank and Kasson, 1968). Although estrogen is an integral component of the natural luteolytic mechanism, the effect of exogenous estrogen is variable and it should not be considered equipotent to the putative luteolysin, PGF2 (Burke et al., 2000).
The second injection of estrogen was administered to induce an LH surge and subsequently reduced the period necessary to detect estrus. Further, the second injection of EB reduced the time to the next estrus so that more nonpregnant cows returned to estrus sooner (Macmillan et al., 1999). Thus, in the present experiments, in both P4 + estrogen treatments, most returns to estrus occurred earlier than expected, probably due to the second injection of estrogen. In cattle, an increased titer of endogenous estradiol normally promotes the preovulatory LH surge by stimulating the number of GnRH receptors in the anterior pituitary while concentrations of P4 are basal (Hansel and Convey, 1983). Therefore, administration of exogenous estrogen after luteolysis may induce an LH surge. A dose of 1 mg of EB has been shown to be sufficient to elicit behavioral signs of estrus in anestrous cows (Fike et al., 1997). It has been demonstrated that 1 or 0.5 mg of ECP induces an LH surge in lactating dairy cows (Pancarci et al., 2002; Stevenson et al., 2002) and dairy heifers (Lopes et al., 2000) when given 24 h after a luteolytic dose of PGF2. Administration of EB at CIDR removal on d 20 of the cycle has been used to reduce variability in timing of the LH surge (Hanlon et al., 1996). When administering EB 24 h after CIDR removal, an LH surge occurred approximately 24 h later, with females exhibiting estrus and ovulating earlier than those with no EB injection after insert removal (Hanlon et al., 1996). Based on our results in Exp. 3 and 4, a greater percentage of returns to estrus occurred 8 d after EB or ECP injection on d 13, which was 1 d earlier than predicted based on the latter report. We suggest that initial injections of ECP probably induced emergence of a new wave 1 d later than EB, consistent with delayed estrus in ECP- vs. EB-treated cows in Exp. 3. Therefore, expected day of estrus occurred 10 to 11 d after ECP administration in Exp. 3 and 4 (following a single injection of ECP on d 13). In contrast, peak estrus occurred on d 9 after ECP or 2 d after CIDR removal when ECP injection was combined with CIDR inserts.
A third purpose of our experiments was to assess fertility (conception rates) at the resynchronized estrus. In Exp. 1 and 2, use of P4 or MGA plus ECP either tended to reduce or reduced fertility at the resynchronized estrus, respectively. In Exp. 3 and 4, females in those treatments had numerically lower conception rates. Other research (Purvis and Whittier, 1997) has shown that conception rate of beef heifers (second AI) after resynchronization with MGA does not differ from controls, but is numerically less in MGA-treated heifers. Lower conception rates in the MGA and MGA + ECP heifers indicated that some persistent follicles might have developed in heifers assigned to those treatments (Chenault et al., 1990). Based on previous results with EB (Burke et al., 2000), it was expected that the ECP injection on d 13 would initiate a new wave of follicular growth if a majority of heifers had two follicular waves. A greater variation in cycle duration in yearling heifers may make attempts to resynchronize estrus more difficult. This variability is partly due to a greater percentage of heifers with three follicular waves, and the existing dominant follicle on d 13 after AI was neither LH-dependent nor responsive to the loss of LH pulses (turn over and lose its dominance) after an estrogen injection (Burke et al., 2000). With these preliminary studies, we can only conclude that our protocols might reduce conception, but more work is warranted to see if significant reductions occur when adequate numbers of females are tested in the same experiment.
One report indicated that the EB + CIDR treatments increased fertility of dairy cows as a consequence of promoting three follicular waves (Macmillan et al., 1999). This was evident when conception rates were less in cows in which fertilized oocytes were derived from the second (58%) compared to the third (95%) follicular wave of the estrous cycle in beef (Ahmad et al., 1997) and dairy cows (30 vs. 68%; Townson et al., 2002).
The final and most important practical goal of estrus-synchronization programs is to facilitate and increase usage of AI. Less than 6% of beef cows in the United States are artificially inseminated annually (NAHMS, 1997). According to that survey of cattle producers, the most common reasons for not utilizing estrus-synchronization programs and AI include lack of time and labor (37%), difficulty (too complicated) (20%), other (20%), cost (13%), lack of facilities (8%), and because the programs "do not work" (2%). Resynchronization treatments based on estrogen and used CIDR inserts tended to increase pregnancy rates after two inseminations in lactating cows during the early breeding season in Exp. 3, but not in Exp. 4, and also were inexpensive to apply. From an economic standpoint, if the second estrus can be resynchronized with little additional cost, costs associated with the first estrus can be distributed across two inseminations by realizing more AI pregnancies and the resulting genetic gain from use of genetically superior AI sires. This may represent a strategic tool to increase utilization of AI and increase profits of cow-calf operations.
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Implications
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Resynchronization of estrus beginning 13 d following insemination by feeding melengestrol acetate, inserting an intravaginal progesterone-releasing insert, or providing an insert + estrogen injection increased synchrony of estrus and visual detection of estrus (when estradiol cypionate was added) of nonpregnant heifers. In lactating beef cows, incorporation of either estradiol benzoate or estradiol cypionate and an insert to resynchronize repeat estrus increased artificial insemination resubmission rate, tended to increase pregnancy rates after two inseminations in one of two experiments, and reduced the period needed for detection of estrus. In both heifers and lactating beef cows, resynchronization treatments had no negative effects on established pregnancies, but seemed to decrease conception rates in heifers after resynchronization of estrus. Resynchronization may provide another tool for cow-calf enterprises to facilitate use of artificial insemination and make greater genetic progress in their herds.
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Footnotes
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1 Contribution No. 03-104-J, Kansas Agric. Exp. Stn., Manhattan, KS. We acknowledge T. J. Marple and assistance of the student workers at the KSU Purebred Beef Unit and the animal technicians at the KSU Dairy Teaching and Research Center for their care of cattle used in these studies. We thank the owners of Losey Land and Cattle (Agra, KS; Exp. 2), DarLynn Ranch (Pierz, MN), and Thielen Ranch (Dorrance, KS; Exp. 3) for cooperation and use of their cattle. We express appreciation to B. A. Hensley for her expert laboratory assistance. Appreciation is expressed to Select Sires (Plain City, OH) for funds provided to support some of this research. 
Received for publication October 8, 2002.
Accepted for publication March 25, 2003.
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Abstract
Introduction
