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* Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506-0201
Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-2054
North Florida Research and Education Center, University of Florida, Marianna 32446-7906
North Central Research and Outreach Center, University of Minnesota, Grand Rapids 55744
# Northwest Research and Outreach Center, University of Minnesota, Crookston 56716
|| Department of Animal Science, Michigan State University, East Lansing 48824
¶ Department of Dairy Science, University of Wisconsin, Madison 53706
** Department of Animal Science, University of Missouri, Columbia 65211
2 Corresponding author: jss{at}k-state.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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14 d apart), with the second injection administered 14 d before initiating the Ovsynch protocol (injection of GnRH 7 d before and 48 h after PGF2 injection, with timed AI at 0 or 24 h after the second GnRH injection). Heatmount detectors were applied to cows just before the first Presynch injection, assessed 14 d later at the second Presynch injection (replaced when activated or missing), and reassessed at initiation of the Ovsynch protocol. Ovaries were examined for the presence of a corpus luteum (CL) by ultrasound before the initiation of treatment. Treatments were assigned to cows based on the presence or absence of a CL detected by ultrasound: 1) no CL + no CIDR; 2) no CL + CIDR insert for 7 d; and 3) CL present. Further, alternate cows within the 3 treatments were assigned to be inseminated concurrent with the second GnRH injection of Ovsynch (0 h) or 24 h later. Pregnancy was diagnosed at 33 and 61 d after the second GnRH injection. By using low (<1 ng/mL) concentrations of progesterone in serum as the standard for noncycling status, heatmount detectors were activated on a large percentage of noncycling cows (>60%), whereas the single ultrasound examination incorrectly classified noncycling cows only 21% of the time. Conversely, cycling cows (progesterone 
1 ng/mL) were correctly identified 70 to 78% of the time by heatmount detectors, but 85 to 92% were correctly identified by ultrasound. Overall accuracy of heatmount detectors and ultrasound was 71 and 84%, respectively. Application of progesterone to cows without a CL at the time of the first injection of GnRH reduced the incidence of ovulation but increased the proportions of pregnancies per AI at d 33 or 61 compared with nontreated cows without a CL at the onset of the Ovsynch protocol. Percentages of cows pregnant and pregnancy survival did not differ for cows having a CL before treatment compared with those not having a CL and treated with progesterone. Compared with no response, when a follicle ovulated in response to the first GnRH injection, percentage of cows becoming pregnant after the timed AI increased from 33.3 to 41.6%. Timing of AI at 0 or 24 h after the second GnRH injection did not alter pregnancies per AI, but cows having luteal activity before treatment had improved pregnancies per AI compared with noncycling cows. We conclude that identifying noncycling cows by ultrasound was more accurate than by heatmount detectors. Subsequent progesterone treatment of previously cycling cows not having a CL at the onset of Ovsynch increased the proportion of pregnant cows, equal to that of cows having a CL but not treated with progesterone.
Key Words: anovulation • controlled internal drug release • Ovsynch • pregnancy per artificial insemination
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Previous studies produced inconsistent results after supplementing progesterone via a progesterone-releasing intravaginal controlled internal drug-release (CIDR) insert during various timed AI (TAI) protocols. For example, our previous regional research project reported numerically greater P/AI measured 28 d after TAI for cows treated with progesterone during the Ovsynch protocol (injection of GnRH 7 d before and 48 h after PGF2 injection, with TAI between 0 and 24 h after the second GnRH injection; Stevenson et al., 2006). In that study, P/AI were greater for both cycling and noncycling cows treated with the CIDR insert compared with no CIDR treatment, but only at 4 of the 6 locations at 28 d and at 3 of 6 locations at 56 d after TAI. This inconsistent response is corroborated by other large-scale studies (El-Zarkouny et al., 2004; Galvão et al., 2004; Moreira et al., 2004a,b).
Estrus-detection aids, including tail paint, chalk, and heatmount detectors, are inexpensive tools that may aid in the detection of noncycling cows before first AI. Likewise, examination of ovaries by transrectal palpation (Morrow et al., 1966) or ultrasonography (Silva et al., 2007) is a means of identifying noncycling cows that may benefit from progesterone supplementation as part of a TAI protocol.
Pregnancies per AI are maximized when the TAI of the Ovsynch protocol is administered at 16 h after the second GnRH injection (Pursley et al., 1998). In practice, this timing is inconvenient and does not correspond to when other management activities occur (e.g., AI, pregnancy diagnosis, vaccinations, and other treatments) while dairy cows are locked up at the feed line after the morning milkings. Further, P/AI was similar when AI occurred concomitant with the second GnRH injection or 24 h later (Nebel and Jobst, 1998; Pursley et al., 1998).
The objectives of the present experiment were to determine 1) the accuracy of detecting potentially noncycling cows by using heatmount detectors applied to cows for 28 d before initiating the Ovsynch protocol or by a single ovarian examination using transrectal ultrasonography at the onset of the Ovsynch protocol; 2) the benefit of applying progesterone (via a CIDR insert) during the first 7 d of the Ovsynch protocol; and 3) P/ AI when timing of AI occurred concurrent with the second GnRH injection of the Ovsynch protocol or 24 h later.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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. Cows were enrolled in a Presynch + Ovsynch protocol. Two 25-mg injections of PGF2 (5 mL of Lutalyse, Pfizer, New York, NY, or 5 mL of Prostamate, IVX Animal Health Inc., St. Joseph, MO) were given i.m. 14 d apart. The Ovsynch protocol was then initiated 14 d after the second Presynch injection by injecting GnRH. Prostaglandin F2 (25 mg) was administered 7 d later and was followed in 48 h by a second injection of GnRH. All injections of GnRH (100 µg) were administered i.m., consisting of 2 mL of Ova-Cyst (IVX Animal Health Inc.).
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Treatments
At the onset of the Ovsynch protocol, ovaries were examined by transrectal ultrasonography. Ultrasonography was conducted by using a transrectal 5.0- or 7.5-MHz linear-array transducer (Aloka 500V; Corometrics Medical Systems Inc., Wallingford, CT). Follicles and luteal structures were mapped and follicles were sized by using internal calipers. Treatments were assigned to cows based on the presence of a CL (CL absent vs. CL present): 1) noncycling (no CL + no CIDR); 2) noncycling (no CL + CIDR insert for 7 d); and 3) cycling (CL present). Further, alternate cows within the 3 treatments were assigned to be inseminated concurrently with the second GnRH injection of Ovsynch (0 h) or 24 h later. Pregnancy was diagnosed at 33 and 61 d after the second GnRH injection. The presence of fluid in the uterus, a CL, and a viable embryo were evidence of pregnancy. For cows that were pregnant at the first diagnosis (d 33), a subsequent ultrasound examination at d 61 was used to determine pregnancy loss.
At 3 locations (Kansas, Michigan, and Minnesota), follicles also were mapped and sized at further ovarian exams performed by ultrasound before the PGF2 and second GnRH injections of Ovsynch, and at 5 d after the second GnRH injection to determine whether ovulation had occurred in response to the second GnRH injection. Blood was collected at all locations before each hormonal injection and again at 5 and 12 d after the second GnRH injection. Concentrations of progesterone were measured to determine luteal activity before and after treatment.
Progesterone Immunoassays
Serum concentrations of progesterone for samples collected at 3 locations were quantified by RIA (Skaggs et al., 1986). For Kansas samples, intra- and interassay coefficients of variation were 6.3 and 7.4%, respectively. Repeated pool samples averaged 3.5 ± 0.1 ng/mL in 11 assays. For Michigan samples, intra- and interassay coefficients of variation were 4.9 and 5.3%, respectively. Repeated pool samples averaged 4.1 ± 0.1 ng/mL in 6 assays. For Minnesota samples, intra- and interassay coefficients of variation were 7.2 and 8.4%, respectively. Repeated pool samples averaged 4.7 ± 0.1 ng/mL in 8 assays.
Serum concentrations of progesterone for Missouri samples were quantified by RIA (Progesterone Coat-a-Count kit, Diagnostic Products Corp., Los Angeles, CA; Kirby et al., 1997). Intra- and interassay coefficients of variation were 3.0 and 10.2%, respectively.
Serum concentrations of progesterone for samples collected at 2 locations (Indiana and Wisconsin) were quantified by ELISA (Rasmussen et al., 1996), with intra- and interassay coefficients of variation of 4.8 and 8.9%, respectively.
Statistical Analyses
Ovulation was assumed to have occurred when concentrations of progesterone were 1 ng/mL in any or all of the 3 samples collected before the first and second Presynch injection and before the onset of treatment. Assessment of prior luteal activity by examining serum concentrations of progesterone was considered to be the standard with which accuracy of the heatmount detectors and the single ultrasound exam were compared. Our first objective was to determine whether noncycling cows could be detected accurately by heat-mount detectors or by a single ultrasound examination of ovarian structures. Prior ovulatory activity and estrous cycles were assumed to have occurred if the heat-mount detector was activated or missing at any or all of the observation times during the 28-d period between the first Presynch injection and the first GnRH injection of Ovsynch. Prior ovulatory activity was assumed to have occurred when a CL was detected by ultrasonography before the onset of the Ovsynch protocol and treatment.
Measures of correct noncycling status, correct luteal activity, false positives, and false negatives were calculated (procedure FREQ; SAS Institute Inc., Cary, NC), in addition to sensitivity, specificity, apparent prevalence, and predictive values positive and negative (Win Episcope 2.0). Kappa values were calculated by using procedure FREQ. Kappa is a measure of repeatability in which a value of 1 indicates perfect agreement and 0 indicates the amount of agreement that occurs by chance alone. A kappa value of 0.4 to 0.5 indicates moderate agreement, 0.5 to 0.6 indicates good agreement, and values >0.6 indicate excellent agreement (Martin et al., 1987).
Characteristics and outcomes of various descriptive traits by location were calculated by using a general linear model procedure (procedure GLM; SAS Institute Inc.), with a model consisting of location, lactation number (1, 2, and 3+), and their interaction. Means were separated by using the least significant difference (LSD) in procedure GLM when a protected F-test (P 
0.05) was detected in the ANOVA. Traits summarized for each location included a standard calving difficulty score (CDS; 1 = no assistance; 2 = slight problem; 3 = needed assistance; 4 = considerable force; and 5 = extreme difficulty); BCS (1 = thin and 5 = fat) at calving and before the first Presynch injection; DIM at TAI; P/ AI at d 33 and 61; pregnancy loss during that interval; luteal activity before treatment based on serum concentrations of progesterone; and regression of the CL (cows having elevated concentrations of progesterone 48 to 72 h before AI, in which blood progesterone was <1 ng/ mL at 48 h after the PGF2 injection of the Ovsynch protocol) in response to the PGF2 of the Ovsynch protocol.
Ovarian characteristics (ovulation incidence in response to first GnRH of Ovsynch), number of CL per cow, proportion of cows having a CL before PGF2 of Ovsynch, diameter of the ovulatory follicle before the second GnRH injection, and ovulation incidence after the second GnRH injection were analyzed by procedure GLM by using a model consisting of treatment (no CL + no CIDR, no CL + CIDR, and CL present), lactation number, location, 2-way interactions of treatment and lactation number and location, CDS, and BCS at the second Presynch injection (covariate). A priori contrasts were constructed to compare treatments (no CL + no CIDR, and no CL + CIDR) vs. cows having a CL present and the effect of absence or presence of a CIDR in cows with no CL.
Pregnancies per AI at d 33 and 61 and pregnancy loss during that interval were analyzed by using a similar model consisting of treatment, lactation number, location, time of AI (0 vs. 24 h), 2-way interactions of treatment and the preceding variables, location x time of AI, sire and technician, each nested within location, and CDS. Similar a priori contrasts of treatments were constructed.
Concentrations of progesterone were compared between CIDR and no-CIDR cows for samples collected before the PGF2 injection of Ovsynch, the second GnRH injection, and at 5 and 12 d after the second GnRH injection. The ANOVA consisted of treatment (no CL + no CIDR, no CL + CIDR, and CL present), cycling status based on progesterone concentrations in blood serum before treatments were applied, lactation number, location, all 2-way interactions with treatment, and CDS. For the latter 2 collection times, concentrations assessed from d 5 and 12 after the second GnRH injection, pregnancy status, and its interaction with treatment also were included in the model.
Ancillary models were applied to determine the effects of cycling status, lactation number, location, CDS, and BCS on the number of ovarian follicles 10 mm in diameter, diameter of the largest follicle, and proportion of cows having a CL before onset of the treatment. In addition, P/AI at d 33 was analyzed according to various progesterone patterns that occurred in blood collected at 3 times before each Presynch and the first GnRH injection. These samples of high (H; 1 ng/mL) and low (L; <1 ng/mL) concentrations formed 8 permutations of progesterone patterns (e.g., LHH, HHH, HLL, HLH, HHL, LLH, LLL, and LHL) that were included in a model consisting of treatment (no CL + no CIDR, no CL + CIDR, and CL present), time of AI, lactation number, location, progesterone pattern, all 2-way interactions with treatment, incidence of ovulation in response to the first GnRH injection and its interaction with progesterone pattern, CDS, and sire and technician, each nested within location. With the addition of progesterone concentration before the PGF2 injection plus the pretreatment cycling status of cows, P/AI at d 33 was compared to determine whether luteal status before or during the Ovsynch protocol was a predictor for the benefit of adding supplemental progesterone via the CIDR insert.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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. A total of 1,072 cows were enrolled in the study in 6 states in the Midwest, but for some traits assessed, data were available for fewer cows. Nearly all traits in Table 2 differed among locations, except for P/AI at d 33. Calving difficulty scores averaged 1.3 ± 0.48 (SD) among locations and ranged from 1.2 to 1.7. Average BCS after calving and before the first Presynch injection (37 ± 7 DIM) were 2.8 ± 0.48 (SD) and 2.6 ± 0.44 (SD), respectively.
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of the Ovsynch protocol averaged 88.5% across locations.
Detection of Noncycling Cows
Our first objective was to determine the accuracy of detecting noncycling cows by means of heatmount detectors or a single ultrasound examination of ovarian structures in comparison with serum concentrations of progesterone. At the first Presynch PGF2 injection, 45.5% (485/1,067) of cows had circulating progesterone concentrations 1 ng/mL. By the time of the second Presynch PGF2 injection, however, another 20% of cows had elevated concentrations of progesterone (694/ 1,067 = 65%). This increased percentage resulted from a total of 282 cows that had low progesterone at the first Presynch injection but then had high progesterone at the second Presynch injection (282/1,067 = 26.4%), with only 6.8% (73/1,067) of cows changing from high (first Presynch injection) to low (second Presynch injection). At the time of the first GnRH injection of Ovsynch, a slightly greater percentage of cows were classified as having high progesterone (768/1,067 = 72%). A small proportion of cows (96/1,067 = 9%) had low progesterone at the first GnRH injection after being high at the second Presynch injection, whereas 15.9% (170/1,067) of cows changed from low (second Presynch injection) to high progesterone (first GnRH injection of Ovsynch). A small proportion of cows (126/1,067 = 11.8%) having low progesterone at both Presynch injections subsequently had high progesterone at the first GnRH injection of Ovsynch (prior noncycling cows that ovulated for 14 d before the start of Ovsynch). Thus, even after both Presynch injections, 28% (299/1,067) of cows continued to have lower circulating progesterone concentrations at the first GnRH injection of Ovsynch. This is consistent with results from other studies using a Presynch protocol before Ovsynch (El-Zarkouny et al., 2004; Galvão et al., 2004).
To assess the accuracy of the ultrasound and heat-mount procedures, we first compared results using only the progesterone concentration at the time of the first GnRH injection of Ovsynch. Our rationale was that the single ultrasound examination conducted at the first GnRH injection of Ovsynch should correspond to concentrations of progesterone [i.e., the presence (high concentrations) or absence (low concentrations) of a CL detected by ultrasound]. In addition, we anticipated that if heatmount detectors were accurate in detecting prior estrus activity after the second Presynch injection, then an activated detector should correspond to the presence of high progesterone at the initiation of Ovsynch.
The relationship between measured serum progesterone concentrations and the presence of an activated heatmount detector or a detected CL is presented in Table 3. The presence of an activated heatmount detector had little relationship with the circulating concentrations of progesterone. Even cows having very low circulating progesterone (<0.5 ng/mL) had a high percentage of activated detectors (141/230 = 61.3%). Further, cows having greater progesterone concentrations (1 ng/mL) showed only a slightly greater percentage of activated detectors (520/753 = 69.1%).
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). For example, among cows having low progesterone concentrations (<0.5 ng/mL), only 20.8% (45/216) had a visible CL. In contrast, cows having greater circulating progesterone (1 ng/mL) at first GnRH were more likely to have a CL detected by ultrasound (646/695 = 92.9%).
Table 4 shows how well a single ultrasound examination or 2 evaluations of heatmount detectors corresponded to whether an individual cow had elevated progesterone concentrations at any of the 3 evaluations (before either Presynch injection and before the first GnRH injection of Ovsynch). Overall, relative to the 3 progesterone measurements assessed for 28 d before Ovsynch, heatmount detectors and a single ultrasound exam of ovarian structures underestimated both anovulation and previous luteal activity (Table 4). Variation among herds for the overall accuracy of assessing prior luteal activity (total correct diagnoses of prior noncycling and cycling activity) was evident (Table 5), and may be related to the competency of operators or the stage of luteal development (i.e., a new CL or regressing CL at the time of the exam). Relative to serum concentrations of progesterone, the overall accuracy of heat-mount detectors among locations ranged from 49.8 to 86.3%, whereas that of ultrasonography ranged from 68.8 to 90%.
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1 ng/mL progesterone) were correctly identified 78% (694/883) of the time by heatmount detectors, but 85% (759/893) were correctly identified by ultrasound. Thus, relative to serum progesterone patterns, the overall accuracy of heatmount detectors was 71.4% and that of a single ultrasound examination was 84%. Ultrasound differed (P < 0.001) from heatmount detectors in every category except for apparent prevalence, including being more (P < 0.001) sensitive and specific than heatmount detectors. Particularly striking was the low specificity of the heatmount detectors (only 33.9%), corresponding to the inaccuracy of this technique for accurately identifying noncycling cows. The kappa coefficient (0.52) for ultrasound was in a range that indicated good agreement with progesterone concentrations, whereas that of the heatmount detectors (0.11) was in a range that indicated a poor predictive value for progesterone status. As expected, heatmount detectors had greater (P < 0.001) rates of false positives (incorrectly identifying prior estrual activity and subsequent luteal activity; 3.1-fold greater) and false negatives (missing prior estrual activity and subsequent luteal activity; 1.4-fold greater) than ultrasound.
Ovarian Characteristics Before Treatment
Various ovarian traits of cows were assessed before treatment was initiated at 5 locations (Kansas, Michigan, Minnesota, Missouri, and Wisconsin). Cows having previous luteal activity (as assessed by serum concentrations of progesterone in 3 previous samples for 28 d) had fewer (P < 0.01) numbers of follicles 10 mm in diameter before the first GnRH injection at the onset of treatment than did noncycling cows (1.4 ± 0.1; n = 682 vs. 1.7 ± 0.1; n = 149). Diameter of the largest follicle at the onset of treatment, however, did not differ (15.6 ± 0.2; n = 630 vs. 15.4 ± 0.3; n = 137) between noncycling and cycling cows, respectively. As expected, the percentage of cows having a CL on the day of treatment was nearly 5-fold greater (P < 0.001) in cycling cows (85.9%) compared with noncycling cows (17.7%). Concentrations of progesterone in cows with no CL averaged 0.6 ± 0.2 ng/mL, whereas those in cows having a CL averaged 2.4 ± 0.2 ng/mL. Although concentrations of progesterone were minimal in noncycling cows, some cows had a visible CL, but may have been misclassified as noncycling because blood progesterone was not detectable at sampling times in cows having a newly formed CL or a regressing CL.
The presence of at least 1 large cystic-like follicle (>25 mm) was noted in 7.3% (61/836) of evaluated cows. Most (72%; 44/61) of these "cystic" cows, however, either had a CL present or had high circulating progesterone concentrations, leaving only 17 cows (2%) with a probable follicular cyst.
Ovarian Characteristics in Response to Progesterone
Our second objective was to determine the effects of progesterone (CIDR insert) in cows identified with or without a CL at the onset of the Ovsynch protocol. This assessment was made (at all 6 locations) by a single ultrasound examination of ovaries. As cited earlier, prior cycling status of some cows was incorrectly categorized by that single examination. The incidence of ovulation in response to the first GnRH injection was less (P < 0.05) in cows with CL absent that were treated with progesterone (CIDR insert) than in cows with CL absent and not receiving a CIDR insert (Table 6). The average incidence of ovulation was less (P < 0.001) for cows having a CL than for those without a CL (Table 6). Concentrations of progesterone for cows before treatment were 0.7 ± 0.2 ng/mL (no CL + no CIDR), 0.6 ± 0.2 ng/mL (no CL + CIDR), and 4.0 ± 0.1 ng/mL (CL present). Because concentrations of progesterone in ovariectomized cows are >1 ng/mL by 5 min and peak 2 h after insertion of a CIDR (Rathbone et al., 2002), GnRH-induced LH release may be attenuated by elevated concentrations of endogenous or exogenous (via a CIDR) progesterone. A reduced incidence of GnRH-induced ovulation in lactating dairy cows was observed previously for cows concurrently treated with GnRH and a CIDR insert compared with cows treated with GnRH (68 vs. 87%, respectively; Galvão et al., 2004). Taken together, that study (Galvão et al., 2004) and the current study indicate that cows having elevated progesterone (CL present) or those administered a progesterone insert concurrently with a GnRH injection may have a reduced incidence of ovulation compared with cows having low progesterone or no CL.
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). The incidence of ovulation of that follicle, as assessed 5 d later (after TAI), did not differ among cows having or not having a CL (Table 6) at the onset of treatment.
Fertility in Response to Progesterone
Pregnancies per AI at d 33 and 61 and pregnancy loss in 1,068 cows (4 were culled before pregnancy diagnosis) during that interval are summarized in Table 6. Treatment with a CIDR in cows with a CL absent increased (P 0.05) P/AI at either 33 or 61 d after AI (Table 6), but did not differ from that of cows with a CL present. Pregnancy loss did not differ for cows with a CL absent that either received or did not receive a CIDR. In contrast, pregnancy loss tended (P < 0.10) to be less for cows with a CL present than for those in which a CL was not detected before treatment. Cows requiring no calving assistance (CDS = 1) subsequently had greater (P < 0.05) P/AI than those requiring some (CDS >1) assistance (42.7%; n = 693 vs. 34.3%; n = 375), respectively.
A CIDR treatment x location interaction tended (P = 0.08) to occur (Table 7) for P/AI at d 33. Pregnancies per AI for cows with a CL absent that were treated with a CIDR insert were numerically greater at 4 of 6 locations than for cows with a CL absent that were not treated with progesterone.
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). Herd had no significant effect in the statistical model for P/AI at d 33. A tendency (P = 0.11) was detected for a herd x time of AI interaction. In contrast, the timing of AI had no effect on P/AI (P = 0.57). The largest numerical difference in P/AI between the 2 AI times was observed in cows with a CL present [0 h = 35.4% (142/401) vs. 24 h = 40.8% (161/395)].
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). When ovulation occurred in response to the first GnRH injection, P/AI was 25% greater (P < 0.05) than when ovulation did not occur (41.6 vs. 33.3%, respectively). In 6 of the 8 pretreatment progesterone patterns, P/AI were greater when ovulation had occurred in response to the first GnRH injection (Table 9).
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) of the 8 permutations. Three of these 6 patterns in which P/AI were numerically greater for CIDR-treated cows included cows with low progesterone at the onset of Ovsynch, whereas the other 3 patterns included cows that had 1 or 2 low progesterone concentrations at the first or second sampling times, but had elevated progesterone at the onset of treatment. Pregnancies per AI in noncycling cows (LLL; Table 9) were not improved in response to treatment with progesterone.
Further, when cows with a CL absent and having elevated progesterone at the onset of treatment were treated with the CIDR, P/AI did not differ from those of cows with a CL present (first subtotal in Table 9). In contrast, when cows with a CL absent and having low progesterone at the onset of treatment were treated with a CIDR, P/AI did not differ from those of the other 2 treatments (second subtotal in Table 9).
The relationship of pretreatment cycling status and the presence of high vs. low concentrations of progesterone before the PGF2 injection of Ovsynch was examined further (Table 10). Despite the presence of the CIDR insert, 34.7% (30 noncycling and 10 cycling cows; Table 10) of 155 CIDR-treated cows with a CL absent had concentrations of progesterone <1 ng/mL at insert removal, indicating that these cows did not have a functional CL. Concentrations of progesterone in CIDR-treated cows bearing a luteal structure are generally not different before the insert is removed but differ after insert removal when a CL is present (Stevenson et al., 2006). Further, because identifying the cycling status was less accurate by 1 ultrasound examination relative to 3 estimates of serum concentrations of progesterone in these cows, 44.5% (69 cycling cows of 155 cows with CL absent) treated with a CIDR insert had elevated pretreatment concentrations of progesterone at some time during the 28 d before treatment (indicating prior cyclicity). In addition, 56% (65 cycling cows of 116 cows with CL absent) not treated with a CIDR insert met this criterion and were cycling before Ovsynch.
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, and similar effects of truly cycling cows are summarized in the bottom 3 lines of Table 10. Pregnancies per AI in noncycling cows treated or not treated with a CIDR insert did not differ from those in cows with a CL present, regardless of progesterone concentrations at the time of the PGF2 injection (first subtotal in Table 10). In contrast, regardless of progesterone concentrations at the time of the PGF2 injection, cows with a CL absent that were treated with a CIDR and cows with a CL present had greater P/AI than cows with a CL absent that were not treated with a CIDR (second total in Table 10). Overall, cows having prior cycling activity, as assessed by 3 estimates of serum progesterone before treatment, had 53% greater (P < 0.001) P/AI at d 33 than did those without prior luteal activity (37.8 vs. 24.7%; Table 10). This advantage in P/AI for prior cycling cows was consistent for cows with a CL present and for cows with a CL absent that were treated with progesterone, but not for cows with a CL absent that were not treated with progesterone (Table 10).
The CIDR treatment of noncycling cows not having a functional CL before the PGF2 injection improved P/AI in a study by Stevenson et al. (2006), but not in the present study (Table 10) or in another large study (Moreira et al., 2004a). Further, CIDR treatment in previously cycling cows having low concentrations of progesterone before PGF2 injection (early CL regression) had numerically greater P/AI in the present study, as in a previous study (Stevenson et al., 2006). Interpretation of the results in the previous study (Stevenson et al., 2006) suggested that the CIDR insert should improve P/AI in cows having no active CL or low progesterone before the PGF2 injection, regardless of previously cycling or luteal status. In the present study, we could only verify improved P/AI for previously cycling cows having low progesterone (early CL regression), as well as high progesterone at CIDR insert removal.
Concentrations of progesterone in serum 2 d before the second GnRH injection (day of the PGF2 injection of Ovsynch and removal of the CIDR inserts), at the time of the second GnRH injection, and 5 and 12 d later are illustrated in Figure 2. At insert removal, cows with a CL absent that were treated with CIDR inserts had concentrations of progesterone that did not differ from those in cows without inserts, but cows in both treatments had less (P < 0.05) progesterone than did cows with a CL present. This same pattern existed 48 h later when the second GnRH injection was given (0 h). At 5 d after AI, no differences in progesterone concentration were detected among treatments, but at 12 d after the second GnRH injection or at 11 to 12 d after AI, cows with a CL absent that were treated with a CIDR had greater (P < 0.05) concentrations of progesterone than did cows with a CL present. The greater progesterone 12 d later was similar to an earlier report (Melendez et al., 2006) in which cows treated with CIDR inserts during the Ovsynch protocol had greater progesterone concentrations than did control cows, regardless of their pregnancy status. It seems that maturing follicles exposed to supplemental progesterone during CIDR treatment may, after ovulation, differentiate into CL having a greater progesterone secretory capacity.
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General Discussion
Our first objective was to determine the accuracy of using heatmount detectors and ultrasound to identify noncycling cows. Another study (Silva et al., 2007) concluded that applying ultrasound at the onset of the Ovsynch protocol slightly overestimated the proportion of noncycling cows, but because all noncycling cows were identified by ultrasound, it could be used in field conditions to target specialized treatments for noncycling cows. In the present study, only 7% (49/695) of cows having high progesterone at the first GnRH treatment did not have an ultrasonically detected CL, again indicating that most cows with elevated progesterone will have a CL detected by ultrasound (Table 3). This accuracy, however, becomes less impressive when this single ultrasound examination is used to detect cows that had ovulated at any time during the 28 d before initiating Ovsynch, with 15% (134/893) of previously cycling cows having no CL detected (Table 4). Further, in contrast to Silva et al. (2007), not all noncycling cows were correctly identified by ultrasound in our study by using the single progesterone value at the time of first GnRH (75/273 = 27.5% of noncycling cows with CL). Nevertheless, the most striking discrepancy found in our study was when comparisons were made between cyclicity status and activation of the heatmount detectors. Accuracy of heatmount detectors has been reported to be quite variable, ranging from 36 to 80%, and was only 56 to 94% efficient in identifying all estrual activity (Stevenson, 2001). In our study, there was almost no relationship between activation of the heat-mount detectors during the Presynch protocol and the cyclicity status of cows. Indeed, the specificity of this technique was only 33.9%, with a kappa coefficient (0.11) that indicated almost no predictive value of heat-mount activation for cyclicity status. Further, more than 60% of noncycling cows had activated heatmount devices. Breeding of these noncycling cows having activated heatmount devices likely would have resulted in few P/AI. Nevertheless, the heatmount devices correctly predicted cyclicity in 78.5% of cycling cows, and insemination of these cows based on the activated heatmount device may have produced reasonable fertility. These results raise an interesting question of whether a thorough analysis of other estrus-detection methods, such as the ubiquitously used tail chalk, might be equally inaccurate in identifying noncycling cows, producing a reduction in fertility in cows inseminated after "detection" with these methods.
Although ultrasound was not 100% accurate when attempting to determine prior estrual activity, fertility benefits that accrued from applying a CIDR to lactating cows in the present study were limited to those previously cycling cows not having a CL at the onset of treatment. Therefore, the accuracy of using ultrasound eliminated the unneeded use of CIDR inserts in 84% of cows with a CL present (796 of 951 cows; Table 9), in which no P/AI increase likely would have been realized. Pregnancies per AI at d 33 were greater for cows with a CL absent (32.3%) that were treated with progesterone than for cows with a CL absent that were not treated (24.1%; Table 6). It is clear that regardless of progesterone concentrations at the time of the PGF2 injection of Ovsynch, previously cycling cows benefited by treatment with a CIDR compared with those not treated with a CIDR (Table 10).
We (Stevenson et al., 2006) previously reported that P/AI were improved in 2 categories of cows treated with a CIDR insert at the onset of the Ovsynch protocol: 1) noncycling cows before the onset of Ovsynch and no CL induced in response to the first GnRH injection of Ovsynch (33 vs. 17%); and 2) cycling cows in which early luteolysis occurred before the PGF2 injection of Ovsynch (38 vs. 19%), respectively. The present study fails to show any benefit for cows in the former status (see pretreatment noncycling cows having low progesterone before PGF2 injection in Table 10), but corroborates the latter status, in which P/AI for previously cycling cows treated with CIDR inserts and having low concentrations of progesterone before the PGF2 injection of the Ovsynch protocol were improved (Table 10). The latter observation also confirms improvement in P/AI for CIDR-treated cows initiating the Ovsynch protocol in late diestrus (Thatcher et al., 2006), whose CL likely regresses spontaneously during the CIDR treatment. Nevertheless, the CIDR-induced reduction in ovulation to the first GnRH treatment (76.8 vs. 47.1%) raises the interesting possibility that some of the positive effects of progesterone supplementation may be offset by a negative effect of CIDR on GnRH-induced ovulation. Perhaps alternative timing of CIDR insertion is needed to realize the full benefit of this insert.
Another difference between the present study and our previous study (Stevenson et al., 2006) was the addition of Presynch before applying the TAI protocol. We would not expect Presynch to influence noncycling cows because noncycling cows have no CL to regress in response to Presynch PGF2 injections. In contrast, Presynch should have changed the proportion of cows having a CL at the onset of the Ovsynch protocol (El-Zarkouny et al., 2004). Other published reports in which progesterone was applied to cows confirm these inconsistent responses. In one study (experiment 1 of El-Zarkouny et al., 2004), conception rates were improved by adding a CIDR insert compared with cows inseminated at estrus or after the Ovsynch protocol. In a 5-location study conducted during summer in Mexico, in which cows were inseminated by using an Ovsynch protocol, conception rates were improved only in first-lactation cows in which a CIDR insert also was included, but the CIDR insert had no positive effect in noncycling cows (Moreira et al., 2004a). In another study conducted in Mexico, in which estrous cycles were presynchronized (Presynch), with the second Presynch injection administered 12 d before initiating the Ovsynch protocol in all cows, addition of a CIDR insert improved conception rates (Moreira et al., 2004b), whereas no positive effect occurred in 3 other studies applying similar treatments (experiment 2 of El-Zarkouny et al., 2004; Galvão et al., 2004), unless cows were in late diestrus at the time of CIDR insertion (Thatcher et al., 2006).
Our third objective to alter the timing of AI relative to the second GnRH injection of Ovsynch failed to detect any differences in P/AI when cows were inseminated either concurrently with that injection or 24 h later. These findings are consistent with the first report of variable times of AI (Pursley et al., 1998), in which cows inseminated 16 h after GnRH produced greater P/AI than did those cows inseminated at 0, 8, 24, and 32 h. Numerically, the 24-h insemination was 4 percentage points greater than the 0-h insemination in that study and was 3.4 percentage units greater in the present study (Table 8), when all cows were combined. In another study (Portaluppi and Stevenson, 2005), in which GnRH was administered at 48 h after PGF2 and cows were inseminated at 48 or 72 h, P/AI in cows were similar at 23 and 24%, respectively, but improved if both GnRH and AI occurred at 72 h (31%). Similarly, Cornwell et al. (2006) failed to detect any differences in fertility when cows were bred at the same time as the second GnRH or 24 h later. Recently, however, Brusveen et al. (2006) found that cows treated with the second GnRH injection at 56 h after PGF2 and inseminated 16 h later had markedly improved P/AI compared with cows inseminated at 0 or 24 h relative to GnRH administered at 48 h after PGF2.
In summary, identification of noncycling cows was more accurate with a single ultrasound examination than when using heatmount detectors. When lactating dairy cows were found to have no CL (but had evidence of increased concentrations of progesterone in blood serum for 28 d before initiating the Ovsynch protocol) at the onset of the Ovsynch protocol and were treated with progesterone (via a CIDR insert), P/AI were greater than those of similar cows with a CL absent that were not treated with a CIDR insert. Pregnancies per AI of cows with a CL absent that were treated with a CIDR insert did not differ from those of cows having a CL at initiation of the TAI protocol. Our present study and another one (experiment 2 of El-Zarkouny et al., 2004) found no evidence to support 3 previous studies (Galvão et al., 2004; Moreira et al., 2004a; Stevenson et al., 2006) reporting that including progesterone in a TAI protocol for previously noncycling cows is warranted, regardless of luteal status before the PGF2 injection of the Ovsynch protocol. Pregnancy per AI of cows inseminated at 0 vs. 24 h after the second GnRH injection did not differ significantly.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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FOOTNOTES |
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Received for publication November 12, 2007. Accepted for publication March 14, 2008.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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men. 2006. Changes in reproductive physiology of lactating dairy cows due to elevated steroid metabolism. Theriogenology 65:17–29.[CrossRef][Medline]This article has been cited by other articles:
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