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Comparison of two timed artificial insemination system schemes to synchronize estrus and ovulation in Nellore cattle
Accepted Manuscript Comparison of Two Timed AI System Schemes to Synchronize Estrus and Ovulation in Nellore Cattle Marcos Vinicius C. Ferraz Junior, Alexandre V. Pires, Marcos Vinicius Biehl, Marcelo H. Santos, Jose Paulo R. Barroso, José Renato S. Gonçalves, Roberto Sartori, Michael L. Day PII:
S0093-691X(16)30266-7
DOI:
10.1016/j.theriogenology.2016.06.012
Reference:
THE 13722
To appear in:
Theriogenology
Received Date: 6 May 2016 Revised Date:
6 June 2016
Accepted Date: 7 June 2016
Please cite this article as: Ferraz Junior MVC, Pires AV, Biehl MV, Santos MH, Barroso JPR, Gonçalves JRS, Sartori R, Day ML, Comparison of Two Timed AI System Schemes to Synchronize Estrus and Ovulation in Nellore Cattle, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.06.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Comparison of Two Timed AI System Schemes to Synchronize Estrus and
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Ovulation in Nellore Cattle
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Marcos Vinicius C Ferraz Juniora, Alexandre V Piresa,b,*, Marcos Vinicius Biehlb,
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Marcelo H Santosa, Jose Paulo R Barrosoa, José Renato S Gonçalves c, Roberto
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Sartoric, Michael L Day d
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Department of Nutrition and Animal Production, Faculty of Veterinary and Animal
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Science, University of São Paulo, Duque de Caxias north, n 225, Pirassununga, São Paulo, Brazil, Zip: 13635-000. b
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Department of Animal Science, College of Agriculture “Luiz de Queiroz” (ESALQ),
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University of São Paulo, Pádua Dias Avenue, n 11, Piracicaba, São Paulo, Brazil,
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PO Box 09, Zip: 13418-900.
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Experimental Station Hildegard Georgina Von Pritzelwitz, Fazenda Figueira,
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Londrina, Paraná, Brazil. PO Box 8003, Zip: 86010-990. d
Department of Animal Science, University of Wyoming, 1000 East University
Avenue, Laramie, WY, 82071.
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*Corresponding author: Alexandre Vaz Pires. Department of Animal Science,
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ESALQ / USP, Piracicaba, SP, 13418-900, Brazil. Tel.: +55 (19) 3429-4247; Fax:
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+55 (19) 3429-4215 [email protected]
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Abstract
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The aim of this study was to evaluate the reproductive performance of 411
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Nellore cows (198 nulliparous, 80 primiparous and 133 multiparous) submitted to
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the 5dCO-Synch+P4 or 7dEB+P4 systems. The 5dCO-Synch+P4 system
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consisted of insertion of an intravaginal progesterone (P4) insert and 100 µg of
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GnRH (i.m.) on day 0. On day 5, the P4 insert was removed and two doses of 25
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mg of PGF2α (i.m.) were administered 6 hours apart. Cows not detected in estrus
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until 55 hours after insert removal received 100 µg of GnRH i.m. 17 hours later
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(i.e., 72 hours after P4 removal). The 7dEB+P4 system consisted of insertion of a
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P4 insert and 2 mg of estradiol benzoate (i.m.) on day 0. On day 7, the P4 insert
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was removed and 25 mg of PGF2α, 0.6 mg of estradiol cypionate and 300 IU of
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eCG were administered i.m. In both systems, AI was performed according to estrus
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detection (i.e., cows detected in estrus until 55 hours after insert removal were
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inseminated at 55 hours, and cows detected in estrus later, or those not detected
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in estrus were inseminated at 72 hours). Estrus-detection risk was greater (P <
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0.05) in 7dEB+P4 (80.4%) than 5dCO-Synch+P4 system (36.4%). Progesterone
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concentration 10 days after AI was greater (P < 0.05) in 7dEB+P4 than 5dCO-
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Synch+P4 system in primiparous and multiparous, but did not differ between
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systems in nulliparous cows. Pregnancy per AI (P/AI) was greater (P < 0.05) in
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7dEB+P4 (49.7%) than 5dCO-Synch+P4 (35.4%) system. Primiparous had lower
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estrus-detection risk (25.0%), ovulation risk (76.6%) and P/AI (28.7%) than
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multiparous or nulliparous cows. In conclusion, reproductive performance was
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reduced with the 5dCO-Synch+P4 in comparison to the 7dEB+P4 system in
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Nellore cows. Moreover, the reproductive traits observed for primiparous cows
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indicate that more attention is required when timed AI programs is started early
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after calving.
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Key words: Bos indicus; Estradiol; Fixed timed-AI; GnRH; Progesterone.
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1. Introduction
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Artificial insemination is the main method for genetic improvement of
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commercial beef herds. However, the use of AI on a large scale in beef cattle is
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somewhat impractical because of difficulties in detecting estrus in cows that are
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grazing large pastures. Adequate detection of estrus requires much labor and time
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to watch cows, gather and sort calves before AI and often does not match typical
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working hours. Furthermore, lack of detectable estrus behavior may occur,
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especially in Bos indicus cows [1]. Thus, synchronizing ovulation with hormonal
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treatments and using fixed-time AI is an efficient method to improve reproductive
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efficiency and genetic merit of commercial beef herds [2].
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The 5dCO-Synch compared with the 7-d CO-Synch system decreases the
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time the dominant follicle is under the influence of circulating progesterone (P4) by
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2 days, which could potentially increase follicle steroidogenesis and therefore
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pregnancy per AI (P/AI) [3,4]. Younger follicles have more steroidogenic capacity
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when compared with older follicles [5]. In addition, this system allows for a longer
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proestrus. For example, Bridges et al. [4], using a short system (P4 for 5 days),
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showed that an increase in 12 hours between luteolysis (PGF2α treatment and P4
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removal) and AI, increased P/AI in about 15 percentage points in comparison to a
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longer system (P4 for 7 days).
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In South America, the most common system used in beef cattle uses P4 and estradiol benzoate (EB) to synchronize emergence of a new follicular wave, and
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estradiol cypionate (EC) to synchronize ovulation at the end of the system.
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Moreover, eCG is used to increase the percentage of cows ovulating to a timed-AI
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system [2]. No studies have applied the 5-d system in Nellore cows under tropical
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conditions, and it is not known if this system may increase P/AI in Nellore cows as
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suggested in Angus cows [4]. Therefore, the aim of this study was to evaluate the
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reproductive performance of nulliparous, primiparous and multiparous Nellore cows
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submitted to the 5dCO-Synch+P4 in comparison to the well-established 7dEB+P4
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system for Bos indicus cattle.
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2. Materials and methods
The Animal Care and Use Committee from the University of São Paulo
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approved all procedures with animals. The experiment was carried out at the
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Experimental Station Hildegard Georgina Von Pritzelwiltz, in Londrina, PR, Brazil.
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Four hundred and eleven Nellore cows were stratified in two systems
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according to parity [nulliparous (n = 198), primiparous (n = 80), and multiparous (n
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= 133)]. All primiparous and multiparous had a calf during the experiment. Days
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postpartum, age, cyclicity (primiparous and multiparous cows), body weight and
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body condition score (BCS) were collected at the beginning of the protocol and
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from them females in each parity were sorted between systems (Table 1). To
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access cyclicity of cows, ultrasound exam of the ovaries was done once at the
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beginning of the protocol and cows were considered cycling when CL was present.
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In nulliparous cows, cyclicity evaluation was done the same way, however only
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nulliparous with CL were used afterwards.
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For the 7dEB+P4 system, cows received an intravaginal P4 insert (CIDR®, Zoetis, São Paulo, Brazil) and 2 mg EB i.m. (Estrogin®, Biofarm, Jaboticabal,
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Brazil) on day 0. On day 7, P4 insert was removed, and 25 mg of PGF2α
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(Lutalyse®, Zoetis), 0.6 mg of EC (ECP®, Zoetis) and 300 IU of eCG (Folligon®,
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MSD, São Paulo, Brasil) were administered i.m. (Figure 1). For the 5dCO-
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Synch+P4 system, cows received an intravaginal P4 insert (CIDR®) and 100 µg of
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GnRH i.m. (Fertagyl®, Intervet) on day 0. On day 5, P4 insert was removed and
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two treatments of 25 mg PGF2α i.m. were given (Lutalyse®) 6 hours apart. Cows
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that did not show estrus until 55 hours after removal of P4 insert received 100 µg of
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GnRH at 72 hours to induce ovulation, and were inseminated immediately after
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GnRH treatment.
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Every cow received an estrus detection patch (Estrotect®, IVP Brasil, São
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Paulo, Brazil) at the time of P4 insert removal. Cows were observed for estrus
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twice a day (AM and PM) until 80 hours days after P4 insert removal. Cows were
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considered in estrus if more than half of the estrus detection patch (Estrotect)
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coating was removed.
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For both systems, when cows were detected in estrus by 55 hours after
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removal of the P4 insert they were inseminated at 55 hours, whereas the remaining
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cows were inseminated at 72 hours (Figure 1).
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Ovulation risk was estimated by measuring circulating P4 concentration at
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10 days after AI. Cows were considered not ovulating to the system if circulating
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P4 concentration was < 1 ng/mL. Progesterone concentration was measured by
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chemiluminescent test using commercial IMMULITE® 1000 kits (Siemens
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Healthcare Diagnostics, Deerfield, IL, USA). All analysis were performed in the
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Nutrition and Animal Reproduction Laboratory – LZT/ESALQ/USP. The coefficients
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of variation were 2.0% and 4.3% to low adjuster and high adjuster, respectively.
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Statistical analyses were performed using SAS procedures (Statistical Analysis System, Version 9.1 for Windows; SAS Institute, Cary, NC, USA). Cows
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were stratified by days postpartum, age, BCS, weight and cyclicity (multiparous)
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within the system. The systems (5dCO-Synch+P4, or 7dEB+P4) and parity of the
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cow (nulliparous, primiparous, or multiparous) were analyzed by 2 x 3 factorial
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design. Binomials variables (estrus-detection risk, ovulation risk and P/AI) were
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analyzed by the GLIMMIX procedure using the binomial option. Circulating P4
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concentration after AI and time to estrus were analyzed by the MIXED procedure.
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Before analyzing by the MIXED procedure, data were analyzed by normality
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(Shapiro-Wilk) and homogeneity of variance (Welch) tests. Discrepant data
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(outliers – studentized residual > 3 or < -3) were removed from the analyses.
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Progesterone concentration after AI, body weight and BCS effects on
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probability of P/AI were analyzed by logistic regression (LIFETEST procedure)
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regarding timed-AI system and parity of the cow. The ODDSRATIO statement was
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used to compare the effect of parity on the relationship between P4 concentration
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after AI and probability of P/AI. Logistic regression curves were constructed using
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the coefficient generated by the Interactive Data Analysis of SAS [Logit = intercept
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+ slope * (P4)]. Probability curves were constructed by Y = [EXP (logit) / 1 + EXP
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(logit)] * 100. Significant differences were considered when P < 0.05.
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3. Results
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Estrus-detection risk (P < 0.0001) and P/AI (P = 0.023) were greater in cows
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treated with the 7dEB+P4 than 5dCO-Synch+P4 system (Table 2). Moreover, more
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cows submitted to the 7dEB+P4 system were detected in estrus earlier (P <
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0.0001) than cows treated with the 5dCO-Synch+P4 system (Table 2; Figure 2).
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Another difference between systems was that P/AI of cows in estrus (54.0%) was
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greater (P = 0.01) than of cows not expressing estrus (32.5%) in the 7dEB+P4
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system. In contrast, P/AI in cows in estrus (33.9%) and not expressing estrus
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(38.1%) did not differ (P = 0.53) if cows had been treated with the 5dCO-Synch+P4
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system.
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There was an interaction between systems and parity of the cow for circulating P4 concentration after AI. Primiparous and multiparous cows had
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greater (P < 0.0001) P4 concentration if submitted to the 7dEB+P4 system than to
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the 5dCO-Synch+P4 system. In contrast, P4 concentration was similar for both
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systems in nulliparous cows. Likewise, a relationship was detected between P4
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concentration after AI and probability of P/AI. In both systems, increased P4
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concentration after AI was associated with greater P/AI (Figure 3A), but no
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difference was detected between systems (P = 0.1945). In addition, there was a
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relationship between P4 concentration and probability of P/AI in the three parity of
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cows, but the magnitude of this relationship was not similar among parities.
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Nulliparous had greater probability of P/AI (P = 0.0037) than primiparous and
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multiparous cows at a similar circulating P4 concentration after AI (Figure 3B). Primiparous cows had lesser estrus-detection risk (P < 0.0001), ovulation
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risk (P = 0.0003), and P/AI (P = 0.0230) than nulliparous and multiparous cows.
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However, there was no effect of initial body weight (P = 0.12) or BCS (P = 0.51) on
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probability of P/AI.
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4. Discussion
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The objective of this study was to evaluate the use of the 5dCO-Synch+P4
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timed-AI system in Nellore cows. Cows exposed to the system used as a control,
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the 7dEB+P4 system, however, had a better reproductive performance than cows
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exposed to the 5dCO-Synch+P4 system, likely, due to the combination of eCG and
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EC that are used as growth promoter of the ovulatory follicle and ovulation inducer,
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respectively. Both hormones administrated before ovulation induce an increase in
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estradiol concentration during proestrus [6,7]. Moreover, eCG improves ovulation
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risk and diameter of the ovulatory follicle [2,8], and P/AI in Bos indicus cows
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(reviewed by Baruselli et al. [2]). In our study, use of the 5dCO-Synch+P4 system
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resulted in poorer estrus-detection risk and less P4 concentration 10 days after AI
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in primiparous and multiparous cows. A study with cows in a similar condition to
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our experiment showed that treatment with GnRH induced high ovulation risk [9],
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comparable with what occurred in our study. In contrast, cows had smaller (8.5
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mm) follicle diameter at AI [9]. Follicles at that diameter have ovulatory capacity
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[10], however result in lower P/AI when induced to ovulate [9,11]. Therefore, this
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might be one of the reasons why fertility was compromised in cows bred to the
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5dCO-Synch+P4 system. Primiparous and multiparous cows may have been in negative energy
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balance (NEB) at the onset of the study because of their relatively short days
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postpartum. Those cows had a very low incidence of estrus (near 0%; data not
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shown) if submitted to the 5dCO-Synch+P4 system. In contrast, an experiment
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using the same 5dCO-Synch+P4 system in which 43% of suckled Angus cows
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were anovular described about 80% of cows in estrus in response to the protocol
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[4]. Moreover, another experiment from the same series of studies, reported that
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although 90% of the cows exposed to the 5dCO-Synch+P4 system were anovular,
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P/AI was 80% [4]. Thus, it is reasonable to suppose that cows from the present
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study were deeper in NEB/anovular condition. Moreover, multiparous and
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primiparous cows synchronized with the 5dCO-Synch+P4 system had lower
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circulating P4 concentration 10 days after AI, associated with lower P/AI. Pugliesi
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et al. [12] showed that supplementing 150 mg of long-acting P4 from day 4 post-AI
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increased P/AI in cows that had ovulated smaller, but not larger follicles. In our
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study, greater P4 concentration at 10 days after AI enhanced the probability of
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P/AI, independent of timed-AI system or parity of the cow. It has been shown that
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circulating P4 concentration after AI is associated positively with embryo
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development [13] and affects embryonic survival [14].
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There was no relationship between body weight and BCS with probability of
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P/AI. Body condition score of the primiparous and multiparous cows in this study
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varied between 2.5 and 3.5. Obviously, BCS and body weight are important for
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P/AI, but perhaps, more important than BCS at the time of AI is the expected high
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BCS loss between calving and the time of AI [15].
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In cows exposed to the 7dEB+P4 system, incidence of estrus was clustered between 48 and 55 hours after P4 insert removal. These results are similar to
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systems that used a P4 insert for 8 days [16]. In this kind of system, the LH peak
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occurs at about 50 hours after EC administration, and ovulation occurs at
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approximately 72 hours after P4 removal [16,17]. In cows exposed to the 5dCO-
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Synch+P4 system, incidence of estrus was more concentrated near 80 hours after
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P4 insert removal. This result with Nellore heifers were not similar to what has
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been reported by Bridges et al. [4], who described a high concentration of estrus
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between 48 and 60 hours in Angus cows. In contrast, our results are similar to
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those of Abreu et al. [18,19], who observed that most cows with a younger follicle
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at removal of the P4 insert exhibited estrus between 72 and 80 hours after removal
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of the P4 insert when the 5dCO-Synch+P4 system was employed. This delay in
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estrus expression after removal of the P4 insert may be due a higher sensitivity of
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the hypothalamus to elevated circulating P4 concentrations in Bos indicus cattle. It
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has been reported that Nellore heifers had a slower development of the
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dominant/preovulatory follicle when circulating P4 concentrations were elevated
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[20]. The greater sensitivity of the hypothalamus to circulating P4 in addition to
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lower ovulatory efficiency to the first GnRH treatment described in heifers [21] may
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increase the possibility of an inadequate synchronization to the 5dCO-Synch+P4
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system. Moreover, it has been reported that the analog of GnRH (gonadorelin)
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used in this study induces a lower LH surge compared with other analogs of
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GnRH, such as lecirelin, fertirelin, or buserelin [22,23]. In this study 34% of the
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heifers showed estrus at 80 hours after GnRH administration. Therefore, this group
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of cows probably did not have an estrous cycle synchronized, and failed to respond
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to the second GnRH of the system. When submitted to the 7dEB+P4 system, cows detected in estrus had
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greater P/AI than cows not detected in estrus. In addition, most of cows submitted
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to 7dEB+P4 system showed estrus before AI, contrasting with cows treated with
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5dCO-Synch+P4 which majority was submitted to AI before estrus or with no
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expressed estrus. These observations may help to explain the low P/AI in cows
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that showed estrus after AI, since the best scheme for AI is when cows are
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submitted to AI about 12 hours after detected estrus [24].
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5. Conclusion
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The 7dEB+P4 system increased estrus-detection risk, P/AI and circulating P4 10 days after AI in comparison with the 5dCO-Synch+P4 system. However,
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further research is needed in Bos indicus cows submitted to the 5dCO-Synch+P4
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system, especially regarding the time to perform AI in the 5dCO-Synch+P4 system.
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Acknowledgments
This study was financially supported by the Fundação de Amparo à
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Pesquisa do Estado de São Paulo (FAPESP 2012/01345-9). We are grateful of
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Fazenda Figueira - Experimental Station Hildegard Georgina Von Pritzelwiltz
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(FEALQ) for allowing the use of cattle and supplies for this study.
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[17] Colazo MG, Kastelic JP, Mapletoft RJ. Effects of estradiol cypionate (ECP) on
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ovarian follicular dynamics, synchrony of ovulation, and fertility in P4-based, fixed-
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time AI programs in beef heifers. Theriogenology 2003;60:855-65.
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[18] Abreu FM, Geary TW, Cruppe LH, Madsen CA, Jinks EM, Pohler KG,
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Vasconcelos JL, Day ML. The effect of follicle age on pregnancy rate in beef cows.
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J Anim Sci 2014;92:1015-21.
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[19] Abreu FM, Cruppe LH, Maquivar M, Utt MD, Madsen CA, Vasconcelos JL,
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Mussard ML, Day ML, Geary TW. Effect of follicle age on conception rate in beef
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heifers. J Anim Sci 2014;92:1022-8.
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[20] Carvalho JBP, Carvalho NAT, Reis EL, Nichi M, Souza AH, Baruselli PS.
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Effect of early luteolysis in progesterone-based timed AI protocols in Bos indicus,
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Bos indicus × Bos taurus, and Bos taurus heifers. Theriogenology 2008;69:167-75.
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[21] Cruppe LH, Day ML, Abreu FM, Kruse S, Lake SL, Biehl MV, Cipriano RS,
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Mussard ML, Bridges GA. The requirement of GnRH at the beginning of the five-
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day CO-Synch + controlled internal drug release protocol in beef heifers. J Anim
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Sci 2014;92:4198-203.
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[22] Picard-Hagen N, Lhermie G, Florentin S, Merle D, Frein P, Gayrard V. Effect
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of gonadorelin, lecirelin, and buserelin on LH surge, ovulation, and progesterone in
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cattle. Theriogenology 2015;84:177-83.
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[23] Chenault JR, Kratzer DD, Rzepkowski RA, Goodwin MC. LH and FSH
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response of Holstein heifers to fertirelin acetate, gonadorelin and buserelin.
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Theriogenology 1990; 34:81–98
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[24] Trimberg W, Davis HP. Conception rate in dairy cattle by artificial insemination
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at various stages of estrus. Neb Agric Exp Sta Res Bull 1943;129:1-14.
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Table 1. Characterization of cows at the beginning of systems. Parity
n
DPP (day)
Age (year)
CL (%)
BW (kg)
BCS (1-5)
Nulliparous
198
.
2.1 ± 0.01
100
342 ± 2
.
Primiparous
80
39 ± 0.5
3.9 ± 0.03
0
391 ± 3
2.7 ± 0.01
Multiparous
133
39 ± 0.5
7.5 ± 0.20
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448 ± 4
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2.8 ± 0.02
N: Number; DPP: days postpartum; CL: percentage of cows with corpus luteum;
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BW: body weight; BCS: body condition score. All the variables (mean ± SE) shown
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in this table were collected at the beginning of systems and all multiparous and
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primiparous had a calf.
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Table 2. Reproductive performance of Nellore cows according to the timed-AI system (7dEB+P4 or 5dCO-Synch+P4) and parity
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(nulliparous, primiparous, or multiparous).
Pregnancy per AI, %
49.7 (101/203)
35.4 (73/206)
46.2a (91/197)
Estrus-detection risk, %
80.4 (164/204)
36.4 (75/206)
80.8a (160/198)
Primiparous
a
Multiparous
S
Parity
S*P
28.7b (23/80)
45.4a (60/132)
-
0.009
0.023
0.312
25.0c (20/80)
44.7b (59/132)
-
<.0001
<.0001
0.579
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b
a
Ovulation risk, %
89.4 (178/199)
90.2 (185/205)
92.3 (183/198)
76.6 (59/77)
93.9 (124/132)
-
0.769
0.0003
0.248
Time to estrus, h
54.1 (165)
70.8 (75)
59.7 (160)
58.6 (20)
58.4 (60)
0.85
<.0001
0.090
0.236
P4 concentration after AI1, ng/mL
5.6 (161)
4.9 (174)
4.5 (165)
5.6 (51)
6.2 (119)
0.1
<.0001
<.0001
0.0308
1
Considering only cows with progesterone (P4) concentration ≥ 1 ng/mL at 10 d after AI. EB – Estradiol benzoate; (n/n) – ratio for
each variable in percentage; (n) Number of cows in each variable. Means followed by different letters in each line differ (P < 0.05).
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5 d (GnRH)
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7 d (EB)
P-value SEM
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Parity (P)
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System (S)
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Figure captions
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Figure 1. Scheme of systems. 7dEB+P4 system, Day 0 - cows received
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progesterone (P4) insert and 2 mg of estradiol benzoate (EB). Day 7 - P4 insert
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was removed and cows received 25 mg of PGF2α, 0.6 mg of estradiol cypionate
368
(EC) and 300 IU of eCG. 5dCO-Synch+P4 system, Day 0 - cows received P4 insert
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and 100 µg of GnRH. Day 5 - P4 insert was removed and cows received two doses
370
of 25 mg of PGF2α with 6 hours apart. Cows that not expressing estrus until 55
371
hours received 100 µg of GnRH at 72 hours. All cows were fitted with estrus-
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detection patches (Estrotect) when P4 inserts were removed to aid in detecting
373
estrus. Estrus detection was evaluated at 32, 48, 55, 72 and 80 hours after P4
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insert removal. Artificial insemination was performed according to estrus detection,
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and cows in estrus until 55 hours were submitted to AI at this point. Cows in estrus
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at 72 hours or that not expressing estrus were submitted to AI at 72 hours.
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Figure 2. Percentage of cows in estrus according to each system (7dEB+P4 and
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5dCO-Synch+P4). Cows were observed twice a day from 32 to 80 hours after
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progesterone (P4) insert removal. Cows were determined to be in estrus when
381
more than 50% of the patch (Estrotect) was rubbed. Numbers above the bars
382
represent the percentage of cows.
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Figure
3. Probability of pregnancy per AI (P/AI) for each system and each parity of
385
the cow according to progesterone (P4) concentration 10 days after AI. A –
386
Probability of P/AI for each system: 7dEB+P4 [Probability of P/AI = exp (−1.2075 +
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0.2366*P4)/1 + exp (−1.2075 + 0.2366*P4); P = 0.0002]. 5dCO-Synch+P4
388
[Probability of P/AI = exp (−1.9983 + 0.3042*P4)/1 + exp (−1.9983 + 0.3042*P4); P
389
= 0.0002]. B – Probability of P/AI for each parity of the cow. Nulliparous [Probability
390
of P/AI = exp (−1.3809 + 0.2901*P4)/1 + exp (−1.3809 + 0.2901*P4); P = 0.0006].
391
Primiparous [Probability of P/AI = exp (−3.6201 + 0.5184*P4)/1 + exp (−3.6201 +
392
0.5184*P4); P = 0.0001]. Multiparous [Probability of P/AI = exp (−1.4468 +
393
0.2210*P4)/1 + exp (− 1.4468 + 0.2210*P4); P = 0.01). a,b = Equation of
394
nulliparous was different (P = 0.0037) regarding to primiparous and multiparous
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cows.
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ACCEPTED MANUSCRIPT Highlights
Reproductive performance was reduced with the 5dCO-Synch+P4 in Nellore cows.
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Primiparous and multiparous cows had greater P4 concentration in 7dEB+P4 protocol. In both protocols, increased P4 concentration after AI was associated with greater P/AI.
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Primiparous cows had lower performance, and require more attention after calving.
S0093-691X(16)30266-7
DOI:
10.1016/j.theriogenology.2016.06.012
Reference:
THE 13722
To appear in:
Theriogenology
Received Date: 6 May 2016 Revised Date:
6 June 2016
Accepted Date: 7 June 2016
Please cite this article as: Ferraz Junior MVC, Pires AV, Biehl MV, Santos MH, Barroso JPR, Gonçalves JRS, Sartori R, Day ML, Comparison of Two Timed AI System Schemes to Synchronize Estrus and Ovulation in Nellore Cattle, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.06.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Revised 1
Comparison of Two Timed AI System Schemes to Synchronize Estrus and
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Ovulation in Nellore Cattle
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Marcos Vinicius C Ferraz Juniora, Alexandre V Piresa,b,*, Marcos Vinicius Biehlb,
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Marcelo H Santosa, Jose Paulo R Barrosoa, José Renato S Gonçalves c, Roberto
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Sartoric, Michael L Day d
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Department of Nutrition and Animal Production, Faculty of Veterinary and Animal
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a
Science, University of São Paulo, Duque de Caxias north, n 225, Pirassununga, São Paulo, Brazil, Zip: 13635-000. b
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Department of Animal Science, College of Agriculture “Luiz de Queiroz” (ESALQ),
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University of São Paulo, Pádua Dias Avenue, n 11, Piracicaba, São Paulo, Brazil,
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PO Box 09, Zip: 13418-900.
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Experimental Station Hildegard Georgina Von Pritzelwitz, Fazenda Figueira,
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c
Londrina, Paraná, Brazil. PO Box 8003, Zip: 86010-990. d
Department of Animal Science, University of Wyoming, 1000 East University
Avenue, Laramie, WY, 82071.
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*Corresponding author: Alexandre Vaz Pires. Department of Animal Science,
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ESALQ / USP, Piracicaba, SP, 13418-900, Brazil. Tel.: +55 (19) 3429-4247; Fax:
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+55 (19) 3429-4215 [email protected]
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Abstract
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The aim of this study was to evaluate the reproductive performance of 411
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Nellore cows (198 nulliparous, 80 primiparous and 133 multiparous) submitted to
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the 5dCO-Synch+P4 or 7dEB+P4 systems. The 5dCO-Synch+P4 system
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consisted of insertion of an intravaginal progesterone (P4) insert and 100 µg of
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GnRH (i.m.) on day 0. On day 5, the P4 insert was removed and two doses of 25
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mg of PGF2α (i.m.) were administered 6 hours apart. Cows not detected in estrus
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until 55 hours after insert removal received 100 µg of GnRH i.m. 17 hours later
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(i.e., 72 hours after P4 removal). The 7dEB+P4 system consisted of insertion of a
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P4 insert and 2 mg of estradiol benzoate (i.m.) on day 0. On day 7, the P4 insert
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was removed and 25 mg of PGF2α, 0.6 mg of estradiol cypionate and 300 IU of
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eCG were administered i.m. In both systems, AI was performed according to estrus
38
detection (i.e., cows detected in estrus until 55 hours after insert removal were
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inseminated at 55 hours, and cows detected in estrus later, or those not detected
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in estrus were inseminated at 72 hours). Estrus-detection risk was greater (P <
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0.05) in 7dEB+P4 (80.4%) than 5dCO-Synch+P4 system (36.4%). Progesterone
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concentration 10 days after AI was greater (P < 0.05) in 7dEB+P4 than 5dCO-
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Synch+P4 system in primiparous and multiparous, but did not differ between
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systems in nulliparous cows. Pregnancy per AI (P/AI) was greater (P < 0.05) in
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7dEB+P4 (49.7%) than 5dCO-Synch+P4 (35.4%) system. Primiparous had lower
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estrus-detection risk (25.0%), ovulation risk (76.6%) and P/AI (28.7%) than
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multiparous or nulliparous cows. In conclusion, reproductive performance was
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reduced with the 5dCO-Synch+P4 in comparison to the 7dEB+P4 system in
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Nellore cows. Moreover, the reproductive traits observed for primiparous cows
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indicate that more attention is required when timed AI programs is started early
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after calving.
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Key words: Bos indicus; Estradiol; Fixed timed-AI; GnRH; Progesterone.
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1. Introduction
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Artificial insemination is the main method for genetic improvement of
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commercial beef herds. However, the use of AI on a large scale in beef cattle is
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somewhat impractical because of difficulties in detecting estrus in cows that are
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grazing large pastures. Adequate detection of estrus requires much labor and time
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to watch cows, gather and sort calves before AI and often does not match typical
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working hours. Furthermore, lack of detectable estrus behavior may occur,
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especially in Bos indicus cows [1]. Thus, synchronizing ovulation with hormonal
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treatments and using fixed-time AI is an efficient method to improve reproductive
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efficiency and genetic merit of commercial beef herds [2].
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The 5dCO-Synch compared with the 7-d CO-Synch system decreases the
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time the dominant follicle is under the influence of circulating progesterone (P4) by
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2 days, which could potentially increase follicle steroidogenesis and therefore
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pregnancy per AI (P/AI) [3,4]. Younger follicles have more steroidogenic capacity
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when compared with older follicles [5]. In addition, this system allows for a longer
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proestrus. For example, Bridges et al. [4], using a short system (P4 for 5 days),
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showed that an increase in 12 hours between luteolysis (PGF2α treatment and P4
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removal) and AI, increased P/AI in about 15 percentage points in comparison to a
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longer system (P4 for 7 days).
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In South America, the most common system used in beef cattle uses P4 and estradiol benzoate (EB) to synchronize emergence of a new follicular wave, and
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estradiol cypionate (EC) to synchronize ovulation at the end of the system.
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Moreover, eCG is used to increase the percentage of cows ovulating to a timed-AI
79
system [2]. No studies have applied the 5-d system in Nellore cows under tropical
80
conditions, and it is not known if this system may increase P/AI in Nellore cows as
81
suggested in Angus cows [4]. Therefore, the aim of this study was to evaluate the
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reproductive performance of nulliparous, primiparous and multiparous Nellore cows
83
submitted to the 5dCO-Synch+P4 in comparison to the well-established 7dEB+P4
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system for Bos indicus cattle.
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2. Materials and methods
The Animal Care and Use Committee from the University of São Paulo
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approved all procedures with animals. The experiment was carried out at the
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Experimental Station Hildegard Georgina Von Pritzelwiltz, in Londrina, PR, Brazil.
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Four hundred and eleven Nellore cows were stratified in two systems
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according to parity [nulliparous (n = 198), primiparous (n = 80), and multiparous (n
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= 133)]. All primiparous and multiparous had a calf during the experiment. Days
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postpartum, age, cyclicity (primiparous and multiparous cows), body weight and
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body condition score (BCS) were collected at the beginning of the protocol and
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from them females in each parity were sorted between systems (Table 1). To
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access cyclicity of cows, ultrasound exam of the ovaries was done once at the
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beginning of the protocol and cows were considered cycling when CL was present.
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In nulliparous cows, cyclicity evaluation was done the same way, however only
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nulliparous with CL were used afterwards.
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For the 7dEB+P4 system, cows received an intravaginal P4 insert (CIDR®, Zoetis, São Paulo, Brazil) and 2 mg EB i.m. (Estrogin®, Biofarm, Jaboticabal,
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Brazil) on day 0. On day 7, P4 insert was removed, and 25 mg of PGF2α
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(Lutalyse®, Zoetis), 0.6 mg of EC (ECP®, Zoetis) and 300 IU of eCG (Folligon®,
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MSD, São Paulo, Brasil) were administered i.m. (Figure 1). For the 5dCO-
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Synch+P4 system, cows received an intravaginal P4 insert (CIDR®) and 100 µg of
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GnRH i.m. (Fertagyl®, Intervet) on day 0. On day 5, P4 insert was removed and
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two treatments of 25 mg PGF2α i.m. were given (Lutalyse®) 6 hours apart. Cows
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that did not show estrus until 55 hours after removal of P4 insert received 100 µg of
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GnRH at 72 hours to induce ovulation, and were inseminated immediately after
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GnRH treatment.
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Paulo, Brazil) at the time of P4 insert removal. Cows were observed for estrus
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twice a day (AM and PM) until 80 hours days after P4 insert removal. Cows were
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considered in estrus if more than half of the estrus detection patch (Estrotect)
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coating was removed.
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For both systems, when cows were detected in estrus by 55 hours after
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removal of the P4 insert they were inseminated at 55 hours, whereas the remaining
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cows were inseminated at 72 hours (Figure 1).
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Ovulation risk was estimated by measuring circulating P4 concentration at
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10 days after AI. Cows were considered not ovulating to the system if circulating
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P4 concentration was < 1 ng/mL. Progesterone concentration was measured by
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chemiluminescent test using commercial IMMULITE® 1000 kits (Siemens
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Healthcare Diagnostics, Deerfield, IL, USA). All analysis were performed in the
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Nutrition and Animal Reproduction Laboratory – LZT/ESALQ/USP. The coefficients
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of variation were 2.0% and 4.3% to low adjuster and high adjuster, respectively.
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Statistical analyses were performed using SAS procedures (Statistical Analysis System, Version 9.1 for Windows; SAS Institute, Cary, NC, USA). Cows
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were stratified by days postpartum, age, BCS, weight and cyclicity (multiparous)
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within the system. The systems (5dCO-Synch+P4, or 7dEB+P4) and parity of the
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cow (nulliparous, primiparous, or multiparous) were analyzed by 2 x 3 factorial
132
design. Binomials variables (estrus-detection risk, ovulation risk and P/AI) were
133
analyzed by the GLIMMIX procedure using the binomial option. Circulating P4
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concentration after AI and time to estrus were analyzed by the MIXED procedure.
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Before analyzing by the MIXED procedure, data were analyzed by normality
136
(Shapiro-Wilk) and homogeneity of variance (Welch) tests. Discrepant data
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(outliers – studentized residual > 3 or < -3) were removed from the analyses.
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Progesterone concentration after AI, body weight and BCS effects on
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probability of P/AI were analyzed by logistic regression (LIFETEST procedure)
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regarding timed-AI system and parity of the cow. The ODDSRATIO statement was
141
used to compare the effect of parity on the relationship between P4 concentration
142
after AI and probability of P/AI. Logistic regression curves were constructed using
143
the coefficient generated by the Interactive Data Analysis of SAS [Logit = intercept
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+ slope * (P4)]. Probability curves were constructed by Y = [EXP (logit) / 1 + EXP
145
(logit)] * 100. Significant differences were considered when P < 0.05.
146
3. Results
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Estrus-detection risk (P < 0.0001) and P/AI (P = 0.023) were greater in cows
150
treated with the 7dEB+P4 than 5dCO-Synch+P4 system (Table 2). Moreover, more
151
cows submitted to the 7dEB+P4 system were detected in estrus earlier (P <
152
0.0001) than cows treated with the 5dCO-Synch+P4 system (Table 2; Figure 2).
153
Another difference between systems was that P/AI of cows in estrus (54.0%) was
154
greater (P = 0.01) than of cows not expressing estrus (32.5%) in the 7dEB+P4
155
system. In contrast, P/AI in cows in estrus (33.9%) and not expressing estrus
156
(38.1%) did not differ (P = 0.53) if cows had been treated with the 5dCO-Synch+P4
157
system.
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There was an interaction between systems and parity of the cow for circulating P4 concentration after AI. Primiparous and multiparous cows had
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greater (P < 0.0001) P4 concentration if submitted to the 7dEB+P4 system than to
161
the 5dCO-Synch+P4 system. In contrast, P4 concentration was similar for both
162
systems in nulliparous cows. Likewise, a relationship was detected between P4
163
concentration after AI and probability of P/AI. In both systems, increased P4
164
concentration after AI was associated with greater P/AI (Figure 3A), but no
165
difference was detected between systems (P = 0.1945). In addition, there was a
166
relationship between P4 concentration and probability of P/AI in the three parity of
167
cows, but the magnitude of this relationship was not similar among parities.
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Nulliparous had greater probability of P/AI (P = 0.0037) than primiparous and
169
multiparous cows at a similar circulating P4 concentration after AI (Figure 3B). Primiparous cows had lesser estrus-detection risk (P < 0.0001), ovulation
171
risk (P = 0.0003), and P/AI (P = 0.0230) than nulliparous and multiparous cows.
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However, there was no effect of initial body weight (P = 0.12) or BCS (P = 0.51) on
173
probability of P/AI.
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4. Discussion
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The objective of this study was to evaluate the use of the 5dCO-Synch+P4
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timed-AI system in Nellore cows. Cows exposed to the system used as a control,
179
the 7dEB+P4 system, however, had a better reproductive performance than cows
180
exposed to the 5dCO-Synch+P4 system, likely, due to the combination of eCG and
181
EC that are used as growth promoter of the ovulatory follicle and ovulation inducer,
182
respectively. Both hormones administrated before ovulation induce an increase in
183
estradiol concentration during proestrus [6,7]. Moreover, eCG improves ovulation
184
risk and diameter of the ovulatory follicle [2,8], and P/AI in Bos indicus cows
185
(reviewed by Baruselli et al. [2]). In our study, use of the 5dCO-Synch+P4 system
186
resulted in poorer estrus-detection risk and less P4 concentration 10 days after AI
187
in primiparous and multiparous cows. A study with cows in a similar condition to
188
our experiment showed that treatment with GnRH induced high ovulation risk [9],
189
comparable with what occurred in our study. In contrast, cows had smaller (8.5
190
mm) follicle diameter at AI [9]. Follicles at that diameter have ovulatory capacity
191
[10], however result in lower P/AI when induced to ovulate [9,11]. Therefore, this
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might be one of the reasons why fertility was compromised in cows bred to the
193
5dCO-Synch+P4 system. Primiparous and multiparous cows may have been in negative energy
195
balance (NEB) at the onset of the study because of their relatively short days
196
postpartum. Those cows had a very low incidence of estrus (near 0%; data not
197
shown) if submitted to the 5dCO-Synch+P4 system. In contrast, an experiment
198
using the same 5dCO-Synch+P4 system in which 43% of suckled Angus cows
199
were anovular described about 80% of cows in estrus in response to the protocol
200
[4]. Moreover, another experiment from the same series of studies, reported that
201
although 90% of the cows exposed to the 5dCO-Synch+P4 system were anovular,
202
P/AI was 80% [4]. Thus, it is reasonable to suppose that cows from the present
203
study were deeper in NEB/anovular condition. Moreover, multiparous and
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primiparous cows synchronized with the 5dCO-Synch+P4 system had lower
205
circulating P4 concentration 10 days after AI, associated with lower P/AI. Pugliesi
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et al. [12] showed that supplementing 150 mg of long-acting P4 from day 4 post-AI
207
increased P/AI in cows that had ovulated smaller, but not larger follicles. In our
208
study, greater P4 concentration at 10 days after AI enhanced the probability of
209
P/AI, independent of timed-AI system or parity of the cow. It has been shown that
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circulating P4 concentration after AI is associated positively with embryo
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development [13] and affects embryonic survival [14].
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There was no relationship between body weight and BCS with probability of
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P/AI. Body condition score of the primiparous and multiparous cows in this study
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varied between 2.5 and 3.5. Obviously, BCS and body weight are important for
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P/AI, but perhaps, more important than BCS at the time of AI is the expected high
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BCS loss between calving and the time of AI [15].
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In cows exposed to the 7dEB+P4 system, incidence of estrus was clustered between 48 and 55 hours after P4 insert removal. These results are similar to
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systems that used a P4 insert for 8 days [16]. In this kind of system, the LH peak
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occurs at about 50 hours after EC administration, and ovulation occurs at
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approximately 72 hours after P4 removal [16,17]. In cows exposed to the 5dCO-
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Synch+P4 system, incidence of estrus was more concentrated near 80 hours after
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P4 insert removal. This result with Nellore heifers were not similar to what has
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been reported by Bridges et al. [4], who described a high concentration of estrus
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between 48 and 60 hours in Angus cows. In contrast, our results are similar to
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those of Abreu et al. [18,19], who observed that most cows with a younger follicle
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at removal of the P4 insert exhibited estrus between 72 and 80 hours after removal
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of the P4 insert when the 5dCO-Synch+P4 system was employed. This delay in
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estrus expression after removal of the P4 insert may be due a higher sensitivity of
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the hypothalamus to elevated circulating P4 concentrations in Bos indicus cattle. It
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has been reported that Nellore heifers had a slower development of the
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dominant/preovulatory follicle when circulating P4 concentrations were elevated
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[20]. The greater sensitivity of the hypothalamus to circulating P4 in addition to
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lower ovulatory efficiency to the first GnRH treatment described in heifers [21] may
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increase the possibility of an inadequate synchronization to the 5dCO-Synch+P4
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system. Moreover, it has been reported that the analog of GnRH (gonadorelin)
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used in this study induces a lower LH surge compared with other analogs of
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GnRH, such as lecirelin, fertirelin, or buserelin [22,23]. In this study 34% of the
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heifers showed estrus at 80 hours after GnRH administration. Therefore, this group
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of cows probably did not have an estrous cycle synchronized, and failed to respond
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to the second GnRH of the system. When submitted to the 7dEB+P4 system, cows detected in estrus had
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greater P/AI than cows not detected in estrus. In addition, most of cows submitted
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to 7dEB+P4 system showed estrus before AI, contrasting with cows treated with
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5dCO-Synch+P4 which majority was submitted to AI before estrus or with no
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expressed estrus. These observations may help to explain the low P/AI in cows
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that showed estrus after AI, since the best scheme for AI is when cows are
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submitted to AI about 12 hours after detected estrus [24].
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5. Conclusion
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The 7dEB+P4 system increased estrus-detection risk, P/AI and circulating P4 10 days after AI in comparison with the 5dCO-Synch+P4 system. However,
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further research is needed in Bos indicus cows submitted to the 5dCO-Synch+P4
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system, especially regarding the time to perform AI in the 5dCO-Synch+P4 system.
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Acknowledgments
This study was financially supported by the Fundação de Amparo à
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Pesquisa do Estado de São Paulo (FAPESP 2012/01345-9). We are grateful of
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Fazenda Figueira - Experimental Station Hildegard Georgina Von Pritzelwiltz
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(FEALQ) for allowing the use of cattle and supplies for this study.
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[17] Colazo MG, Kastelic JP, Mapletoft RJ. Effects of estradiol cypionate (ECP) on
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ovarian follicular dynamics, synchrony of ovulation, and fertility in P4-based, fixed-
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Vasconcelos JL, Day ML. The effect of follicle age on pregnancy rate in beef cows.
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[19] Abreu FM, Cruppe LH, Maquivar M, Utt MD, Madsen CA, Vasconcelos JL,
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Mussard ML, Day ML, Geary TW. Effect of follicle age on conception rate in beef
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[20] Carvalho JBP, Carvalho NAT, Reis EL, Nichi M, Souza AH, Baruselli PS.
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Effect of early luteolysis in progesterone-based timed AI protocols in Bos indicus,
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Bos indicus × Bos taurus, and Bos taurus heifers. Theriogenology 2008;69:167-75.
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[21] Cruppe LH, Day ML, Abreu FM, Kruse S, Lake SL, Biehl MV, Cipriano RS,
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Mussard ML, Bridges GA. The requirement of GnRH at the beginning of the five-
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day CO-Synch + controlled internal drug release protocol in beef heifers. J Anim
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Sci 2014;92:4198-203.
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[22] Picard-Hagen N, Lhermie G, Florentin S, Merle D, Frein P, Gayrard V. Effect
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of gonadorelin, lecirelin, and buserelin on LH surge, ovulation, and progesterone in
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cattle. Theriogenology 2015;84:177-83.
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[23] Chenault JR, Kratzer DD, Rzepkowski RA, Goodwin MC. LH and FSH
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response of Holstein heifers to fertirelin acetate, gonadorelin and buserelin.
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Theriogenology 1990; 34:81–98
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[24] Trimberg W, Davis HP. Conception rate in dairy cattle by artificial insemination
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at various stages of estrus. Neb Agric Exp Sta Res Bull 1943;129:1-14.
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Table 1. Characterization of cows at the beginning of systems. Parity
n
DPP (day)
Age (year)
CL (%)
BW (kg)
BCS (1-5)
Nulliparous
198
.
2.1 ± 0.01
100
342 ± 2
.
Primiparous
80
39 ± 0.5
3.9 ± 0.03
0
391 ± 3
2.7 ± 0.01
Multiparous
133
39 ± 0.5
7.5 ± 0.20
23
448 ± 4
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2.8 ± 0.02
N: Number; DPP: days postpartum; CL: percentage of cows with corpus luteum;
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BW: body weight; BCS: body condition score. All the variables (mean ± SE) shown
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in this table were collected at the beginning of systems and all multiparous and
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primiparous had a calf.
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Table 2. Reproductive performance of Nellore cows according to the timed-AI system (7dEB+P4 or 5dCO-Synch+P4) and parity
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(nulliparous, primiparous, or multiparous).
Pregnancy per AI, %
49.7 (101/203)
35.4 (73/206)
46.2a (91/197)
Estrus-detection risk, %
80.4 (164/204)
36.4 (75/206)
80.8a (160/198)
Primiparous
a
Multiparous
S
Parity
S*P
28.7b (23/80)
45.4a (60/132)
-
0.009
0.023
0.312
25.0c (20/80)
44.7b (59/132)
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<.0001
<.0001
0.579
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Nulliparous
b
a
Ovulation risk, %
89.4 (178/199)
90.2 (185/205)
92.3 (183/198)
76.6 (59/77)
93.9 (124/132)
-
0.769
0.0003
0.248
Time to estrus, h
54.1 (165)
70.8 (75)
59.7 (160)
58.6 (20)
58.4 (60)
0.85
<.0001
0.090
0.236
P4 concentration after AI1, ng/mL
5.6 (161)
4.9 (174)
4.5 (165)
5.6 (51)
6.2 (119)
0.1
<.0001
<.0001
0.0308
1
Considering only cows with progesterone (P4) concentration ≥ 1 ng/mL at 10 d after AI. EB – Estradiol benzoate; (n/n) – ratio for
each variable in percentage; (n) Number of cows in each variable. Means followed by different letters in each line differ (P < 0.05).
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7 d (EB)
P-value SEM
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Figure captions
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Figure 1. Scheme of systems. 7dEB+P4 system, Day 0 - cows received
366
progesterone (P4) insert and 2 mg of estradiol benzoate (EB). Day 7 - P4 insert
367
was removed and cows received 25 mg of PGF2α, 0.6 mg of estradiol cypionate
368
(EC) and 300 IU of eCG. 5dCO-Synch+P4 system, Day 0 - cows received P4 insert
369
and 100 µg of GnRH. Day 5 - P4 insert was removed and cows received two doses
370
of 25 mg of PGF2α with 6 hours apart. Cows that not expressing estrus until 55
371
hours received 100 µg of GnRH at 72 hours. All cows were fitted with estrus-
372
detection patches (Estrotect) when P4 inserts were removed to aid in detecting
373
estrus. Estrus detection was evaluated at 32, 48, 55, 72 and 80 hours after P4
374
insert removal. Artificial insemination was performed according to estrus detection,
375
and cows in estrus until 55 hours were submitted to AI at this point. Cows in estrus
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at 72 hours or that not expressing estrus were submitted to AI at 72 hours.
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Figure 2. Percentage of cows in estrus according to each system (7dEB+P4 and
379
5dCO-Synch+P4). Cows were observed twice a day from 32 to 80 hours after
380
progesterone (P4) insert removal. Cows were determined to be in estrus when
381
more than 50% of the patch (Estrotect) was rubbed. Numbers above the bars
382
represent the percentage of cows.
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Figure
3. Probability of pregnancy per AI (P/AI) for each system and each parity of
385
the cow according to progesterone (P4) concentration 10 days after AI. A –
386
Probability of P/AI for each system: 7dEB+P4 [Probability of P/AI = exp (−1.2075 +
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0.2366*P4)/1 + exp (−1.2075 + 0.2366*P4); P = 0.0002]. 5dCO-Synch+P4
388
[Probability of P/AI = exp (−1.9983 + 0.3042*P4)/1 + exp (−1.9983 + 0.3042*P4); P
389
= 0.0002]. B – Probability of P/AI for each parity of the cow. Nulliparous [Probability
390
of P/AI = exp (−1.3809 + 0.2901*P4)/1 + exp (−1.3809 + 0.2901*P4); P = 0.0006].
391
Primiparous [Probability of P/AI = exp (−3.6201 + 0.5184*P4)/1 + exp (−3.6201 +
392
0.5184*P4); P = 0.0001]. Multiparous [Probability of P/AI = exp (−1.4468 +
393
0.2210*P4)/1 + exp (− 1.4468 + 0.2210*P4); P = 0.01). a,b = Equation of
394
nulliparous was different (P = 0.0037) regarding to primiparous and multiparous
395
cows.
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Reproductive performance was reduced with the 5dCO-Synch+P4 in Nellore cows.
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Primiparous and multiparous cows had greater P4 concentration in 7dEB+P4 protocol. In both protocols, increased P4 concentration after AI was associated with greater P/AI.
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Primiparous cows had lower performance, and require more attention after calving.