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Double-Ovsynch, compared with presynch with or without GnRH, improves fertility in heat-stressed lactating dairy cows
Accepted Manuscript Double-Ovsynch, compared to Presynch with or without GnRH, improves fertility in heat-stressed lactating dairy cows E. Dirandeh, A. Rezaei Roodbari, M.G. Colazo PII:
S0093-691X(14)00554-8
DOI:
10.1016/j.theriogenology.2014.10.011
Reference:
THE 12953
To appear in:
Theriogenology
Received Date: 5 August 2014 Revised Date:
5 October 2014
Accepted Date: 6 October 2014
Please cite this article as: Dirandeh E, Roodbari AR, Colazo MG, Double-Ovsynch, compared to Presynch with or without GnRH, improves fertility in heat-stressed lactating dairy cows, Theriogenology (2014), doi: 10.1016/j.theriogenology.2014.10.011. 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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Double-Ovsynch, compared to Presynch with or without GnRH, improves fertility in
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heat-stressed lactating dairy cows
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E. Dirandeha*, A. Rezaei Roodbarib, and M.G. Colazoc
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Department of Animal Science, Sari Agricultural Sciences and Natural Resources University,
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P.O. Box 578, Sari, Mazandaran, Iran
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Department of Animal Science, University of Tehran, P.O. Box 4111, Karaj, Alborz, Iran c
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Livestock Research Branch, Alberta Agriculture and Rural Development, Edmonton, AB, T6H 5T6, Canada
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*Corresponding author: Tel: +98-1133822741; Fax: +98-1133822565
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E-mail: [email protected] (Essa Dirandeh)
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Abstract The objective was to compare three timed-AI (TAI) protocols in lactating dairy cows
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during heat stress. Multiparous Holstein cows yielding (mean ± SEM) 29.4 ± 0.3 kg of milk/d
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were randomly assigned to one of three TAI protocols at 34 ± 5.1 d in milk: 1) Double-Ovsynch
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(DO; n=486), the cows received GnRH-7d-PGF2α-3d-GnRH and Ovsynch56 (GnRH-7d-
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PGF2α-56h-GnRH-16h-AI) was initiated 7 d later; 2) Presynch-GnRH-Ovsynch (PGO; n = 453),
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the cows received PGF2α-14d-PGF2α-2d-GnRH and Ovsynch56 was initiated 7 d later; and 3)
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Presynch-Ovsynch (PO; n = 435) the cows received PGF2α-14d-PGF2α and Ovsynch56 was
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initiated 12 d later. The ovulatory response to the first GnRH of Ovsynch56 was higher in DO
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(65.0%) compared to PGO (53.2%) and PO (45.5%). Luteolytic response to PGF2α of Ovsynch
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was similar among TAI protocols (90.1, 87.1, and 86.2% for DO, PGO and PO, respectively).
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Synchronization rate was greater in DO (86.2%) than in PGO (78.1%) and PO (72.1%)
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protocols. Irrespective of the TAI protocol, cows that ovulated in response to first GnRH had
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greater response to PGF2α (92.7 vs 77.1%). Mean (± SEM) diameter (mm) of ovulatory follicle
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at TAI was larger in DO (16.1 ± 0.3) than PGO (15.6 ± 0.21) and PO (15.2 ± 0.12). Cows
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subjected to DO had greater P/AI at 32 d and at 60 d after TAI (26.6 and 24.4%) compared to
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those in PGO (21.4 and 20.0%) and PO (17.2 and 15.9%). However, TAI protocol had no
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significant effect on the incidence of pregnancy loss (6.1, 6.6, and 7.4% for DO, GO and PO,
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respectively). In summary, cows in the DO protocol had a greater ovulation rate to the first
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GnRH and a higher synchronization rate, larger ovulatory follicles and greater P/AI. Of the three
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protocols used, DO yield the best reproductive performance in heat-stressed, lactating dairy
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cows.
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Keywords: Double-Ovsynch; Presynch-Ovsynch; Timed-AI protocols; Dairy cows; Heat stress
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1.
Introduction
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There are several GnRH-based protocols that increase insemination risk for dairy cattle, as they
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facilitate timed-AI (TAI) without the necessity to detect estrus [1]. The Ovsynch protocol,
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commonly used to synchronize ovulation for TAI in dairy cattle, consists of two GnRH
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treatments given 9 d apart, with prostaglandin F2α (PGF2α) given 7 d after the first GnRH [2].
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Over the past few years, the Ovsynch protocol has been modified to further improve pregnancy
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per AI (P/AI). A presynchronization strategy that involves two injections of prostaglandin F2α
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(PGF2α), each given 14 d apart, increased P/AI when Ovsynch was initiated 11 [3] or 12 d [4,5]
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after the last PGF2α of the Presynch protocol (PO). However, acyclic cows are unlikely to
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benefit from presynchronization with PGF2α as they do not have a CL. Hence, alternate
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presynchronization protocols that integrate GnRH into the Presynch have been proposed to
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optimize follicular development in acyclic cows [6]. A presynchronization protocol that
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combines a single GnRH treatment with PGF2α (PGO) decreased the percentage of acyclic cows
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over the Presynch protocol [3]. Most recently, a new presynchronization protocol that included
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PGF2α and GnRH (Double Ovsynch; DO) improved P/AI compared to the Presynch-Ovsynch
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(PO) protocol in primiparous cows (65.2 versus 45.2%) [7]. In addition, Ayres et al. [8] reported
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that DO compared to PO increased the percentage of cows with a CL at initial GnRH and
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improved most aspects of synchronization during an Ovsynch protocol.
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It is well known that lactating dairy cows inseminated during heat stress have impaired
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ovarian follicular dynamics and decreased fertility [9,10]. These negative effects on follicular
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steroidogenesis and oocyte quality were still manifest during early autumn [11,12] with fertility
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not restored until early winter [13] when temperatures had moderated. Recently, Akbarabadi et
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al. [14] reported that service per conception tended to increase during a warm season (1.95 ±
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0.14) compared to a cold season (1.62 ± 0.17) in dairy cows subjected to a PO protocol.
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Therefore, there is a need to identify new approaches to improve fertility in lactating dairy cows
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exposed to heat stress. One of those approaches could be elimination of compromised follicles
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with hormonal treatments that induce ovulation, synchronize emergence of a new follicular
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wave, and allow TAI [12,15].
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We hypothesized that cows with their estrous cycle presynchronized with GnRH and
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PGF2α (DO and PGO) have greater cyclicity and P/AI than cows with their estrous cycle
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presynchronized with only PGF2α (PO). The objectives of this study were to compare ovarian
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response, P/AI and pregnancy loss of multiparous Holstein cows subjected to three TAI
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protocols (DO, PGO or PO) during heat stress.
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2.
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2.1.
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Materials and methods
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Animals and management
This experiment was conducted at a commercial dairy farm between July and Nov 2013
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(Temperature Humidity Index [THI] = 78-84). A total of 1,374 multiparous lactating Holstein
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cows (yielding 29.4 ± 0.3 kg of milk/d) were enrolled. Cows were housed in free-stall barns with
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fans and bedded with sand. Cows received a TMR formulated for lactating dairy cows producing
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40 kg of 3.5% fat milk according to NRC [16] guidelines and had free access to water. Diets
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were fed twice daily (07:00 and 16:00) for ad libitum intake (10% of refusals on as fed basis).
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Main ingredients were silage (corn and alfalfa), grain (barley or corn), hay (alfalfa or grass), and
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mineral supplements. All cows participating in this experiment were milked thrice daily at
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approximately 8-h intervals, and monitored daily for signs of diseases. If any health issues
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occurred, animals were moved to hospital pens and appropriate treatments were performed
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(following standard treatment protocols) until total recovery.
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2.2. TAI protocols
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Weekly, a cohort of cows at 34 ± 5.1 d in milk (DIM) was randomly assigned to receive
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one of three TAI protocols: Double-Ovsynch (DO; n = 486), Presynch-GnRH-Ovsynch (PGO; n
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= 453) or Presynch-Ovsynch (PO; n = 435). The DO cows received GnRH (100 µg gonadorelin
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acetate, im; Parnell Technologies PTY. LTD., Alexandria, Australia) followed by PGF2α (500
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µg cloprostenol, im; Parnell Technologies) 7 d later and GnRH 3 d after PGF2α, then began the
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Ovsynch protocol 7 d later. The PGO cows received two injections of PGF2α 14 d apart,
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followed by GnRH 2 d after last PGF2α, then began the Ovsynch 12 d later. The PO cows
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received two injections of PGF2α 14 d apart, and then began the Ovsynch protocol 12 d later. All
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cows received the same Ovsynch protocol (so called Ovsynch56) [17] that consists of GnRH
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followed by PGF2α 7 d later and a second GnRH treatment administered 56 h after PGF2α.
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Cows were timed-inseminated 16 h after second GnRH treatment. Estrus detection was
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performed by visual observation (three times daily for at least 30 min each) from PGF2α to the
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second GnRH treatment of Ovsynch56. The following symptoms were used to characterize
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estrus: vaginal mucous discharge, bellowing, increased nervousness and activity, walking the
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fence line, swelling and reddening of the vulva, mounting other cows, or observed in standing
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estrus. Cows that showed signs of estrus were inseminated approximately 12 h after onset of
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estrus and were considered to have an early AI.
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Two professional AI technicians performed all inseminations, with semen from three
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commercially available sires equally balanced among the three experimental groups. Treatment
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protocols and activities during this study are shown (Fig. 1).
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2.3. Ultrasonographic examinations
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Ovarian examinations were performed by transrectal ultrasonography (BCF equipped with
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a 6-8 MHz linear transducer; Ultrasound Australas, Victoria, Australia) during the Ovsynch56 (at
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first GnRH injection, at PGF2α injection, at TAI, and 7 d after TAI) in all cows.
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Cycling status and proportion of cows ovulating to initial GnRH treatment were determined
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as previously described [18]. Ultrasonography at TAI was used to determine diameter of the
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ovulatory follicle and along with ultrasonography 7 d after TAI were used to determine ovulation
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to second GnRH. Ovulation was confirmed by the presence of a CL 7 d after TAI in the same
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ovary, which had the largest follicle at TAI.
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Cows with early ovulation included those with no dominant follicle ≥10 mm at TAI and
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those that were inseminated before TAI. Response to PGF2α was considered attained when the
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functional CL regressed. Synchronization rate was defined as the number of cows responding to
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PGF2α and ovulating after second GnRH over the total number of cows [19].
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Pregnancy diagnosis was performed by ultrasonography at 32 d after AI. Pregnancy was
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characterized by the presence of fluid, an embryo, and a heartbeat. Cows diagnosed pregnant at
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32 d were re-examined at 60 ± 5 d after AI to confirm pregnancy. Pregnancy loss was considered
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to have occurred when a cow was diagnosed pregnant at 32 d after TAI and not pregnant at 60 d.
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2.4. Statistical analyses
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All data were analyzed using SAS (version 9.1 for Windows; SAS Institute Inc., Cary, NC).
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Binomial data were analyzed using generalized estimating equations (GEE) of the GENMOD
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procedure, as described [20]. For analysis of pregnancy status at 32 and 60 d after TAI, the
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model included the effects of TAI protocol, response to first GnRH of Ovsynch56 and their
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interactions in all cows. For analysis of ovarian responses to Ovsynch56, the model considered
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TAI protocol, parity, and their interactions. Variables and interactions remaining in the final
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multivariable model at P < 0.05, based on the robust empirical standard errors produced by the
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GEE analysis, were considered significant. The main-effects model was assessed for first-order
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interactions, where treatments remained in the model. Probability values 0.05 were considered
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significant, whereas those between 0.051 and 0.10 were considered trends.Diameter of the
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ovulatory follicle was analyzed using PROC MIXED. Differences between means were tested by
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the PDIFF option and adjusted using the Tukey procedure. Probability values ≤ 0.05 were
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considered significant, whereas those from 0.051 to 0.1 were considered trends.
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3. Results
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Ovarian response data are presented in Table 1. More (P = 0.04) cows in the DO protocol
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had a CL at first GnRH of Ovsynch56 compared to those in PGO or PO. However, there were no
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differences among TAI protocols in the percentage of cows having a CL at the PGF2α treatment
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of the Ovsynch56. The proportion of cows that responded to the first GnRH injection of the
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Ovsynch56 was greater (P = 0.01) for DO than PGO and PO protocol, and it was greater for
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PGO than PO (P = 0.04). Regardless of TAI protocol, cows that ovulated in response to first
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GnRH had greater response to PGF2α (92.7 vs 77.1%). However, the percentage of cows with
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luteal regression after PGF2α and early ovulations did not differ among TAI protocols.
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Ovulatory response to second GnRH injection of Ovsynch56 was greater (P = 0.02) in the DO
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protocol compared to PGO and PO (Table 1).
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The proportion of cows that showed signs of estrus and were inseminated before TAI
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(Early AI) did not differ between PGO (19/453, 4.1%) and PO (15/435, 3.4%) but it was greater
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(P = 0.04) in DO (34/486, 7.0%). Mean (± SEM) diameter (mm) of ovulatory follicle at the time
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of TAI was larger (P = 0.04) in DO (16.1 ± 0.3) than PGO (15.6 ± 0.21) and PO (15.2 ± 0.12)
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groups, but did not differ (P = 0.38) between PGO and PO (Table 1).
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Timed-AI protocol affected the percentage of cows diagnosed pregnant at 32 and 60 d after
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AI (Table 2). A greater (P = 0.02) percentage of DO cows were pregnant at 32 d after AI
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compared to PGO and PO cows. The proportion of pregnant cows was also greater (P = 0.03) for
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PGO than PO at 32 d after AI. At second pregnancy exam (60 ± 5 d after AI), P/AI was greater
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(P = 0.03) for DO than for PGO and PO protocols. Proportion of pregnant cows was also greater
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(P = 0.04) for PGO than PO at second pregnancy exam. However, the risk of pregnancy loss
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between 32 and 60 d after AI did not differ among TAI protocols.
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When only synchronized cows (n = 1092) were considered, a greater P/AI at 32 and 60 d
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after AI was still observed in DO cows compared to PGO and PO cows. Among cows that
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responded to first GnRH injection of Ovsynch56, P/AI at 32 d after AI was greatest in DO group
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compared to PGO and PO groups (P = 0.03, Fig 2).
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4.
Discussion
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The use of exogenous hormones to synchronize emergence of a new follicular wave and
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allow TAI has been proposed as one approach to reduce heat stress infertility [12]. The current
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study compared ovarian response, P/AI and pregnancy loss of multiparous Holstein cows
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subjected to three TAI protocols during heat stress. Pregnancy per AI diagnosed at 32 and 60 d
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after AI was greater in cows subjected to DO than in cows subjected to PGO or PO protocols,
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but pregnancy loss did not differ among TAI protocols. Similarly, Souza et al. [7] compared DO
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versus PO at first TAI and reported that DO resulted in a higher P/AI in primiparous (65.2 vs
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45.2%, P < 0.05) but not multiparous (37.5 vs 39.3%) cows. Astiz and Fargas, [21] also reported
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higher P/AI in primiparous cows subjected to DO than that in those subjected to G6G (i.e.
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PGF2α-2d-GnRH and the Ovsynch56 is initiated 6 d later). The success of ovulation
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synchronization protocols has been related to ovulatory response to first GnRH injection of
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Ovsynch [22-24] and progesterone concentrations prior to PGF2α injection of Ovsynch [25-28].
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The greatest P/AI at 32 d occurred in DO cows that ovulated to first GnRH injection of Ovsynch.
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Therefore, the overall greater P/AI obtained in cows in the DO protocol may be attributed to a
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better ovarian response to treatments. In fact, a greater proportion of cows in the DO group
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ovulated following first GnRH of Ovysnch56. In addition, more cows in this group responded to
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PGF2α treatment and ovulated after second GnRH.
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The impetus for incorporating an injection of GnRH to the PO protocol used in this study
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was to induce ovulation, synchronize a new follicular wave and induce cyclicity in anovular
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cows. Hence, cows that ovulated to this injection of GnRH would have had a large dominant
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follicle more likely to respond to first GnRH of Ovsynch56. Indeed, addition of a GnRH
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injection before first GnRH of Heatsynch (a modification of the 7-d Ovsynch protocol in which
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the second GnRH treatment is replaced by estradiol) in cows presynchronized with PGF2α
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decreased the percentage of anovular cows, but did not significantly increase P/AI [3]. In the
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present study, cows subjected to the PGO protocol had improved ovarian response and better
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P/AI than those subjected to PO. Although, we expected that incorporation of GnRH into a PO
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protocol would improve P/AI in Holstein cows during heat stress, this improvement was below
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that attained in the DO protocol.
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In a previous study, pregnancy outcome to Ovsynch was highly associated with the
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ovulatory response to initial GnRH treatment [23]. Similarly, Chebel et al. [24] reported that
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P/AI was 10.0% greater in cows responding to first GnRH compared to those that did not
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respond. A lack of follicle turnover due to failure to respond to the initial GnRH administration
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might compromise the quality of embryos and consequently reduce P/AI. Cerri et al. [29]
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demonstrated that embryo quality but not fertilization rate was compromised when follicle
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turnover occurred in a very low percentage of cow subjected to a TAI protocol. Embryos from
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those cows had fewer blastomeres and a lower proportion of live blastomeres [29]. In the present
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study, the difference in P/AI by ovulatory response to first GnRH depended on the TAI protocol.
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In this regard, the difference in P/AI between cows that ovulated versus those that did not
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ovulate was similar to that reported by Chebel et al. [24] and was significantly different in cows
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subjected to DO, but smaller and not significant in cows subjected to PGO or PO.
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Cows with greater plasma progesterone concentrations at first GnRH injection of Ovsynch56
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were more fertile [24,30], which was associated with decreased LH pulsatility and improved
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oocyte competence [31]. Conversely, lower progesterone concentrations at intitiation of Ovsynch
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in presynchronized cows resulted in lower P/AI after first postpartum AI [32]. Although
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progesterone concentrations were not determined in this study, we speculated that more cows in
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DO had elevated circulating progesterone concentrations during Ovsynch, as more cows in this
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group had a CL at initiation of TAI protocol and ovulated following first GnRH. It is well known
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that anovular cows at the first postpartum AI have reduced P/AI [33] and increased late
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embryonic mortality [28]. In that regard, Herlihy et al. [26] reported that a DO protocol was
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effective at inducing cyclicity in most anovular cows and improved P/AI at the first postpartum
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service compared to PO (46.3 vs 38.2%). In our study, the incidence of cows without a CL at
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first GnRH of Ovsynch was 11.5, 19.9 and 23.0% for DO, PGO and PO respectively consistent
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with the suggestion that DO is effective in inducing cyclicity in anovular cows. Although the
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majority of anovular cows ovulate after initial GnRH of Ovsynch and form a CL [17,34], low
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progesterone concentrations during development of the ovulatory follicle appear to contribute to
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reduced fertility in anovular cows [32]. This might be exacerbated in heat-stressed cows as some
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of the reproductive losses during summer are associated with decreased expression of estrus and
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reduced luteal progesterone secretion during the estrous cycle [35-37]. Fan cooling of lactating
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dairy cows during summer increased estrous response rates and area under the luteal phase curve
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was significantly higher than for controls [35]. In lactating, cyclic Holstein cows the
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concentration of circulating progesterone during the estrous cycle was lower during summer
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compared to spring or winter [36,37]. Therefore, in addition to better synchronization, it seems
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likely that the DO protocol increased circulating progesterone concentrations prior to
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administration of PGF2α and subsequently improved P/AI. Indeed, Ayres et al. [14] reported
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that presynchronization with DO compared to PO increased the percentage of cows with high
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(>3.0 ng/mL) circulating P4 at PGF2α of Ovsynch (88.0 vs 76.3%).
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In synchronized cows, P/AI was also greater in DO cows; hence, another potential reason
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for the improved P/AI in this group was ovulation of larger follicles compared to those in cows
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in the PGO and PO groups. Premature ovulation of a dominant follicle with GnRH reduced
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ovulatory follicle size and decreased fertility in both beef [38,39] and dairy cattle [23,40].
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Wiltbank et al. [41] suggested that lower fertility in cows with a small ovulatory follicle was
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associated with reduced serum estradiol concentrations before AI and ovulation of a less mature
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oocyte. Reducing the size of the ovulatory follicle also reduced subsequent CL volume and
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serum P4 concentrations [40]. Plasma estradiol concentration is already reduced during heat
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stress; combined with ovulation of small follicle, this would be expected to further compromise
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oocyte quality and postovulatory CL function.
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In conclusion, results from this study supported use of a DO protocol to increase P/AI in
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lactating dairy cows during heat stress. Cows subjected to the DO protocol had a greater
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ovulation rate to first GnRH of Ovsynch, improved overall synchronization rate, ovulated larger
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follicles and had greater P/AI. Although P/AI was below what would normally be attained during
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a cold season, DO can be recommended as a reproductive management tool to maximize fertility
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in lactating dairy cows during heat stress.
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Acknowledgments
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The authors thank Dr. John Kastelic (University of Calgary) for critical review of the
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manuscript. Our gratitude is also extended to the owners and personnel of Mahdasht Meat &
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Milk Company (Northern Iran) for allowing us access to their cows and facilities to conduct this
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research and for covering the cost of hormonal treatments.
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insemination (AI) during Ovsynch affects pregnancies per AI in lactating dairy cows. J Dairy Sci
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[18] Colazo MG, Gordon MB, Rajamahendran R, Mapletoft RJ, Ambrose DJ. Pregnancy rates to
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timed-AI in dairy cows treated with gonadotropin releasing hormone or porcine luteinizing
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hormone. Theriogenology 2009;72:262–70.
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[19] Yilmazbas-Mecitoglu G, Karakaya E, Keskin A, Alkan A, Gumen A. Reducing the duration
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between gonadotropin-releasing hormone (GnRH) and prostaglandin F2α treatment in the
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Ovsynch protocol to 6 days improved ovulation to second GnRH treatment, but inclined to reduce
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fertility. J Dairy Sci 2013;96:3817–24.
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[20] Colazo MG, Ponce-Barajas P, Ambrose DJ. Pregnancy per artificial insemination in lactating
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dairy cows subjected to 2 different intervals from presynchronization to initiation of Ovsynch
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protocol. J Dairy Sci 2013;96:7640–8.
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[21] Astiz S, Fargas O. Pregnancy per AI differences between primiparous and multiparous high-
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yield dairy cows after using Double Ovsynch or G6G synchronization protocols. Theriogenology
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2013;79:1065–70.
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[22] Vasconcelos JL, Silcox RW, Rosa GJ, Pursley JR, Wiltbank MC. Synchronization rate, size
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of the ovulatory follicle, and pregnancy rate after synchronization of ovulation beginning on
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different days of the estrous cycle in lactating dairy cows. Theriogenology1999;52:1067–78.
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[23] Bello NM, Steibel JP, Pursley JR. Optimizing ovulation to first GnRH improved outcomes to
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each hormonal injection of Ovsynch in lactating dairy cows. J Dairy Sci 2006;89:3413–24.
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2006;89:4205–19.
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[25] Martins JPN, Policelli RK, Neuder LM, Raphael W, Pursley JR. Effects of cloprostenol
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sodium at final prostaglandin F2α of Ovsynch on complete luteolysis and pregnancy per artificial
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insemination in lactating dairy cows. J Dairy Sci 2011;94:2815–24.
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[26] Herlihy MM, Giordano JO, Souza AH, Ayres H, Ferreira RM, Keskin A, Nascimento AB,
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Guenther JN, Gaska JM, Kacuba SJ, Crowe MA, Butler ST, Wiltbank MC. Presynchronization
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with Double-Ovsynch improves fertility at first postpartum artificial insemination in lactating
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dairy cows. J Dairy Sci 2012;95:7003–14.
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[27] Giordano JO, Wiltbank MC, Guenther JN, Pawlisch R, Bas S, Cunha AP, Fricke PM.
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Increased fertility in lactating dairy cows resynchronized with Double-Ovsynch compared with
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Ovsynch initiated 32 d after timed artificial insemination. J Dairy Sci 2012;95:639–53.
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[28] Colazo MG, Dourey A, Rajamahendran R, Ambrose DJ. Progesterone supplementation
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before timed AI increased ovulation synchrony and pregnancy per AI, and supplementation after
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timed AI reduced pregnancy losses in lactating dairy cows. Theriogenology 2013;79:833–41.
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[29] Cerri RL, Rutigliano HM, Chebel RC, Santos JE. Period of dominance of the ovulatory
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follicle influences embryo quality in lactating dairy cows. Reproduction 2009;137:813–23.
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[30] Stevenson JS, Pulley SL.Characteristics and retention of luteal structures, extended
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postinsemination cycle, progesterone, and pregnancy-specific protein B in serum after human
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chorionic gonadotropin treatment of dairy cows. J Dairy Sci 2012;95:4396–409.
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[31] Mihm M, Baguisi A, Poland MP, Roche JF. Association between the duration of dominance
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of the ovulatory follicle and pregnancy rate in beef heifers. J Reprod Fertil 1994;102:123–30.
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[32] Bisinotto RS, Chebel RC, Santos JEP. Follicular wave of the ovulatory follicle and not cyclic
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status influences fertility of dairy cows. J Dairy Sci 2010;93:3578–87.
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[33] Santos JEP, Rutigliano HM, SáFilho MF. Risk factors for resumption of postpartum estrous
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cycles and embryonic survival in lactating dairy cows. Anim Reprod Sci 2009;110:207–21.
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[34] Gümen A, Guenther JN, Wiltbank MC. Follicular size and response to Ovsynch versus
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detection of estrus in anovular and ovular lactating dairy cows. J Dairy Sci 2003;86:3184-94.
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[35] Younas M, Fuquay J W, Smith A E, Moore A B. Estrous and endocrine responses of lactating
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Holsteins to forced ventilation during summer. J Dairy Sci 1993;76:430-36.
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[36] Rosenberg M, Herz Z, Davidson M, Folman Y. Seasonal variations in post-partum plasma
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progesterone levels and conception in primiparous and multiparous dairy cows. J Reprod Fertil
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1977; 51:363-67.
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[37] Howell JL , Fuquay JW, Smith AE. Corpus luteum growth and function in lactating Holstein
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cows during spring and summer. J Dairy Sci 1994;77:735-39.
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[38] Perry GA, Smith MF, Lucy MC, Green JA, Parks TE, MacNeil MD, Roberts AJ, Geary TW.
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Relationship between follicular size at insemination and pregnancy success. Proc Natl Acad Sci
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USA 2005;102:5268–73.
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[39] Mussard ML, Burke CR, Behlke EJ, Gasser CL, Day ML. Influence of premature induction
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of a luteinizing hormone surge with gonadotropin-releasing hormone on ovulation, luteal function,
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and fertility in cattle. J Anim Sci 2007;85:937–43.
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[40] Vasconcelos JLM, Sartori R, Oliveira HN, Guenther JG, Wiltbank MC. Reduction in size of
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the ovulatory follicle reduces subsequent luteal size and pregnancy rate. Theriogenology
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2001;56:307–14.
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[41] Wiltbank MC, Sartori R, Herlihy MM, Vasconcelos JLM, Nascimento AB, Souza AH, Ayres
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H, Cunha AP, Keskin A, Guenther JN, Gumen A. Managing the dominant follicle in lactating
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dairy cows. Theriogenology 2011;76:1568–82.
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Table 1. Number and percentage of lactating Holstein cows that had a CL at first GnRH and PGF2α, ovulated after first GnRH, ovulated before timed-AI (TAI), responded to PGF2α treatment, and ovulated after AI in the three TAI protocols. TAI protocols PGO
486
453
Cows with a CL at first GnRH of Ovsynch56, % (no.)
88.5a (430)
80.1b (363)
Cows with a CL at PGF2α of Ovsynch56, % (no.)
96.2 (468)
94.7 (429)
Cows that ovulated to first GnRH of Ovsynch56, % (no.)1
65.0a (316)
Cows that had luteal regression after PGF2α of Ovsynch56, % (no.)2 Cows with early ovulation, % (no.)3 Cows that ovulated after AI, % (no.)4
P-Value
435
0.04
92.6 (403)
0.60
53.2b (241)
45.5c (198)
0.01
93.5 (438)
87.1 (395)
86.2 (375)
0.71
8.2 (40) 87.2a (424)
7.7 (35) 78.1b (354)
7.6 (33) 72.1c (314)
0.83
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77.0b (335)
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TAI protocols: DO = Double-Ovsynch; PGO = Presynch-GnRH-Ovsynch; PO = Presynch-Ovsynch 1 Ovulation was determined retrospectively by recording the location, number, and size of follicles and CL at first GnRH, and comparing those data to ovarian structures on the day of PGF2α of Ovsynch56. 2 Response to PGF2α was considered attained when the CL diameter decreased from PGF2α treatment to a diameter of 10 mm or less at TAI. 3 Cows with early ovulation included those with no dominant follicle ≥ 10 mm at TAI and those that were inseminated before TAI 4 Ovulation was confirmed by the presence of a CL 7 d after TAI in the same ovary, which had the largest follicle at TAI
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Table 2. Effect of timed-AI (TAI) protocols on pregnancy per AI (P/AI) and pregnancy loss in all (n=1374) and synchronized (n=1092) lactating Holstein cows.
TAI protocols
486
453
P/AI at 32 d after AI, % (no.)
23.2 (113) a
16.7 (76) b
[% (no. / total no.)] P/AI at 60 d after AI, % (no.)
21.6 (105) a
15.6 (71) b
[% (no. / total Pregnancy lossno.)] between 32 and 60 d after AI, % (no. not pregnant at 60 d/ no. pregnant at 32 d)
6.1 (8/113)
6.6 (5/76)
No. synchronized cows1
424
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PO
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PGO
435
12.4 (54) c
0.02
11.4 (50) c
0.03
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DO
7.4 (4/54)
354
314
0.67
P/AI at 32 d after AI, % (no.)
26.6 (113) a
21.4 (76) b
17.2 (54) c
0.03
[% (no. / total no.)] P/AI at 60 d after AI, % (no.)
24.4 (105) a
20.0 (71) b
15.9 (50) c
0.04
TAI(no. protocols: = Double-Ovsynch; PGO = Presynch-GnRH-Ovsynch; PO = Presynch-Ovsynch [% / totalDO no.)]
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A cow was considered synchronized if she responded to PGF2α and ovulated after second GnRH of Ovsynch56
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3d
7d
DO GnRH
PGF2α
PGF2α 14 d
2d
PGF2α 7d U/S
GnRH
PGF2α 7d
U/S
14 d
12 d
PO
PGF2α
U/S
TAI
16 h
56 h
7d
U/S
U/S
GnRH
U/S
GnRH
PGF2α
PGF2α
7d
U/S
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56 h
U/S
7d
GnRH
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7d
GnRH
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GnRH
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GnRH
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U/S
GnRH 16 h
56 h
TAI 7d
U/S
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Fig. 1. Diagram of activities and treatments during the study. Lactating Holstein cows (n=1374) were randomly assigned to one of three timed-AI protocols; Double-Ovsynch (DO), PresynchGnRH-Ovsynch (PGO) or Presynch-Ovsynch (PO). Mean ± SEM days in milk at initiation of the protocol were 38 ± 3, 32 ± 2, and 29 ± 2 d for DO, PGO, and PO, respectively. Transrectal ultrasonography (U/S) was performed during the Ovsynch56 to determine ovarian response to treatments.
U/S
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40 33.7 a
24.2 ab 21.8 ab
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18.2 b
18.1 b 16.2 b
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Fig. 2. Effect of timed-AI (TAI) protocol and ovulatory response to first GnRH of Ovsynch56 on pregnancy per AI (P/AI) at 32 d in synchronized cows (n=1092). A cow was considered synchronized if she responded to PGF2α and ovulated after second GnRH of Ovsynch56. Open bars represent cows that ovulated after first GnRH of Ovsynch56; solid bars represent cows that failed to ovulate after first GnRH of Ovsynch56. TAI protocols: DO = Double-Ovsynch; PGO = Presynch-GnRH-Ovsynch; PO = Presynch-Ovsynch. a,b Bars with different superscript letters differ (P=0.03).
S0093-691X(14)00554-8
DOI:
10.1016/j.theriogenology.2014.10.011
Reference:
THE 12953
To appear in:
Theriogenology
Received Date: 5 August 2014 Revised Date:
5 October 2014
Accepted Date: 6 October 2014
Please cite this article as: Dirandeh E, Roodbari AR, Colazo MG, Double-Ovsynch, compared to Presynch with or without GnRH, improves fertility in heat-stressed lactating dairy cows, Theriogenology (2014), doi: 10.1016/j.theriogenology.2014.10.011. 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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Revised 1
Double-Ovsynch, compared to Presynch with or without GnRH, improves fertility in
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heat-stressed lactating dairy cows
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E. Dirandeha*, A. Rezaei Roodbarib, and M.G. Colazoc
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Department of Animal Science, Sari Agricultural Sciences and Natural Resources University,
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P.O. Box 578, Sari, Mazandaran, Iran
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Department of Animal Science, University of Tehran, P.O. Box 4111, Karaj, Alborz, Iran c
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Livestock Research Branch, Alberta Agriculture and Rural Development, Edmonton, AB, T6H 5T6, Canada
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*Corresponding author: Tel: +98-1133822741; Fax: +98-1133822565
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E-mail: [email protected] (Essa Dirandeh)
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Abstract The objective was to compare three timed-AI (TAI) protocols in lactating dairy cows
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during heat stress. Multiparous Holstein cows yielding (mean ± SEM) 29.4 ± 0.3 kg of milk/d
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were randomly assigned to one of three TAI protocols at 34 ± 5.1 d in milk: 1) Double-Ovsynch
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(DO; n=486), the cows received GnRH-7d-PGF2α-3d-GnRH and Ovsynch56 (GnRH-7d-
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PGF2α-56h-GnRH-16h-AI) was initiated 7 d later; 2) Presynch-GnRH-Ovsynch (PGO; n = 453),
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the cows received PGF2α-14d-PGF2α-2d-GnRH and Ovsynch56 was initiated 7 d later; and 3)
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Presynch-Ovsynch (PO; n = 435) the cows received PGF2α-14d-PGF2α and Ovsynch56 was
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initiated 12 d later. The ovulatory response to the first GnRH of Ovsynch56 was higher in DO
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(65.0%) compared to PGO (53.2%) and PO (45.5%). Luteolytic response to PGF2α of Ovsynch
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was similar among TAI protocols (90.1, 87.1, and 86.2% for DO, PGO and PO, respectively).
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Synchronization rate was greater in DO (86.2%) than in PGO (78.1%) and PO (72.1%)
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protocols. Irrespective of the TAI protocol, cows that ovulated in response to first GnRH had
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greater response to PGF2α (92.7 vs 77.1%). Mean (± SEM) diameter (mm) of ovulatory follicle
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at TAI was larger in DO (16.1 ± 0.3) than PGO (15.6 ± 0.21) and PO (15.2 ± 0.12). Cows
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subjected to DO had greater P/AI at 32 d and at 60 d after TAI (26.6 and 24.4%) compared to
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those in PGO (21.4 and 20.0%) and PO (17.2 and 15.9%). However, TAI protocol had no
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significant effect on the incidence of pregnancy loss (6.1, 6.6, and 7.4% for DO, GO and PO,
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respectively). In summary, cows in the DO protocol had a greater ovulation rate to the first
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GnRH and a higher synchronization rate, larger ovulatory follicles and greater P/AI. Of the three
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protocols used, DO yield the best reproductive performance in heat-stressed, lactating dairy
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cows.
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Keywords: Double-Ovsynch; Presynch-Ovsynch; Timed-AI protocols; Dairy cows; Heat stress
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1.
Introduction
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There are several GnRH-based protocols that increase insemination risk for dairy cattle, as they
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facilitate timed-AI (TAI) without the necessity to detect estrus [1]. The Ovsynch protocol,
47
commonly used to synchronize ovulation for TAI in dairy cattle, consists of two GnRH
48
treatments given 9 d apart, with prostaglandin F2α (PGF2α) given 7 d after the first GnRH [2].
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Over the past few years, the Ovsynch protocol has been modified to further improve pregnancy
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per AI (P/AI). A presynchronization strategy that involves two injections of prostaglandin F2α
51
(PGF2α), each given 14 d apart, increased P/AI when Ovsynch was initiated 11 [3] or 12 d [4,5]
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after the last PGF2α of the Presynch protocol (PO). However, acyclic cows are unlikely to
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benefit from presynchronization with PGF2α as they do not have a CL. Hence, alternate
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presynchronization protocols that integrate GnRH into the Presynch have been proposed to
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optimize follicular development in acyclic cows [6]. A presynchronization protocol that
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combines a single GnRH treatment with PGF2α (PGO) decreased the percentage of acyclic cows
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over the Presynch protocol [3]. Most recently, a new presynchronization protocol that included
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PGF2α and GnRH (Double Ovsynch; DO) improved P/AI compared to the Presynch-Ovsynch
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(PO) protocol in primiparous cows (65.2 versus 45.2%) [7]. In addition, Ayres et al. [8] reported
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that DO compared to PO increased the percentage of cows with a CL at initial GnRH and
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improved most aspects of synchronization during an Ovsynch protocol.
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It is well known that lactating dairy cows inseminated during heat stress have impaired
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ovarian follicular dynamics and decreased fertility [9,10]. These negative effects on follicular
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steroidogenesis and oocyte quality were still manifest during early autumn [11,12] with fertility
65
not restored until early winter [13] when temperatures had moderated. Recently, Akbarabadi et
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al. [14] reported that service per conception tended to increase during a warm season (1.95 ±
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0.14) compared to a cold season (1.62 ± 0.17) in dairy cows subjected to a PO protocol.
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Therefore, there is a need to identify new approaches to improve fertility in lactating dairy cows
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exposed to heat stress. One of those approaches could be elimination of compromised follicles
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with hormonal treatments that induce ovulation, synchronize emergence of a new follicular
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wave, and allow TAI [12,15].
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We hypothesized that cows with their estrous cycle presynchronized with GnRH and
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PGF2α (DO and PGO) have greater cyclicity and P/AI than cows with their estrous cycle
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presynchronized with only PGF2α (PO). The objectives of this study were to compare ovarian
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response, P/AI and pregnancy loss of multiparous Holstein cows subjected to three TAI
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protocols (DO, PGO or PO) during heat stress.
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2.
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2.1.
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Materials and methods
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Animals and management
This experiment was conducted at a commercial dairy farm between July and Nov 2013
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(Temperature Humidity Index [THI] = 78-84). A total of 1,374 multiparous lactating Holstein
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cows (yielding 29.4 ± 0.3 kg of milk/d) were enrolled. Cows were housed in free-stall barns with
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fans and bedded with sand. Cows received a TMR formulated for lactating dairy cows producing
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40 kg of 3.5% fat milk according to NRC [16] guidelines and had free access to water. Diets
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were fed twice daily (07:00 and 16:00) for ad libitum intake (10% of refusals on as fed basis).
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Main ingredients were silage (corn and alfalfa), grain (barley or corn), hay (alfalfa or grass), and
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mineral supplements. All cows participating in this experiment were milked thrice daily at
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approximately 8-h intervals, and monitored daily for signs of diseases. If any health issues
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occurred, animals were moved to hospital pens and appropriate treatments were performed
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(following standard treatment protocols) until total recovery.
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2.2. TAI protocols
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Weekly, a cohort of cows at 34 ± 5.1 d in milk (DIM) was randomly assigned to receive
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one of three TAI protocols: Double-Ovsynch (DO; n = 486), Presynch-GnRH-Ovsynch (PGO; n
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= 453) or Presynch-Ovsynch (PO; n = 435). The DO cows received GnRH (100 µg gonadorelin
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acetate, im; Parnell Technologies PTY. LTD., Alexandria, Australia) followed by PGF2α (500
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µg cloprostenol, im; Parnell Technologies) 7 d later and GnRH 3 d after PGF2α, then began the
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Ovsynch protocol 7 d later. The PGO cows received two injections of PGF2α 14 d apart,
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followed by GnRH 2 d after last PGF2α, then began the Ovsynch 12 d later. The PO cows
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received two injections of PGF2α 14 d apart, and then began the Ovsynch protocol 12 d later. All
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cows received the same Ovsynch protocol (so called Ovsynch56) [17] that consists of GnRH
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followed by PGF2α 7 d later and a second GnRH treatment administered 56 h after PGF2α.
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Cows were timed-inseminated 16 h after second GnRH treatment. Estrus detection was
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performed by visual observation (three times daily for at least 30 min each) from PGF2α to the
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second GnRH treatment of Ovsynch56. The following symptoms were used to characterize
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estrus: vaginal mucous discharge, bellowing, increased nervousness and activity, walking the
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fence line, swelling and reddening of the vulva, mounting other cows, or observed in standing
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estrus. Cows that showed signs of estrus were inseminated approximately 12 h after onset of
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estrus and were considered to have an early AI.
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Two professional AI technicians performed all inseminations, with semen from three
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commercially available sires equally balanced among the three experimental groups. Treatment
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protocols and activities during this study are shown (Fig. 1).
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2.3. Ultrasonographic examinations
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Ovarian examinations were performed by transrectal ultrasonography (BCF equipped with
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a 6-8 MHz linear transducer; Ultrasound Australas, Victoria, Australia) during the Ovsynch56 (at
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first GnRH injection, at PGF2α injection, at TAI, and 7 d after TAI) in all cows.
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Cycling status and proportion of cows ovulating to initial GnRH treatment were determined
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as previously described [18]. Ultrasonography at TAI was used to determine diameter of the
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ovulatory follicle and along with ultrasonography 7 d after TAI were used to determine ovulation
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to second GnRH. Ovulation was confirmed by the presence of a CL 7 d after TAI in the same
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ovary, which had the largest follicle at TAI.
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Cows with early ovulation included those with no dominant follicle ≥10 mm at TAI and
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those that were inseminated before TAI. Response to PGF2α was considered attained when the
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functional CL regressed. Synchronization rate was defined as the number of cows responding to
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PGF2α and ovulating after second GnRH over the total number of cows [19].
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Pregnancy diagnosis was performed by ultrasonography at 32 d after AI. Pregnancy was
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characterized by the presence of fluid, an embryo, and a heartbeat. Cows diagnosed pregnant at
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32 d were re-examined at 60 ± 5 d after AI to confirm pregnancy. Pregnancy loss was considered
133
to have occurred when a cow was diagnosed pregnant at 32 d after TAI and not pregnant at 60 d.
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2.4. Statistical analyses
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All data were analyzed using SAS (version 9.1 for Windows; SAS Institute Inc., Cary, NC).
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Binomial data were analyzed using generalized estimating equations (GEE) of the GENMOD
139
procedure, as described [20]. For analysis of pregnancy status at 32 and 60 d after TAI, the
140
model included the effects of TAI protocol, response to first GnRH of Ovsynch56 and their
141
interactions in all cows. For analysis of ovarian responses to Ovsynch56, the model considered
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TAI protocol, parity, and their interactions. Variables and interactions remaining in the final
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multivariable model at P < 0.05, based on the robust empirical standard errors produced by the
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GEE analysis, were considered significant. The main-effects model was assessed for first-order
145
interactions, where treatments remained in the model. Probability values 0.05 were considered
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significant, whereas those between 0.051 and 0.10 were considered trends.Diameter of the
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ovulatory follicle was analyzed using PROC MIXED. Differences between means were tested by
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the PDIFF option and adjusted using the Tukey procedure. Probability values ≤ 0.05 were
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considered significant, whereas those from 0.051 to 0.1 were considered trends.
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3. Results
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Ovarian response data are presented in Table 1. More (P = 0.04) cows in the DO protocol
154
had a CL at first GnRH of Ovsynch56 compared to those in PGO or PO. However, there were no
155
differences among TAI protocols in the percentage of cows having a CL at the PGF2α treatment
156
of the Ovsynch56. The proportion of cows that responded to the first GnRH injection of the
157
Ovsynch56 was greater (P = 0.01) for DO than PGO and PO protocol, and it was greater for
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PGO than PO (P = 0.04). Regardless of TAI protocol, cows that ovulated in response to first
159
GnRH had greater response to PGF2α (92.7 vs 77.1%). However, the percentage of cows with
160
luteal regression after PGF2α and early ovulations did not differ among TAI protocols.
161
Ovulatory response to second GnRH injection of Ovsynch56 was greater (P = 0.02) in the DO
162
protocol compared to PGO and PO (Table 1).
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The proportion of cows that showed signs of estrus and were inseminated before TAI
164
(Early AI) did not differ between PGO (19/453, 4.1%) and PO (15/435, 3.4%) but it was greater
165
(P = 0.04) in DO (34/486, 7.0%). Mean (± SEM) diameter (mm) of ovulatory follicle at the time
166
of TAI was larger (P = 0.04) in DO (16.1 ± 0.3) than PGO (15.6 ± 0.21) and PO (15.2 ± 0.12)
167
groups, but did not differ (P = 0.38) between PGO and PO (Table 1).
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Timed-AI protocol affected the percentage of cows diagnosed pregnant at 32 and 60 d after
169
AI (Table 2). A greater (P = 0.02) percentage of DO cows were pregnant at 32 d after AI
170
compared to PGO and PO cows. The proportion of pregnant cows was also greater (P = 0.03) for
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PGO than PO at 32 d after AI. At second pregnancy exam (60 ± 5 d after AI), P/AI was greater
172
(P = 0.03) for DO than for PGO and PO protocols. Proportion of pregnant cows was also greater
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(P = 0.04) for PGO than PO at second pregnancy exam. However, the risk of pregnancy loss
174
between 32 and 60 d after AI did not differ among TAI protocols.
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When only synchronized cows (n = 1092) were considered, a greater P/AI at 32 and 60 d
176
after AI was still observed in DO cows compared to PGO and PO cows. Among cows that
177
responded to first GnRH injection of Ovsynch56, P/AI at 32 d after AI was greatest in DO group
178
compared to PGO and PO groups (P = 0.03, Fig 2).
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181
4.
Discussion
182
The use of exogenous hormones to synchronize emergence of a new follicular wave and
184
allow TAI has been proposed as one approach to reduce heat stress infertility [12]. The current
185
study compared ovarian response, P/AI and pregnancy loss of multiparous Holstein cows
186
subjected to three TAI protocols during heat stress. Pregnancy per AI diagnosed at 32 and 60 d
187
after AI was greater in cows subjected to DO than in cows subjected to PGO or PO protocols,
188
but pregnancy loss did not differ among TAI protocols. Similarly, Souza et al. [7] compared DO
189
versus PO at first TAI and reported that DO resulted in a higher P/AI in primiparous (65.2 vs
190
45.2%, P < 0.05) but not multiparous (37.5 vs 39.3%) cows. Astiz and Fargas, [21] also reported
191
higher P/AI in primiparous cows subjected to DO than that in those subjected to G6G (i.e.
192
PGF2α-2d-GnRH and the Ovsynch56 is initiated 6 d later). The success of ovulation
193
synchronization protocols has been related to ovulatory response to first GnRH injection of
194
Ovsynch [22-24] and progesterone concentrations prior to PGF2α injection of Ovsynch [25-28].
195
The greatest P/AI at 32 d occurred in DO cows that ovulated to first GnRH injection of Ovsynch.
196
Therefore, the overall greater P/AI obtained in cows in the DO protocol may be attributed to a
197
better ovarian response to treatments. In fact, a greater proportion of cows in the DO group
198
ovulated following first GnRH of Ovysnch56. In addition, more cows in this group responded to
199
PGF2α treatment and ovulated after second GnRH.
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The impetus for incorporating an injection of GnRH to the PO protocol used in this study
201
was to induce ovulation, synchronize a new follicular wave and induce cyclicity in anovular
202
cows. Hence, cows that ovulated to this injection of GnRH would have had a large dominant
203
follicle more likely to respond to first GnRH of Ovsynch56. Indeed, addition of a GnRH
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injection before first GnRH of Heatsynch (a modification of the 7-d Ovsynch protocol in which
205
the second GnRH treatment is replaced by estradiol) in cows presynchronized with PGF2α
206
decreased the percentage of anovular cows, but did not significantly increase P/AI [3]. In the
207
present study, cows subjected to the PGO protocol had improved ovarian response and better
208
P/AI than those subjected to PO. Although, we expected that incorporation of GnRH into a PO
209
protocol would improve P/AI in Holstein cows during heat stress, this improvement was below
210
that attained in the DO protocol.
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In a previous study, pregnancy outcome to Ovsynch was highly associated with the
212
ovulatory response to initial GnRH treatment [23]. Similarly, Chebel et al. [24] reported that
213
P/AI was 10.0% greater in cows responding to first GnRH compared to those that did not
214
respond. A lack of follicle turnover due to failure to respond to the initial GnRH administration
215
might compromise the quality of embryos and consequently reduce P/AI. Cerri et al. [29]
216
demonstrated that embryo quality but not fertilization rate was compromised when follicle
217
turnover occurred in a very low percentage of cow subjected to a TAI protocol. Embryos from
218
those cows had fewer blastomeres and a lower proportion of live blastomeres [29]. In the present
219
study, the difference in P/AI by ovulatory response to first GnRH depended on the TAI protocol.
220
In this regard, the difference in P/AI between cows that ovulated versus those that did not
221
ovulate was similar to that reported by Chebel et al. [24] and was significantly different in cows
222
subjected to DO, but smaller and not significant in cows subjected to PGO or PO.
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Cows with greater plasma progesterone concentrations at first GnRH injection of Ovsynch56
224
were more fertile [24,30], which was associated with decreased LH pulsatility and improved
225
oocyte competence [31]. Conversely, lower progesterone concentrations at intitiation of Ovsynch
226
in presynchronized cows resulted in lower P/AI after first postpartum AI [32]. Although
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progesterone concentrations were not determined in this study, we speculated that more cows in
228
DO had elevated circulating progesterone concentrations during Ovsynch, as more cows in this
229
group had a CL at initiation of TAI protocol and ovulated following first GnRH. It is well known
230
that anovular cows at the first postpartum AI have reduced P/AI [33] and increased late
231
embryonic mortality [28]. In that regard, Herlihy et al. [26] reported that a DO protocol was
232
effective at inducing cyclicity in most anovular cows and improved P/AI at the first postpartum
233
service compared to PO (46.3 vs 38.2%). In our study, the incidence of cows without a CL at
234
first GnRH of Ovsynch was 11.5, 19.9 and 23.0% for DO, PGO and PO respectively consistent
235
with the suggestion that DO is effective in inducing cyclicity in anovular cows. Although the
236
majority of anovular cows ovulate after initial GnRH of Ovsynch and form a CL [17,34], low
237
progesterone concentrations during development of the ovulatory follicle appear to contribute to
238
reduced fertility in anovular cows [32]. This might be exacerbated in heat-stressed cows as some
239
of the reproductive losses during summer are associated with decreased expression of estrus and
240
reduced luteal progesterone secretion during the estrous cycle [35-37]. Fan cooling of lactating
241
dairy cows during summer increased estrous response rates and area under the luteal phase curve
242
was significantly higher than for controls [35]. In lactating, cyclic Holstein cows the
243
concentration of circulating progesterone during the estrous cycle was lower during summer
244
compared to spring or winter [36,37]. Therefore, in addition to better synchronization, it seems
245
likely that the DO protocol increased circulating progesterone concentrations prior to
246
administration of PGF2α and subsequently improved P/AI. Indeed, Ayres et al. [14] reported
247
that presynchronization with DO compared to PO increased the percentage of cows with high
248
(>3.0 ng/mL) circulating P4 at PGF2α of Ovsynch (88.0 vs 76.3%).
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In synchronized cows, P/AI was also greater in DO cows; hence, another potential reason
250
for the improved P/AI in this group was ovulation of larger follicles compared to those in cows
251
in the PGO and PO groups. Premature ovulation of a dominant follicle with GnRH reduced
252
ovulatory follicle size and decreased fertility in both beef [38,39] and dairy cattle [23,40].
253
Wiltbank et al. [41] suggested that lower fertility in cows with a small ovulatory follicle was
254
associated with reduced serum estradiol concentrations before AI and ovulation of a less mature
255
oocyte. Reducing the size of the ovulatory follicle also reduced subsequent CL volume and
256
serum P4 concentrations [40]. Plasma estradiol concentration is already reduced during heat
257
stress; combined with ovulation of small follicle, this would be expected to further compromise
258
oocyte quality and postovulatory CL function.
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In conclusion, results from this study supported use of a DO protocol to increase P/AI in
260
lactating dairy cows during heat stress. Cows subjected to the DO protocol had a greater
261
ovulation rate to first GnRH of Ovsynch, improved overall synchronization rate, ovulated larger
262
follicles and had greater P/AI. Although P/AI was below what would normally be attained during
263
a cold season, DO can be recommended as a reproductive management tool to maximize fertility
264
in lactating dairy cows during heat stress.
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Acknowledgments
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The authors thank Dr. John Kastelic (University of Calgary) for critical review of the
269
manuscript. Our gratitude is also extended to the owners and personnel of Mahdasht Meat &
270
Milk Company (Northern Iran) for allowing us access to their cows and facilities to conduct this
271
research and for covering the cost of hormonal treatments.
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Ovsynch protocol to 6 days improved ovulation to second GnRH treatment, but inclined to reduce
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[25] Martins JPN, Policelli RK, Neuder LM, Raphael W, Pursley JR. Effects of cloprostenol
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[26] Herlihy MM, Giordano JO, Souza AH, Ayres H, Ferreira RM, Keskin A, Nascimento AB,
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[27] Giordano JO, Wiltbank MC, Guenther JN, Pawlisch R, Bas S, Cunha AP, Fricke PM.
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[28] Colazo MG, Dourey A, Rajamahendran R, Ambrose DJ. Progesterone supplementation
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timed AI reduced pregnancy losses in lactating dairy cows. Theriogenology 2013;79:833–41.
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[29] Cerri RL, Rutigliano HM, Chebel RC, Santos JE. Period of dominance of the ovulatory
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follicle influences embryo quality in lactating dairy cows. Reproduction 2009;137:813–23.
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[30] Stevenson JS, Pulley SL.Characteristics and retention of luteal structures, extended
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[31] Mihm M, Baguisi A, Poland MP, Roche JF. Association between the duration of dominance
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of the ovulatory follicle and pregnancy rate in beef heifers. J Reprod Fertil 1994;102:123–30.
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status influences fertility of dairy cows. J Dairy Sci 2010;93:3578–87.
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[33] Santos JEP, Rutigliano HM, SáFilho MF. Risk factors for resumption of postpartum estrous
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cycles and embryonic survival in lactating dairy cows. Anim Reprod Sci 2009;110:207–21.
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[34] Gümen A, Guenther JN, Wiltbank MC. Follicular size and response to Ovsynch versus
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detection of estrus in anovular and ovular lactating dairy cows. J Dairy Sci 2003;86:3184-94.
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[35] Younas M, Fuquay J W, Smith A E, Moore A B. Estrous and endocrine responses of lactating
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Holsteins to forced ventilation during summer. J Dairy Sci 1993;76:430-36.
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[36] Rosenberg M, Herz Z, Davidson M, Folman Y. Seasonal variations in post-partum plasma
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[37] Howell JL , Fuquay JW, Smith AE. Corpus luteum growth and function in lactating Holstein
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[38] Perry GA, Smith MF, Lucy MC, Green JA, Parks TE, MacNeil MD, Roberts AJ, Geary TW.
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Relationship between follicular size at insemination and pregnancy success. Proc Natl Acad Sci
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USA 2005;102:5268–73.
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[39] Mussard ML, Burke CR, Behlke EJ, Gasser CL, Day ML. Influence of premature induction
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[40] Vasconcelos JLM, Sartori R, Oliveira HN, Guenther JG, Wiltbank MC. Reduction in size of
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the ovulatory follicle reduces subsequent luteal size and pregnancy rate. Theriogenology
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2001;56:307–14.
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[41] Wiltbank MC, Sartori R, Herlihy MM, Vasconcelos JLM, Nascimento AB, Souza AH, Ayres
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H, Cunha AP, Keskin A, Guenther JN, Gumen A. Managing the dominant follicle in lactating
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dairy cows. Theriogenology 2011;76:1568–82.
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Table 1. Number and percentage of lactating Holstein cows that had a CL at first GnRH and PGF2α, ovulated after first GnRH, ovulated before timed-AI (TAI), responded to PGF2α treatment, and ovulated after AI in the three TAI protocols. TAI protocols PGO
486
453
Cows with a CL at first GnRH of Ovsynch56, % (no.)
88.5a (430)
80.1b (363)
Cows with a CL at PGF2α of Ovsynch56, % (no.)
96.2 (468)
94.7 (429)
Cows that ovulated to first GnRH of Ovsynch56, % (no.)1
65.0a (316)
Cows that had luteal regression after PGF2α of Ovsynch56, % (no.)2 Cows with early ovulation, % (no.)3 Cows that ovulated after AI, % (no.)4
P-Value
435
0.04
92.6 (403)
0.60
53.2b (241)
45.5c (198)
0.01
93.5 (438)
87.1 (395)
86.2 (375)
0.71
8.2 (40) 87.2a (424)
7.7 (35) 78.1b (354)
7.6 (33) 72.1c (314)
0.83
SC
77.0b (335)
M AN U
Total no. cows
PO
RI PT
DO
0.02
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TAI protocols: DO = Double-Ovsynch; PGO = Presynch-GnRH-Ovsynch; PO = Presynch-Ovsynch 1 Ovulation was determined retrospectively by recording the location, number, and size of follicles and CL at first GnRH, and comparing those data to ovarian structures on the day of PGF2α of Ovsynch56. 2 Response to PGF2α was considered attained when the CL diameter decreased from PGF2α treatment to a diameter of 10 mm or less at TAI. 3 Cows with early ovulation included those with no dominant follicle ≥ 10 mm at TAI and those that were inseminated before TAI 4 Ovulation was confirmed by the presence of a CL 7 d after TAI in the same ovary, which had the largest follicle at TAI
ACCEPTED MANUSCRIPT
Table 2. Effect of timed-AI (TAI) protocols on pregnancy per AI (P/AI) and pregnancy loss in all (n=1374) and synchronized (n=1092) lactating Holstein cows.
TAI protocols
486
453
P/AI at 32 d after AI, % (no.)
23.2 (113) a
16.7 (76) b
[% (no. / total no.)] P/AI at 60 d after AI, % (no.)
21.6 (105) a
15.6 (71) b
[% (no. / total Pregnancy lossno.)] between 32 and 60 d after AI, % (no. not pregnant at 60 d/ no. pregnant at 32 d)
6.1 (8/113)
6.6 (5/76)
No. synchronized cows1
424
M AN U
Total no, cows
PO
P-Value
RI PT
PGO
435
12.4 (54) c
0.02
11.4 (50) c
0.03
SC
DO
7.4 (4/54)
354
314
0.67
P/AI at 32 d after AI, % (no.)
26.6 (113) a
21.4 (76) b
17.2 (54) c
0.03
[% (no. / total no.)] P/AI at 60 d after AI, % (no.)
24.4 (105) a
20.0 (71) b
15.9 (50) c
0.04
TAI(no. protocols: = Double-Ovsynch; PGO = Presynch-GnRH-Ovsynch; PO = Presynch-Ovsynch [% / totalDO no.)]
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A cow was considered synchronized if she responded to PGF2α and ovulated after second GnRH of Ovsynch56
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3d
7d
DO GnRH
PGF2α
PGF2α 14 d
2d
PGF2α 7d U/S
GnRH
PGF2α 7d
U/S
14 d
12 d
PO
PGF2α
U/S
TAI
16 h
56 h
7d
U/S
U/S
GnRH
U/S
GnRH
PGF2α
PGF2α
7d
U/S
M AN U
PGO
TAI
16 h
56 h
U/S
7d
GnRH
RI PT
7d
GnRH
SC
GnRH
PGF2α
GnRH
7d
U/S
U/S
GnRH 16 h
56 h
TAI 7d
U/S
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Fig. 1. Diagram of activities and treatments during the study. Lactating Holstein cows (n=1374) were randomly assigned to one of three timed-AI protocols; Double-Ovsynch (DO), PresynchGnRH-Ovsynch (PGO) or Presynch-Ovsynch (PO). Mean ± SEM days in milk at initiation of the protocol were 38 ± 3, 32 ± 2, and 29 ± 2 d for DO, PGO, and PO, respectively. Transrectal ultrasonography (U/S) was performed during the Ovsynch56 to determine ovarian response to treatments.
U/S
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35
RI PT
40 33.7 a
24.2 ab 21.8 ab
20
18.2 b
18.1 b 16.2 b
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P/AI (%)
25
SC
30
15 10
0
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DO
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5
PGO
PO
AC C
Fig. 2. Effect of timed-AI (TAI) protocol and ovulatory response to first GnRH of Ovsynch56 on pregnancy per AI (P/AI) at 32 d in synchronized cows (n=1092). A cow was considered synchronized if she responded to PGF2α and ovulated after second GnRH of Ovsynch56. Open bars represent cows that ovulated after first GnRH of Ovsynch56; solid bars represent cows that failed to ovulate after first GnRH of Ovsynch56. TAI protocols: DO = Double-Ovsynch; PGO = Presynch-GnRH-Ovsynch; PO = Presynch-Ovsynch. a,b Bars with different superscript letters differ (P=0.03).