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Presynchronization of lactating dairy cows with PGF2α and GnRH simultaneously, 7 days before Ovsynch have similar outcomes compared to G6G
Accepted Manuscript Pre-synchronization of lactating dairy cows with PGF2α and GnRH simultaneously, 7 days prior to Ovsynch have similar outcomes compared to G6G Muhammad Rizwan Yousuf, João Paulo N. Martins, Nasim Ahmad, Kerry Nobis, J. Richard Pursley PII:
S0093-691X(16)30221-7
DOI:
10.1016/j.theriogenology.2016.05.021
Reference:
THE 13690
To appear in:
Theriogenology
Received Date: 21 January 2016 Revised Date:
19 May 2016
Accepted Date: 19 May 2016
Please cite this article as: Yousuf MR, Martins JPN, Ahmad N, Nobis K, Pursley JR, Pre-synchronization of lactating dairy cows with PGF2α and GnRH simultaneously, 7 days prior to Ovsynch have similar outcomes compared to G6G, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.05.021. 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
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Pre-synchronization of lactating dairy cows with PGF2α and GnRH simultaneously, 7
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days prior to Ovsynch have similar outcomes compared to G6G.
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Muhammad Rizwan Yousufab1, João Paulo N. Martinsa1, Nasim Ahmadb, Kerry
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Nobisc, and J. Richard Pursleya2
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a
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b
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54000 Pakistan
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c
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Authors contributed equally to this manuscript.
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Corresponding author: J. Richard Pursley, 1230 Anthony Hall, Michigan State University,
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East Lansing, MI 48824. E-mail: [email protected]
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Department of Animal Science, Michigan State University, East Lansing, MI 48824
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Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore,
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Nobis Dairy Farm, St. Johns, MI 48879
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ABSTRACT
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The overarching objective of this study was to develop an alternative strategy for first and
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greater services that will improve fertility in lactating dairy cows for dairy operations
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limited by labor or other logistical constraints that make it difficult to utilize Presynch-11,
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G6G, or Double Ovsynch. Our overall hypothesis was that simplification of a Presynch
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program through the combination of PGF2α and GnRH on the same day (PG+G), 7 d before
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the 1st GnRH of Ovsynch, would allow for similar ovulation and luteolysis rate and
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pregnancies per AI (P/AI) compared to G6G. Lactating dairy cows 58 to 64 DIM (1st
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service; n=114) and cows diagnosed not pregnant 39 d after previous AI (2nd+ service;
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n=122) were blocked by parity and service and randomly assigned to control or PG+G.
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Control cows received G6G (n=116) that consisted of PGF2α – 2d – GnRH – 6d – GnRH –
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7d – PGF2α − 56h – GnRH − 16h – AI. Treated cows (PG+G; n=121) received PGF2α and
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GnRH – 7d – GnRH – 7d – PGF2α − 56h – GnRH − 16h – AI. All cows received a 2nd
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PGF2α 24 h after the PGF2α of Ovsynch. First service cows received AI between 76 and 82
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DIM and 2nd+ service received AI 57 d after previous AI. Pregnancies/AI (n=230) were
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similar in controls compared with treated cows on d 35 (57 vs. 50%; P=0.27) and d 49 (54
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vs. 47%; P=0.33), respectively. Percent of cows ovulating following GnRH of the
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presynchronization was greater (P=0.002) for controls vs. treated (80 vs. 58%); however,
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ovulation following 1st GnRH of Ovsynch was similar (67 vs. 68%; P=0.86). Serum
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concentrations of progesterone were similar (P=0.78) at time of 1st GnRH of Ovsynch for
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control and treated cows (2.22 vs. 2.14 ng/mL). However, serum progesterone at time of
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PGF2α of Ovsynch was greater (P=0.002) for control cows compared to treated cows (5.75
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vs. 4.64 ng/mL). In summary, administering both PGF2α and GnRH on the same day, 7 d
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before the start of Ovsynch, appears to be a simple alternative that results in acceptable
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P/AI but potentially less progesterone during growth of the ovulatory follicle.
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Keywords: Pre-synchronization, Lactating dairy cows, Progesterone, PGF2α, GnRH,
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ovulation
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1. Introduction
Fertility of lactating dairy cows treated with Ovsynch was enhanced when the first
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GnRH of the program induced ovulation [1]. This injection caused ovulation in
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approximately 60% of lactating dairy cows when administered during random stages of the
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estrous cycle [1,2]. In comparison, ovulatory response was 80% or more when the first
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GnRH of Ovsynch was administered on d 6 or 7 of the estrous cycle [1,2]. Administering
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GnRH on d 6 or 7 of the estrous cycle compared to random stages resulted in greater
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percentages of cows with a new follicular wave, a new accessory corpus luteum (CL), a
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functional CL at the PGF2α of Ovsynch, and subsequent control of luteolysis [3]. In
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addition, initiating Ovsynch near d 6 or 7 of the estrous cycle increases the chances for
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pregnancy following Ovsynch [1,2]. Three pre-synchronization strategies increased the
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chances for Ovsynch to be initiated near d 6 or 7 of the estrous cycle (e.g., G6G [2,4,5],
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Double-Ovsynch [6-8] and Presynch-11 [9]) and increased pregnancy per artificial
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insemination (P/AI) compared to only Ovsynch or Presynch-14 or -12 and are now referred
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to as “fertility programs” [2,6-9].
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Although fertility programs successfully improve reproductive performance, they are
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logistically challenging due the number of injections and the possibility for compliance
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problems. Farms that are restricted to administering injections on two days of the week due
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to labor constraints are limited to strategies that limit fertility outcomes (e.g., Ovsynch [10-
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12] and Presynch-14 or -12/Ovsynch [13,14]). Creating a pre-synchronization strategy that
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can be administered on two days of the week with fertility outcomes greater that Ovsynch
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or Presynch-14/Ovsynch would be advantageous for dairy producers. Stevens et al. (1993) [15] compared the effect of the administering PGF2α and GnRH
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simultaneously vs. PGF2α with saline in lactating dairy cows on d 8 or 10 of the estrous
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cycle to determine the effect of luteolysis. Administration of PGF2α and GnRH
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simultaneously did not affect the luteolytic actions of PGF2α. Peters and Pursley (2003) [16]
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tested the effect of combining the final GnRH of Ovsynch with the PGF2α of Ovsynch.
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There was no effect on luteolysis or ovulation rate compared to administering GnRH 36 h
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after PGF2α. Yet, P/AI was decreased and there was a trend for greater percentage of short
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cycles when the final GnRH was combined with the PGF2α of Ovsynch.
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The objective of this study was to develop a presynchronization strategy for Ovsynch
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that would limit treatments of GnRH and PGF2α to two days per week that would produce
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fertility outcomes similar to that of G6G/Ovsynch. The hypothesis was combining PGF2α
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and GnRH in a presynchronization strategy one week before Ovsynch would result in
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similar luteal and follicular outcomes following Ovsynch treatments, and P/AI compared to
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G6G/Ovsynch.
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2. Materials and methods
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2.1 Cows, housing, feeding and products
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This experiment was conducted from May and August of 2013 in a commercial
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dairy farm (Nobis Dairy Farm, St. Johns, MI) that milked approximately 900 dairy cows 3
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times daily. Herd milk production during this period averaged approximately 40 kg/cow/d.
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Cows were housed in free stall barns, fed a TMR once daily, and had free access to feed
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and water. The TMR consisted of corn, wheat and alfalfa silages and corn-soybean meal-
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based concentrates formulated to meet or exceed nutrient recommendations for lactating
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dairy cows (NRC, 2001). All treatments of PGF2α (25 mg dinoprost tromethamine, Lutalyse, Zoetis) and
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GnRH (86 µg gonadorelin acetate, 2 mL of Fertagyl, Merck Animal Helath) were
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administered with single dose syringes in semimembranosus or semitendinosus muscles of
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cows by trained personnel from our laboratory. The Institutional Animal Care and Use
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Committee at Michigan State University approved all animal handling and procedures
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described in this manuscript.
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2.2 Experimental design
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Healthy Holstein lactating dairy cows between 58 and 64 DIM (1st service; n=114)
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and cows diagnosed not pregnant 39 d after previous AI (2nd and greater service; n=122)
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were blocked by parity and service and randomly assigned to control (G6G [2]; n=116) or
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treatment (PG+G; n=121; see Fig. 1). Controls were treated with PGF2α followed in 2 d
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with GnRH, then 6 d later Ovsynch [10](GnRH – 7d – PGF2α – 24 h – PGF2α – 32 h –
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GnRH - 16 h - AI) was initiated. Treatment cows received PGF2α and GnRH in different
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sites seconds apart 7 d prior to the first GnRH of Ovsynch. All cows received timed-AI 16
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h after the final GnRH of Ovsynch at 76 to 82 DIM (first service) or 57 d after previous AI
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(second or greater services). Artificial insemination was performed by three AI technicians
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blind to treatments and using commercial semen from multiple sires purchased by the farm.
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Only one sire was used to inseminate weekly cohorts of cows; therefore, treatments were
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not confounded by an effect of service sire.
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2.3 Ovarian ultrasonography, pregnancy diagnoses and blood samples Ovarian structures (follicles and corpora lutea) were mapped and measured at time
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of the pre-synchrony GnRH and 1st GnRH of Ovsynch as previously described by Martins
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et al. [17] using a MicroMaxx Sonosite ultrasound machine with a linear array transducer
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utilizing 10 MHz frequency (Sonosite Inc., Bothell, WA). Ovulation to each GnRH
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injection was determined by the disappearance of one dominant or co-dominant follicles (≥
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10 mm) previously visualized at the d of GnRH injection and the presence of a new CL 2 d
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later. Cows without a CL 2 d following the pre-synchrony GnRH but had a CL at time of 1st
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GnRH of Ovsynch were considered to have ovulated during the 5 d period before the 1st
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GnRH of Ovsynch.
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Diagnoses of pregnancy were conducted by farm veterinarians blind to treatment
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performing transrectal ultrasonography with an Ibex Pro ultrasound machine with a 5-8
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MHz linear array transducer (E.I. Medical Imaging, Loveland, CO) on d 35 and 49 after AI.
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Pregnancy was confirmed by embryo presence and heartbeat.
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Blood samples were collected by coccygeal venipuncture using vacutainer tubes
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without anticoagulant (BD Vacutainer, Franklin Lakes, NJ) on day of pre-synchronization
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PGF2α (n=235) and 2 d later (n=236) to determine cows’ cycling status and luteolytic
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response for the pre-synchronization PGF2α injection. Blood was also sampled at each
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injection of Ovsynch (1st GnRH, n=234; PGF2α, n=235; and final GnRH, n=234).
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Following collection, samples were refrigerated, and transported to the laboratory and
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maintained at 4 °C overnight. To assess circulating P4 concentrations, serum was separated
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within 24 h after collection by centrifugation at 2000 x g for 20 min at 4 °C and stored at -
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20 °C for later hormonal analyses.
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2.4 Progesterone assays and analysis of luteal function Serum concentrations of progesterone (P4) were quantified with RIA (Coat-a-
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Count, Siemens Diagnostic, Los Angeles, CA). Intra- and inter-assay CV were 7.7 and
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6.5%, respectively. Cows with serum concentrations of P4 ≥ 1.00 ng/mL at the start of
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treatments were considered to have a functional CL. Cows with complete luteolysis were
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defined as cows with serum concentrations of P4 ≥ 1.00 ng/mL (with functional CL) at time
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of PGF2α administration that decreased to < 0.50 ng/mL 2 d later. Cows with functional CL
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at the 1st GnRH of Ovsynch were defined as having at least one of the following: (1)
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ovulation to the pre-synchrony GnRH, (2) incomplete luteolysis after the pre-synchrony
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PGF2α and serum P4 at time of 1st GnRH of Ovsynch ≥ 0.5 ng/mL, or (3) ovulation between
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the 5 d period before the 1st GnRH of Ovsynch and serum P4 at time of 1st GnRH of
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Ovsynch ≥ 0.5 ng/mL. Cows with functional CL at PGF2α of Ovsynch were defined as
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having ovulation to at least one GnRH treatments prior to that, during the 5 d period before
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the 1st GnRH of Ovsynch, and no CL regression prior to PGF2α of Ovsynch. Cows with
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complete luteolysis following PGF2α of Ovsynch were characterized as cows with
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functional CL at time of PGF2α administration that decreased to < 0.50 ng/mL 2 d later.
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2.5 Statistical analyses
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Required total sample size used in the study was calculated using the z test for
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logistic regression of the G*Power software [18]. Power analysis indicated that a total of
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217 subjects were needed to detect a 15 % point difference in P/AI between the 2
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treatments using a two-tail test with α = 0.15 and β = 0.8 in a binomial distribution.
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Binomial variables were analyzed using logistic regression with a generalized linear
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mixed model implemented with the GLIMMIX procedure of SAS (Version 9.4, SAS Inst., 7
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Inc., Cary, NC). The model considered treatment (G6G or PG+G), parity (primiparous or
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multiparous) and service number (first or second and greater) as fixed effects and week as a
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random effect. Two-way interactions were only considered in the model if P < 0.20. There
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were n=6 cows that left the herd between time of AI and 1st pregnancy diagnosis.
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Continuous variables such as concentrations of P4 and ovulatory follicle size were
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analyzed using a linear mixed linear model applying the MIIXED procedure of SAS. The
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model used treatment (G6G or PG+G), parity (primiparous or multiparous) and service
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number (first or second and greater) as fixed effects and week as a random effect. Two-way
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interactions were only considered in the model if P < 0.20. Normal distribution of the
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residuals was tested with studentized residual plots for each variable. Residuals were
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considered normally distributed for all variables. All analysis used a two-tailed test and
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probabilities values were considered different when ≤ 0.05 and tendency for difference
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when ≤ 0.10.
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2.5 Verification of the randomization of cows into treatments Parity (P = 0.81) and service (P = 0.66) number were balanced between treatments.
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Mean ± SEM parity number for all cows was 2.3 ± 0.1, ranging from 1 to 6. Service
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number mean ± SEM was 2.1 ± 0.1, ranging from 1 to 7. Days in milk at AI also did not
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differ (P = 0.99) between treatments. Overall mean ± SEM for DIM at AI was 131± 4,
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ranging from 76 to 351 DIM. Therefore, treatment effects were not confounded by parity,
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service number, and DIM at AI.
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3. Results
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3.1 Effect of combining PGF2α and GnRH on initiation of a new estrous cycle prior to
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Ovsynch Administering PGF2α concurrently with GnRH (PG+G) decreased the percentage of
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cows with complete luteolysis, ovulatory response to GnRH in the 2 d period following
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treatment, and percent of cows with a new estrous cycle compared to cows administered
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PGF2α 2 d before GnRH (G6G; Table 1). A functional CL at time of treatment decreased
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ovulatory response to the GnRH in PG+G (Table 1). Ovulatory response was not different
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between G6G and PG+G for cows without a functional CL at time of treatment (Table 1).
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Of cows that did not ovulate in the 2 d period following pre-synchronization
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treatments of GnRH, there were more (P = 0.03) cows in PG+G (17/50; 34%) compared to
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G6G (1/23; 4%) that ovulated in the 5 d period prior to the 1st GnRH of Ovsynch. All cows
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that ovulated in this period had luteolysis following pre-synchronization PGF2α or no CL at
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that time. Approximately one-half of these cows had double ovulations (8/18).
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3.2 Effect of treatment on ovarian function at, and following, 1st GnRH of Ovsynch Administering PGF2α simultaneously with GnRH decreased (P = 0.03) the
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percentage of cows with functional CL at time of 1st GnRH of Ovsynch (Table 2). Serum
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concentrations of P4 were not different between treatments (Table 2 and Fig. 2). Ovulatory
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follicle diameter was greater (P = 0.002) for PG+G compared to G6G in cows with single
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dominant follicles that ovulated following the 1st GnRH of Ovsynch (Table 2). There were
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no treatment differences in ovulatory response following 1st GnRH of Ovsynch (Table 2).
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Cows that responded to the pre-synchronization treatments and were on d 6 or 7 of the
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cycle (n = 134) at 1st GnRH of Ovsynch had greater ovulatory response (81% vs. 51%; P =
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0.001) and smaller ovulatory follicles (14.6 ± 0.2 vs. 15.6 ± 0.3 mm; P = 0.008) compared
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to cows that did not have luteolysis and/or ovulation following pre-synchronization
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treatment (n = 102). Cows on d 6 of the estrous cycle (G6G) ovulated smaller follicles
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(13.8 ± 0.3 vs. 15.5 ± 0.3 mm; P < 0.001) and had lower serum P4 compared (1.76 ± 0.08
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vs. 2.37 ± 0.14 ng/mL; P < 0.001) to cows on d 7 (PG+G) of the estrous cycle.
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3.3 Effect of treatment on luteal function at time of PGF2α of Ovsynch
Percent of cows with functional CL at time of PGF2α of Ovsynch did not differ
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between treatments (Table 3). Mean serum concentrations of P4 at time of PGF2α of
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Ovsynch was greater (P = 0.002) for cows treated with G6G than cows treated with PG+G
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(Table 3) due to treatment differences in 1st service cows (Fig. 2). When treatments were
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combined, primiparous cows had greater (P = 0.003) serum P4 at PGF2α of Ovsynch
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compared to multiparous cows (5.87 ± 0.33 vs. 4.78 ± 0.22 ng/mL; respectively).
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3.4 Effect of treatment on luteolysis to PGF2α of Ovsynch There was no effect of treatment on percentage of cows with complete luteolysis
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(Table 3). Only 7 cows with functional CL at time of PGF2α of Ovsynch did not have
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complete luteolysis. Six of 7 cows had serum P4 between 0.50 and 1.00 ng/mL. A greater
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(P = 0.02) percent of multiparous cows had complete luteolysis compared to primiparous
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cows (99 % vs. 93 %; respectively).
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3.5 Effect of treatment on pregnancies per AI at 35 and 49 d post-AI
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Treatment did not affect P/AI on 35 or 49 d post-AI (Fig. 3). There was no effect of
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service number on P/AI at 35 (P = 0.67; 51% vs. 55% for first vs. greater services,
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respectively) or 49 d post-AI (P = 0.53; 49% vs. 52% for first vs. greater services, 10
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respectively). There was no effect of parity (P = 0.17) on P/AI at 35d post-AI (60% vs.
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50% for primiparous vs. multiparous, respectively). However, primiparous cows had a
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trend (P = 0.06) for higher P/AI at 49 d post-AI compared to multiparous cows (59% vs.
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46%, respectively). Percentage of cows with pregnancy losses between 35 and 49 d post-AI
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was 4% and was not affected by treatment (P = 0.69), parity (P = 0.27) or service (P =
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0.45).
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4. Discussion
The objective of this study was to develop a presynchronization strategy for
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Ovsynch that limits treatments of GnRH and PGF2α to two days per week and results in
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fertility outcomes similar to that of G6G/Ovsynch. G6G is administered on four different
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days of the week. Presynch-10/11 require three days of the week to administer. P/AI was
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not different between treatments but numbers of cows per treatment were limited resulting
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in approximately a 35% probability of a type-II error. Pregnancies per AI on d 35 and 49
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post-AI for G6G were greater (57% and 54%) than in previous studies that used G6G (35%
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to 50%) [2,4,5]. The high luteolysis rate following the final PGF2α of Ovsynch likely
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influenced P/AI in this experiment. Cows without complete luteolysis after PGF2α of
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Ovsynch have reduced chances of pregnancy [19]. The use of two PGF2α injections 1 d
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apart at final PGF2α was very effective in inducing complete luteolysis in cows treated with
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PG+G and G6G, and is in agreement with previous studies [20]. In spite of the high P/AI of
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G6G, cows treated with PG+G had similar P/AI to G6G treatment, indicating that PG+G
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could be a simpler alternative for timed-AI programs for first or greater services for farms
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that want to use the same weekdays of animal handling that are used for the Ovsynch
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protocol.
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We hypothesized that combining PGF2α and GnRH in a presynchronization strategy
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one week before Ovsynch would result in similar luteal and follicular outcomes following
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Ovsynch treatments compared to G6G/Ovsynch. Results indicated that ovulation to the 1st
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GnRH of Ovsynch were not different between G6G and PG+G. This partially supported
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our main hypothesis. The probability of a type-II error was approximately 24% since values
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are in a part of the curve with more power. Similar ovulation rates to 1st GnRH of Ovsynch
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may help to explain similar percentages of cows pregnant for G6G and PG+G. Ovulation to
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the first GnRH of Ovsynch is a major factor for the success of timed-AI programs [1].
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Previous studies demonstrated that cows that ovulated to the 1st GnRH of Ovsynch had
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higher fertility after timed-AI compared to cows that did not ovulate following this
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injection [2,21,22]. G6G [2] and other fertility programs (Presynch-10 or 11 and Double-
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Ovsynch) [6,9,23] were developed to increase percentage of cows on d 6 or 7 of the estrous
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cycle in order to optimize ovulatory response to the first GnRH of Ovsynch, increase
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circulating concentrations of P4 during the development of the ovulatory follicle and
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improve pregnancies per timed-AI. Although PG+G had similar ovulation rate compared to
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G6G, percentage of cows on d 6 or 7 at the 1st GnRH of Ovsynch was reduced for PG+G
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due to a decrease in the percentage of cows that started a new cycle following PG+G.
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responses following PG+G pre-synchronization program. The underlying mechanism that
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reduced the lutelolysis rate after the PGF2α of the pre-synchronization for cows treated with
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PG+G is not clear. In both treatments, PGF2α was administered on a random day of the
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estrous cycle. Only cows with functional CL were used to analyze percentage of cows with
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complete luteolysis. Serum P4 at time of PGF2α was not different between cows with
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complete luteolysis vs. without luteolysis, and it probably did not interfere in the 12
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effectiveness of PGF2α in contrast with a previous experiment that had lower probability of
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complete luteolysis for cows with lower P4 at time of PGF2α [19]. Interestingly, we could
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not find published data to support the possibility that the LH surge induced by the GnRH
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administration disrupted or delayed the luteolysis process in the PG+G treatment. In
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previous data [15], simultaneous administration of GnRH did not affect PGF2α-induced
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luteolysis in 16 cows on d 8 or 10 of the cycle, and all cows (16/16) reached P4 < 0.5
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ng/mL in 1 d post PGF2α. In this study, only 70% (56/79) of cows treated with PG+G
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reached P4 levels < 0.5 ng/mL 2 d post-injections, and only 2/23 had P4 levels between
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0.50 and 1.00 ng/mL 2 d post- PGF2α. Cows treated with G6G in our experiment appeared
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to have a higher luteolysis rate (89%) to what was previously reported on the first article of
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G6G (77 %, [2]) and in the last PGF2α of Ovsynch (77 %, [19]). Therefore, additional
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studies with larger number of experimental units are necessary to determine whether GnRH
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in combination with PGF2α reduces luteolysis rate or whether a Type II error influenced our
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results.
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The addition of 2 d between the administration of GnRH and PGF2α of G6G
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treatment also improved the ovulatory response of GnRH when compared to PG+G. This
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result may be attributed to the reduction of serum P4 for cows with functional CL. During
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luteolysis, serum P4 decrease induces an increase of LH pulse frequency [24], which allows
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the continuous development of a DF and avoids a DF turnover. Cows with functional CL
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treated with G6G had a greater chance to have a DF at time of GnRH of the pre-
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synchronization compared to PG+G. The lower ovulatory response to the GnRH of PG+G
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for cows with functional CL may be attributed to the reduction of the magnitude of the LH
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surge. Giordano et al. [25,26] showed that high serum P4 significantly suppressed GnRH-
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induced LH surge and reduced the magnitude of the LH surge.
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Presence of a functional CL might also have negatively affected the ovulatory
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response for the 1st GnRH of Ovsynch for cows treated with G6G since they had a greater
302
percentage of functional CL at the 1st GnRH of Ovsynch compared to PG+G. Previous
303
studies already demonstrated that the presence of a functional CL at time of GnRH
304
significantly reduced ovulatory response for 1st GnRH of Ovsynch [26-28]. The ovulation
305
rate to the 1st GnRH to Ovsynch for cows treated with G6G in the present study appeared to
306
be similar to what was previously reported for other studies that used G6G (64 % [29] and
307
69 % [5]), Double-Ovsynch (72 %, [6]), Presynch-11 (61 % [9]) and Presynch-10 (70 %
308
[30]). However, it was lower than previous studies in our laboratory using G6G in first
309
service cows (85%, [2,31]). In agreement with previous studies [1,2], cows on d 6 or 7 of
310
the estrous cycle had greater ovulation rate to the 1st GnRH of Ovsynch compared to cows
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in other times of the estrous cycle.
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Since the percentage of cows with functional CL was reduced in cows treated with
313
PG+G, we were expecting a lower serum P4 at time of 1st GnRH of Ovsynch for cows
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treated with PG+G compared to G6G; however, treatments had similar serum P4. This
315
result appeared to be due a smaller percentage of cows with complete luteolysis following
316
PG+G treatment, which might have resulted in a greater proportion of cows in mid-estrous
317
cycle at time of 1st GnRH of Ovsynch. Cows in mid estrous cycle at 1st GnRH of Ovsynch
318
had lower percentage of cows with functional CL and serum P4 at time of PGF2α of
319
Ovsynch and lower fertility following timed-AI compared to cows between d 5 and 9 of the
320
cycle [32]. In the present study, it appears that this higher percentage of cows in mid
321
estrous cycle might not have influenced the overall outcome of the PG+G program since
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percentage of cows with functional CL at time of PGF2α of Ovsynch and P/AI following
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timed-AI was similar between treatments.
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PG+G might be a good substitution for Presynch-14 or -12 [13,14]. These programs
325
use the same weekdays for injections as Ovsynch but take longer to execute. Additionally,
326
these protocols are commonly used in combination with AI after estrus detection following
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the PGF2α administration of the pre-synchronization and subsequent timed-AI for cows that
328
were not detected in estrus [33-36]. Fertility for these cows following estrus after PGF2α of
329
Presynch has been reported to be similar [33-35] or reduced [36] compared to cows that
330
only received timed-AI. On the other hand, the use of the PG+G program for first and
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subsequent inseminations with the addition to detection of estrus between PG+G pre-
332
synchrony and first GnRH of Ovsynch may be a good strategy for farms that want to limit
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the number of days in the week they are handling cows. PG+G increased the percent of
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cows that ovulated during the 5 d prior to the 1st GnRH of Ovsynch. These cows would
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likely be in early stages of follicle development (pre-deviation) at time of PG+G and may
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have normal fertility following AI after estrus detection [37].
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5. Conclusion
Results from the present experiment indicate that administering both PGF2α and
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GnRH on the same day, 7 d prior to the start of Ovsynch (PG+G) may offer a reasonable
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alternative to more complex fertility programs to enhance P/AI for first and subsequent
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services. The protocol PG+G appeared to provide sufficient ovulatory response to the first
343
GnRH of Ovsynch and concentrations of progesterone at time of PGF2α of Ovsynch. The
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increase in the percent of cows in the PG+G treatment that ovulated during the 5 d prior to
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the 1st GnRH of Ovsynch is a concern and need to be further investigated.
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A special thanks to Ken and Kerry Nobis and all the employees of Nobis Dairy
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Farms (St. Johns, MI) for allowing us the use of their cows and facilities and providing
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Fertagyl and Lutalyse used in this project. J.P. Martins was supported by The National
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Council for Scientific and Technological Development (CNPq - Brazil) fellowship –
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Science without Borders program, and M.R. Yousuf was supported by PAK-US S&T coop.
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program Phase IV.
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Table 1: Percentage of cows with functional corpus luteum (CL) at beginning of treatment, and effect of treatment (G6G vs. PG+G) on pre-synchronization outcomes. PG+G
P-value
Cows with functional CL at beginning of treatments1, % (n/n)
56 (65/117)
66 (79/119)
0.11
Cows with complete luteolysis to the PGF2α of the pre-synchronization2, % (n/n)
89 (58/65)
Ovulation to the GnRH of the pre-synchronization, % (n/n)
71 (56/79)
0.01
80 (93/116)
58 (70/120)
0.002
Cows with functional CL at beginning of treatments1, % (n/n)
95 (62/65)
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G6G (Control)
54 (43/79)
<0.001
Cows without functional CL at beginning of treatments1, % (n/n)
61 (31/51)
65 (26/40)
0.51
16.1 ± 0.4
16.2 ± 0.5
0.89
28 (26/93)
16 (11/70)
0.14
69 (80/116)
45 (54/120)
<0.001
Double ovulation to the GnRH of the presynchronization (%)
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Size of the ovulatory follicle of the GnRH of the presynchronization3, mm ± SEM
Cows with new estrous cycle following presynchronization4, % (n/n) 1
Cows with functional CL = Serum progesterone (P4) ≥ 1.00 ng/mL. Only cows with functional CL were used in this analysis. Complete luteolysis was consider when cows with functional CL at d of PGF2α injection had serum P4 < 0.5 ng/mL 2 d later. 3 Only cows with single ovulations used on calculations. 4
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Cows with a new estrous cycle = (1) cows with a functional CL at beginning of treatment that had complete luteal regression and ovulation for the PGF2α and GnRH presynchronization injections, respectively, or (2) cows without functional CL at beginning of treatment that ovulated after the GnRH injection of the pre-synchronization
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Table 2: Effect of treatment (G6G vs. PG+G) on luteal and follicular functions at first GnRH of Ovsynch and response to first GnRH of Ovsynch.
Serum P4 at the 1st GnRH of the Ovsynch, ng/mL ± SEM Ovulation to the 1st GnRH of Ovsynch, % (n/n) Size of the ovulatory follicle of the 1st GnRH of the Ovsynch2, mm ± SEM Double ovulation to the 1st GnRH of Ovsynch, % (n/n)
P-values
91 (104/115)
76 (90/119)
0.03
2.22 ± 0.18 67 (78/116) 14.3 ± 0.3
2.14 ± 0.18
0.78
68 (82/120)
0.86
15.7 ± 0.3
0.002
22 (17/78)
23 (19/82)
0.79
Cows with functional corpus luteum (CL) at the 1st GnRH of Ovsynch were defined as having at least one of the following: (1) ovulation to the pre-synchrony GnRH, (2) no complete luteolysis after the pre-synchrony PGF2α and serum progesterone (P4) at time of 1st GnRH of Ovsynch ≥ 0.5 ng/mL, or (3) ovulation between the 5 d period before the 1st GnRH of Ovsynch and serum P4 at time of 1st GnRH of Ovsynch ≥ 0.5 ng/mL. 1
2
Only cows with single ovulation.
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PG+G
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Cows with functional CL at the first GnRH of the Ovsynch 1, % (n/n)
G6G (Control)
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Table 3: Effect of treatment (G6G vs. PG+G) on luteal function at PGF2α of Ovsynch and response to PGF2α of Ovsynch. G6G PG+G P-values (Control) Cows with functional CL at the PGF2α of Ovsynch 1, % (n/n) Serum P4 at the PGF2α of Ovsynch, ng/mL ± SEM Cows with complete luteolysis after the PGF2α of Ovsynch2, % (n/n) 1
5.75 ± 0.28 96 (103/107)
93 (111/120)
0.67
4.64 ± 0.24
0.002
97 (108/111)
0.68
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Cows with functional corpus luteum (CL) at PGF2α of Ovsynch were defined as having ovulation to at least one GnRH treatment or during the 5 d period before the 1st GnRH of Ovsynch and no CL regression prior to PGF2α of Ovsynch. 2
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Only cows with functional CL were used in this analysis. Complete luteolysis was consider when cows with functional CL at d of PGF2α injection had serum progesterone (P4) < 0.5 ng/mL 2 d later.
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94 (108/115)
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Fig. 1: Schematic illustration of treatments, blood collections (B) and ultrasonography examinations (US). PG= PGF2α; US= Ultrasonography examination of ovaries; B=Blood sample collection
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389 390 391 392
Fig. 2: Effect of treatment (G6G vs. PG+G) on serum concentrations of progesterone (ng/mL) at first GnRH of Ovsynch and at PGF2α of Ovsynch for first and second or greater service cows. Interaction treatment and service at first GnRH of Ovsynch: P = 0.22 and at PGF2α of Ovsynch: P = 0.03.
8
G6G (control)
6.19 6 4
2.68
2.35
n=55
n=57
2 n=55
First GnRH of Ovsynch 393
G6G (control)
6 4
n=60
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PG+G
P = 0.46
5.35
4.93
n=60
n=62
P = 0.29
1.79 2
PGF2α of Ovsynch
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n=58
Second or greater service cows
1.94
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Serum progesterone (ng/mL)
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P = 0.50
0
n=62
First GnRH of Ovsynch
394
P < 0.001
PG+G
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First service cows
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PGF2α of Ovsynch
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Fig. 3: Effect of treatments (G6G vs. PG+G) on pregnancies per AI (P/AI) at 35 and 49 d postAI. Interaction service by treatment was not significant for 35 d post-AI (P = 0.82) and for 49 d post-AI (P = 0.76). 100 G6G (control) PG+G 90 80 P = 0.27
P/AI (%)
70
P = 0.33
60 50
57
54
50
47
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40 30
n=110
n=120
0 35 d post-AI 398
n=109
n=118
49 d post-AI
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Vasconcelos JL, Silcox RW, Rosa GJ, Pursley JR, Wiltbank MC. Synchronization rate, size of the ovulatory follicle, and pregnancy rate after synchronization of ovulation beginning on different days of the estrous cycle in lactating dairy cows. Theriogenology 1999;52:1067–78. Bello NM, Steibel JP, Pursley JR. Optimizing ovulation to first GnRH improved outcomes to each hormonal injection of ovsynch in lactating dairy cows. J Dairy Sci 2006;89:3413–24. Pursley JR, Martins JPN. Impact of circulating concentrations of progesterone and antral age of the ovulatory follicle on fertility of high-producing lactating dairy cows. Reproduction, Fertility and Development 2011;24:267–71. Astiz S, Fargas O. Pregnancy per AI differences between primiparous and multiparous high-yield dairy cows after using Double Ovsynch or G6G synchronization protocols. Theriogenology 2013;79:1065–70. Dirandeh E, Roodbari AR, Gholizadeh M, Deldar H, Masoumi R, Kazemifard M, et al. Administration of prostaglandin F2α 14 d before initiating a G6G or a G7G timed artificial insemination protocol increased circulating progesterone prior to artificial insemination and reduced pregnancy loss in multiparous Holstein cows. J Dairy Sci 2015;98:5414–21. Souza AH, Ayres H, Ferreira RM, Wiltbank MC. A new presynchronization system (Double-Ovsynch) increases fertility at first postpartum timed AI in lactating dairy cows. Theriogenology 2008;70:208–15. Ayres H, Ferreira RM, Cunha AP, Araújo RR, Wiltbank MC. Double-Ovsynch in high-producing dairy cows: effects on progesterone concentrations and ovulation to GnRH treatments. Theriogenology 2013;79:159–64. Herlihy MM, Giordano JO, Souza AH, Ayres H, Ferreira RM, Keskin A, et al. Presynchronization with Double-Ovsynch improves fertility at first postpartum artificial insemination in lactating dairy cows. J Dairy Sci 2012;95:7003–14. Galvão KN, Sá Filho MF, Santos JEP. Reducing the interval from presynchronization to initiation of timed artificial insemination improves fertility in dairy cows. J Dairy Sci 2007;90:4212–8. Pursley JR, Mee MO, Wiltbank MC. Synchronization of ovulation in dairy cows using PGF2alpha and GnRH. Theriogenology 1995;44:915–23. Pursley JR, Kosorok MR, Wiltbank MC. Reproductive management of lactating dairy cows using synchronization of ovulation. J Dairy Sci 1997;80:301–6. Pursley JR, Wiltbank MC, Stevenson JS, Ottobre JS, Garverick HA, Anderson LL. Pregnancy rates per artificial insemination for cows and heifers inseminated at a synchronized ovulation or synchronized estrus. J Dairy Sci 1997;80:295–300. Navanukraw C, Redmer DA, Reynolds LP, Kirsch JD, Grazul-Bilska AT, Fricke PM. A modified presynchronization protocol improves fertility to timed artificial insemination in lactating dairy cows. J Dairy Sci 2004;87:1551–7. Moreira F, Orlandi C, Risco CA, Mattos R, Lopes F, Thatcher WW. Effects of presynchronization and bovine somatotropin on pregnancy rates to a timed artificial insemination protocol in lactating dairy cows. J Dairy Sci 2001;84:1646–59.
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Highlights A new pre-synchronization strategy was tested for Ovsynch.
Fertility was similar in PG+G compared to G6G.
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PG and GnRH were administered simultaneously one week prior to Ovsynch.
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This strategy may be more logistically feasible for producers to utilize.
S0093-691X(16)30221-7
DOI:
10.1016/j.theriogenology.2016.05.021
Reference:
THE 13690
To appear in:
Theriogenology
Received Date: 21 January 2016 Revised Date:
19 May 2016
Accepted Date: 19 May 2016
Please cite this article as: Yousuf MR, Martins JPN, Ahmad N, Nobis K, Pursley JR, Pre-synchronization of lactating dairy cows with PGF2α and GnRH simultaneously, 7 days prior to Ovsynch have similar outcomes compared to G6G, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.05.021. 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
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Pre-synchronization of lactating dairy cows with PGF2α and GnRH simultaneously, 7
3
days prior to Ovsynch have similar outcomes compared to G6G.
4
Muhammad Rizwan Yousufab1, João Paulo N. Martinsa1, Nasim Ahmadb, Kerry
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Nobisc, and J. Richard Pursleya2
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a
7
b
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54000 Pakistan
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c
10
1
Authors contributed equally to this manuscript.
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2
Corresponding author: J. Richard Pursley, 1230 Anthony Hall, Michigan State University,
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East Lansing, MI 48824. E-mail: [email protected]
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Department of Animal Science, Michigan State University, East Lansing, MI 48824
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Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore,
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Nobis Dairy Farm, St. Johns, MI 48879
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ABSTRACT
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The overarching objective of this study was to develop an alternative strategy for first and
16
greater services that will improve fertility in lactating dairy cows for dairy operations
17
limited by labor or other logistical constraints that make it difficult to utilize Presynch-11,
18
G6G, or Double Ovsynch. Our overall hypothesis was that simplification of a Presynch
19
program through the combination of PGF2α and GnRH on the same day (PG+G), 7 d before
20
the 1st GnRH of Ovsynch, would allow for similar ovulation and luteolysis rate and
21
pregnancies per AI (P/AI) compared to G6G. Lactating dairy cows 58 to 64 DIM (1st
22
service; n=114) and cows diagnosed not pregnant 39 d after previous AI (2nd+ service;
23
n=122) were blocked by parity and service and randomly assigned to control or PG+G.
24
Control cows received G6G (n=116) that consisted of PGF2α – 2d – GnRH – 6d – GnRH –
25
7d – PGF2α − 56h – GnRH − 16h – AI. Treated cows (PG+G; n=121) received PGF2α and
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GnRH – 7d – GnRH – 7d – PGF2α − 56h – GnRH − 16h – AI. All cows received a 2nd
27
PGF2α 24 h after the PGF2α of Ovsynch. First service cows received AI between 76 and 82
28
DIM and 2nd+ service received AI 57 d after previous AI. Pregnancies/AI (n=230) were
29
similar in controls compared with treated cows on d 35 (57 vs. 50%; P=0.27) and d 49 (54
30
vs. 47%; P=0.33), respectively. Percent of cows ovulating following GnRH of the
31
presynchronization was greater (P=0.002) for controls vs. treated (80 vs. 58%); however,
32
ovulation following 1st GnRH of Ovsynch was similar (67 vs. 68%; P=0.86). Serum
33
concentrations of progesterone were similar (P=0.78) at time of 1st GnRH of Ovsynch for
34
control and treated cows (2.22 vs. 2.14 ng/mL). However, serum progesterone at time of
35
PGF2α of Ovsynch was greater (P=0.002) for control cows compared to treated cows (5.75
36
vs. 4.64 ng/mL). In summary, administering both PGF2α and GnRH on the same day, 7 d
37
before the start of Ovsynch, appears to be a simple alternative that results in acceptable
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P/AI but potentially less progesterone during growth of the ovulatory follicle.
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Keywords: Pre-synchronization, Lactating dairy cows, Progesterone, PGF2α, GnRH,
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ovulation
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41 42
1. Introduction
Fertility of lactating dairy cows treated with Ovsynch was enhanced when the first
44
GnRH of the program induced ovulation [1]. This injection caused ovulation in
45
approximately 60% of lactating dairy cows when administered during random stages of the
46
estrous cycle [1,2]. In comparison, ovulatory response was 80% or more when the first
47
GnRH of Ovsynch was administered on d 6 or 7 of the estrous cycle [1,2]. Administering
48
GnRH on d 6 or 7 of the estrous cycle compared to random stages resulted in greater
49
percentages of cows with a new follicular wave, a new accessory corpus luteum (CL), a
50
functional CL at the PGF2α of Ovsynch, and subsequent control of luteolysis [3]. In
51
addition, initiating Ovsynch near d 6 or 7 of the estrous cycle increases the chances for
52
pregnancy following Ovsynch [1,2]. Three pre-synchronization strategies increased the
53
chances for Ovsynch to be initiated near d 6 or 7 of the estrous cycle (e.g., G6G [2,4,5],
54
Double-Ovsynch [6-8] and Presynch-11 [9]) and increased pregnancy per artificial
55
insemination (P/AI) compared to only Ovsynch or Presynch-14 or -12 and are now referred
56
to as “fertility programs” [2,6-9].
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Although fertility programs successfully improve reproductive performance, they are
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logistically challenging due the number of injections and the possibility for compliance
59
problems. Farms that are restricted to administering injections on two days of the week due
60
to labor constraints are limited to strategies that limit fertility outcomes (e.g., Ovsynch [10-
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12] and Presynch-14 or -12/Ovsynch [13,14]). Creating a pre-synchronization strategy that
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can be administered on two days of the week with fertility outcomes greater that Ovsynch
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or Presynch-14/Ovsynch would be advantageous for dairy producers. Stevens et al. (1993) [15] compared the effect of the administering PGF2α and GnRH
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simultaneously vs. PGF2α with saline in lactating dairy cows on d 8 or 10 of the estrous
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cycle to determine the effect of luteolysis. Administration of PGF2α and GnRH
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simultaneously did not affect the luteolytic actions of PGF2α. Peters and Pursley (2003) [16]
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tested the effect of combining the final GnRH of Ovsynch with the PGF2α of Ovsynch.
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There was no effect on luteolysis or ovulation rate compared to administering GnRH 36 h
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after PGF2α. Yet, P/AI was decreased and there was a trend for greater percentage of short
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cycles when the final GnRH was combined with the PGF2α of Ovsynch.
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The objective of this study was to develop a presynchronization strategy for Ovsynch
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that would limit treatments of GnRH and PGF2α to two days per week that would produce
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fertility outcomes similar to that of G6G/Ovsynch. The hypothesis was combining PGF2α
75
and GnRH in a presynchronization strategy one week before Ovsynch would result in
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similar luteal and follicular outcomes following Ovsynch treatments, and P/AI compared to
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G6G/Ovsynch.
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2. Materials and methods
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2.1 Cows, housing, feeding and products
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This experiment was conducted from May and August of 2013 in a commercial
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dairy farm (Nobis Dairy Farm, St. Johns, MI) that milked approximately 900 dairy cows 3
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times daily. Herd milk production during this period averaged approximately 40 kg/cow/d.
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Cows were housed in free stall barns, fed a TMR once daily, and had free access to feed
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and water. The TMR consisted of corn, wheat and alfalfa silages and corn-soybean meal-
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based concentrates formulated to meet or exceed nutrient recommendations for lactating
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dairy cows (NRC, 2001). All treatments of PGF2α (25 mg dinoprost tromethamine, Lutalyse, Zoetis) and
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GnRH (86 µg gonadorelin acetate, 2 mL of Fertagyl, Merck Animal Helath) were
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administered with single dose syringes in semimembranosus or semitendinosus muscles of
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cows by trained personnel from our laboratory. The Institutional Animal Care and Use
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Committee at Michigan State University approved all animal handling and procedures
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described in this manuscript.
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2.2 Experimental design
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Healthy Holstein lactating dairy cows between 58 and 64 DIM (1st service; n=114)
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and cows diagnosed not pregnant 39 d after previous AI (2nd and greater service; n=122)
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were blocked by parity and service and randomly assigned to control (G6G [2]; n=116) or
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treatment (PG+G; n=121; see Fig. 1). Controls were treated with PGF2α followed in 2 d
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with GnRH, then 6 d later Ovsynch [10](GnRH – 7d – PGF2α – 24 h – PGF2α – 32 h –
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GnRH - 16 h - AI) was initiated. Treatment cows received PGF2α and GnRH in different
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sites seconds apart 7 d prior to the first GnRH of Ovsynch. All cows received timed-AI 16
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h after the final GnRH of Ovsynch at 76 to 82 DIM (first service) or 57 d after previous AI
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(second or greater services). Artificial insemination was performed by three AI technicians
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blind to treatments and using commercial semen from multiple sires purchased by the farm.
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Only one sire was used to inseminate weekly cohorts of cows; therefore, treatments were
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not confounded by an effect of service sire.
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2.3 Ovarian ultrasonography, pregnancy diagnoses and blood samples Ovarian structures (follicles and corpora lutea) were mapped and measured at time
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of the pre-synchrony GnRH and 1st GnRH of Ovsynch as previously described by Martins
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et al. [17] using a MicroMaxx Sonosite ultrasound machine with a linear array transducer
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utilizing 10 MHz frequency (Sonosite Inc., Bothell, WA). Ovulation to each GnRH
113
injection was determined by the disappearance of one dominant or co-dominant follicles (≥
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10 mm) previously visualized at the d of GnRH injection and the presence of a new CL 2 d
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later. Cows without a CL 2 d following the pre-synchrony GnRH but had a CL at time of 1st
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GnRH of Ovsynch were considered to have ovulated during the 5 d period before the 1st
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GnRH of Ovsynch.
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Diagnoses of pregnancy were conducted by farm veterinarians blind to treatment
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performing transrectal ultrasonography with an Ibex Pro ultrasound machine with a 5-8
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MHz linear array transducer (E.I. Medical Imaging, Loveland, CO) on d 35 and 49 after AI.
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Pregnancy was confirmed by embryo presence and heartbeat.
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Blood samples were collected by coccygeal venipuncture using vacutainer tubes
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without anticoagulant (BD Vacutainer, Franklin Lakes, NJ) on day of pre-synchronization
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PGF2α (n=235) and 2 d later (n=236) to determine cows’ cycling status and luteolytic
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response for the pre-synchronization PGF2α injection. Blood was also sampled at each
126
injection of Ovsynch (1st GnRH, n=234; PGF2α, n=235; and final GnRH, n=234).
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Following collection, samples were refrigerated, and transported to the laboratory and
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maintained at 4 °C overnight. To assess circulating P4 concentrations, serum was separated
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within 24 h after collection by centrifugation at 2000 x g for 20 min at 4 °C and stored at -
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20 °C for later hormonal analyses.
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2.4 Progesterone assays and analysis of luteal function Serum concentrations of progesterone (P4) were quantified with RIA (Coat-a-
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Count, Siemens Diagnostic, Los Angeles, CA). Intra- and inter-assay CV were 7.7 and
135
6.5%, respectively. Cows with serum concentrations of P4 ≥ 1.00 ng/mL at the start of
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treatments were considered to have a functional CL. Cows with complete luteolysis were
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defined as cows with serum concentrations of P4 ≥ 1.00 ng/mL (with functional CL) at time
138
of PGF2α administration that decreased to < 0.50 ng/mL 2 d later. Cows with functional CL
139
at the 1st GnRH of Ovsynch were defined as having at least one of the following: (1)
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ovulation to the pre-synchrony GnRH, (2) incomplete luteolysis after the pre-synchrony
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PGF2α and serum P4 at time of 1st GnRH of Ovsynch ≥ 0.5 ng/mL, or (3) ovulation between
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the 5 d period before the 1st GnRH of Ovsynch and serum P4 at time of 1st GnRH of
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Ovsynch ≥ 0.5 ng/mL. Cows with functional CL at PGF2α of Ovsynch were defined as
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having ovulation to at least one GnRH treatments prior to that, during the 5 d period before
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the 1st GnRH of Ovsynch, and no CL regression prior to PGF2α of Ovsynch. Cows with
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complete luteolysis following PGF2α of Ovsynch were characterized as cows with
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functional CL at time of PGF2α administration that decreased to < 0.50 ng/mL 2 d later.
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2.5 Statistical analyses
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Required total sample size used in the study was calculated using the z test for
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logistic regression of the G*Power software [18]. Power analysis indicated that a total of
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217 subjects were needed to detect a 15 % point difference in P/AI between the 2
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treatments using a two-tail test with α = 0.15 and β = 0.8 in a binomial distribution.
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Binomial variables were analyzed using logistic regression with a generalized linear
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mixed model implemented with the GLIMMIX procedure of SAS (Version 9.4, SAS Inst., 7
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Inc., Cary, NC). The model considered treatment (G6G or PG+G), parity (primiparous or
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multiparous) and service number (first or second and greater) as fixed effects and week as a
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random effect. Two-way interactions were only considered in the model if P < 0.20. There
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were n=6 cows that left the herd between time of AI and 1st pregnancy diagnosis.
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Continuous variables such as concentrations of P4 and ovulatory follicle size were
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analyzed using a linear mixed linear model applying the MIIXED procedure of SAS. The
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model used treatment (G6G or PG+G), parity (primiparous or multiparous) and service
163
number (first or second and greater) as fixed effects and week as a random effect. Two-way
164
interactions were only considered in the model if P < 0.20. Normal distribution of the
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residuals was tested with studentized residual plots for each variable. Residuals were
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considered normally distributed for all variables. All analysis used a two-tailed test and
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probabilities values were considered different when ≤ 0.05 and tendency for difference
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when ≤ 0.10.
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2.5 Verification of the randomization of cows into treatments Parity (P = 0.81) and service (P = 0.66) number were balanced between treatments.
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Mean ± SEM parity number for all cows was 2.3 ± 0.1, ranging from 1 to 6. Service
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number mean ± SEM was 2.1 ± 0.1, ranging from 1 to 7. Days in milk at AI also did not
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differ (P = 0.99) between treatments. Overall mean ± SEM for DIM at AI was 131± 4,
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ranging from 76 to 351 DIM. Therefore, treatment effects were not confounded by parity,
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service number, and DIM at AI.
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3. Results
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3.1 Effect of combining PGF2α and GnRH on initiation of a new estrous cycle prior to
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Ovsynch Administering PGF2α concurrently with GnRH (PG+G) decreased the percentage of
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cows with complete luteolysis, ovulatory response to GnRH in the 2 d period following
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treatment, and percent of cows with a new estrous cycle compared to cows administered
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PGF2α 2 d before GnRH (G6G; Table 1). A functional CL at time of treatment decreased
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ovulatory response to the GnRH in PG+G (Table 1). Ovulatory response was not different
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between G6G and PG+G for cows without a functional CL at time of treatment (Table 1).
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Of cows that did not ovulate in the 2 d period following pre-synchronization
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treatments of GnRH, there were more (P = 0.03) cows in PG+G (17/50; 34%) compared to
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G6G (1/23; 4%) that ovulated in the 5 d period prior to the 1st GnRH of Ovsynch. All cows
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that ovulated in this period had luteolysis following pre-synchronization PGF2α or no CL at
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that time. Approximately one-half of these cows had double ovulations (8/18).
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3.2 Effect of treatment on ovarian function at, and following, 1st GnRH of Ovsynch Administering PGF2α simultaneously with GnRH decreased (P = 0.03) the
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percentage of cows with functional CL at time of 1st GnRH of Ovsynch (Table 2). Serum
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concentrations of P4 were not different between treatments (Table 2 and Fig. 2). Ovulatory
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follicle diameter was greater (P = 0.002) for PG+G compared to G6G in cows with single
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dominant follicles that ovulated following the 1st GnRH of Ovsynch (Table 2). There were
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no treatment differences in ovulatory response following 1st GnRH of Ovsynch (Table 2).
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Cows that responded to the pre-synchronization treatments and were on d 6 or 7 of the
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cycle (n = 134) at 1st GnRH of Ovsynch had greater ovulatory response (81% vs. 51%; P =
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0.001) and smaller ovulatory follicles (14.6 ± 0.2 vs. 15.6 ± 0.3 mm; P = 0.008) compared
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to cows that did not have luteolysis and/or ovulation following pre-synchronization
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treatment (n = 102). Cows on d 6 of the estrous cycle (G6G) ovulated smaller follicles
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(13.8 ± 0.3 vs. 15.5 ± 0.3 mm; P < 0.001) and had lower serum P4 compared (1.76 ± 0.08
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vs. 2.37 ± 0.14 ng/mL; P < 0.001) to cows on d 7 (PG+G) of the estrous cycle.
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3.3 Effect of treatment on luteal function at time of PGF2α of Ovsynch
Percent of cows with functional CL at time of PGF2α of Ovsynch did not differ
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between treatments (Table 3). Mean serum concentrations of P4 at time of PGF2α of
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Ovsynch was greater (P = 0.002) for cows treated with G6G than cows treated with PG+G
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(Table 3) due to treatment differences in 1st service cows (Fig. 2). When treatments were
214
combined, primiparous cows had greater (P = 0.003) serum P4 at PGF2α of Ovsynch
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compared to multiparous cows (5.87 ± 0.33 vs. 4.78 ± 0.22 ng/mL; respectively).
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3.4 Effect of treatment on luteolysis to PGF2α of Ovsynch There was no effect of treatment on percentage of cows with complete luteolysis
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(Table 3). Only 7 cows with functional CL at time of PGF2α of Ovsynch did not have
220
complete luteolysis. Six of 7 cows had serum P4 between 0.50 and 1.00 ng/mL. A greater
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(P = 0.02) percent of multiparous cows had complete luteolysis compared to primiparous
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cows (99 % vs. 93 %; respectively).
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3.5 Effect of treatment on pregnancies per AI at 35 and 49 d post-AI
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Treatment did not affect P/AI on 35 or 49 d post-AI (Fig. 3). There was no effect of
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service number on P/AI at 35 (P = 0.67; 51% vs. 55% for first vs. greater services,
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respectively) or 49 d post-AI (P = 0.53; 49% vs. 52% for first vs. greater services, 10
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respectively). There was no effect of parity (P = 0.17) on P/AI at 35d post-AI (60% vs.
229
50% for primiparous vs. multiparous, respectively). However, primiparous cows had a
230
trend (P = 0.06) for higher P/AI at 49 d post-AI compared to multiparous cows (59% vs.
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46%, respectively). Percentage of cows with pregnancy losses between 35 and 49 d post-AI
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was 4% and was not affected by treatment (P = 0.69), parity (P = 0.27) or service (P =
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0.45).
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4. Discussion
The objective of this study was to develop a presynchronization strategy for
237
Ovsynch that limits treatments of GnRH and PGF2α to two days per week and results in
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fertility outcomes similar to that of G6G/Ovsynch. G6G is administered on four different
239
days of the week. Presynch-10/11 require three days of the week to administer. P/AI was
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not different between treatments but numbers of cows per treatment were limited resulting
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in approximately a 35% probability of a type-II error. Pregnancies per AI on d 35 and 49
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post-AI for G6G were greater (57% and 54%) than in previous studies that used G6G (35%
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to 50%) [2,4,5]. The high luteolysis rate following the final PGF2α of Ovsynch likely
244
influenced P/AI in this experiment. Cows without complete luteolysis after PGF2α of
245
Ovsynch have reduced chances of pregnancy [19]. The use of two PGF2α injections 1 d
246
apart at final PGF2α was very effective in inducing complete luteolysis in cows treated with
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PG+G and G6G, and is in agreement with previous studies [20]. In spite of the high P/AI of
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G6G, cows treated with PG+G had similar P/AI to G6G treatment, indicating that PG+G
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could be a simpler alternative for timed-AI programs for first or greater services for farms
250
that want to use the same weekdays of animal handling that are used for the Ovsynch
251
protocol.
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We hypothesized that combining PGF2α and GnRH in a presynchronization strategy
253
one week before Ovsynch would result in similar luteal and follicular outcomes following
254
Ovsynch treatments compared to G6G/Ovsynch. Results indicated that ovulation to the 1st
255
GnRH of Ovsynch were not different between G6G and PG+G. This partially supported
256
our main hypothesis. The probability of a type-II error was approximately 24% since values
257
are in a part of the curve with more power. Similar ovulation rates to 1st GnRH of Ovsynch
258
may help to explain similar percentages of cows pregnant for G6G and PG+G. Ovulation to
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the first GnRH of Ovsynch is a major factor for the success of timed-AI programs [1].
260
Previous studies demonstrated that cows that ovulated to the 1st GnRH of Ovsynch had
261
higher fertility after timed-AI compared to cows that did not ovulate following this
262
injection [2,21,22]. G6G [2] and other fertility programs (Presynch-10 or 11 and Double-
263
Ovsynch) [6,9,23] were developed to increase percentage of cows on d 6 or 7 of the estrous
264
cycle in order to optimize ovulatory response to the first GnRH of Ovsynch, increase
265
circulating concentrations of P4 during the development of the ovulatory follicle and
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improve pregnancies per timed-AI. Although PG+G had similar ovulation rate compared to
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G6G, percentage of cows on d 6 or 7 at the 1st GnRH of Ovsynch was reduced for PG+G
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due to a decrease in the percentage of cows that started a new cycle following PG+G.
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The combination of PGF2α and GnRH reduced the effect of both injections
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responses following PG+G pre-synchronization program. The underlying mechanism that
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reduced the lutelolysis rate after the PGF2α of the pre-synchronization for cows treated with
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PG+G is not clear. In both treatments, PGF2α was administered on a random day of the
273
estrous cycle. Only cows with functional CL were used to analyze percentage of cows with
274
complete luteolysis. Serum P4 at time of PGF2α was not different between cows with
275
complete luteolysis vs. without luteolysis, and it probably did not interfere in the 12
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effectiveness of PGF2α in contrast with a previous experiment that had lower probability of
277
complete luteolysis for cows with lower P4 at time of PGF2α [19]. Interestingly, we could
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not find published data to support the possibility that the LH surge induced by the GnRH
279
administration disrupted or delayed the luteolysis process in the PG+G treatment. In
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previous data [15], simultaneous administration of GnRH did not affect PGF2α-induced
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luteolysis in 16 cows on d 8 or 10 of the cycle, and all cows (16/16) reached P4 < 0.5
282
ng/mL in 1 d post PGF2α. In this study, only 70% (56/79) of cows treated with PG+G
283
reached P4 levels < 0.5 ng/mL 2 d post-injections, and only 2/23 had P4 levels between
284
0.50 and 1.00 ng/mL 2 d post- PGF2α. Cows treated with G6G in our experiment appeared
285
to have a higher luteolysis rate (89%) to what was previously reported on the first article of
286
G6G (77 %, [2]) and in the last PGF2α of Ovsynch (77 %, [19]). Therefore, additional
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studies with larger number of experimental units are necessary to determine whether GnRH
288
in combination with PGF2α reduces luteolysis rate or whether a Type II error influenced our
289
results.
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The addition of 2 d between the administration of GnRH and PGF2α of G6G
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treatment also improved the ovulatory response of GnRH when compared to PG+G. This
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result may be attributed to the reduction of serum P4 for cows with functional CL. During
293
luteolysis, serum P4 decrease induces an increase of LH pulse frequency [24], which allows
294
the continuous development of a DF and avoids a DF turnover. Cows with functional CL
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treated with G6G had a greater chance to have a DF at time of GnRH of the pre-
296
synchronization compared to PG+G. The lower ovulatory response to the GnRH of PG+G
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for cows with functional CL may be attributed to the reduction of the magnitude of the LH
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surge. Giordano et al. [25,26] showed that high serum P4 significantly suppressed GnRH-
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induced LH surge and reduced the magnitude of the LH surge.
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Presence of a functional CL might also have negatively affected the ovulatory
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response for the 1st GnRH of Ovsynch for cows treated with G6G since they had a greater
302
percentage of functional CL at the 1st GnRH of Ovsynch compared to PG+G. Previous
303
studies already demonstrated that the presence of a functional CL at time of GnRH
304
significantly reduced ovulatory response for 1st GnRH of Ovsynch [26-28]. The ovulation
305
rate to the 1st GnRH to Ovsynch for cows treated with G6G in the present study appeared to
306
be similar to what was previously reported for other studies that used G6G (64 % [29] and
307
69 % [5]), Double-Ovsynch (72 %, [6]), Presynch-11 (61 % [9]) and Presynch-10 (70 %
308
[30]). However, it was lower than previous studies in our laboratory using G6G in first
309
service cows (85%, [2,31]). In agreement with previous studies [1,2], cows on d 6 or 7 of
310
the estrous cycle had greater ovulation rate to the 1st GnRH of Ovsynch compared to cows
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in other times of the estrous cycle.
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Since the percentage of cows with functional CL was reduced in cows treated with
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PG+G, we were expecting a lower serum P4 at time of 1st GnRH of Ovsynch for cows
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treated with PG+G compared to G6G; however, treatments had similar serum P4. This
315
result appeared to be due a smaller percentage of cows with complete luteolysis following
316
PG+G treatment, which might have resulted in a greater proportion of cows in mid-estrous
317
cycle at time of 1st GnRH of Ovsynch. Cows in mid estrous cycle at 1st GnRH of Ovsynch
318
had lower percentage of cows with functional CL and serum P4 at time of PGF2α of
319
Ovsynch and lower fertility following timed-AI compared to cows between d 5 and 9 of the
320
cycle [32]. In the present study, it appears that this higher percentage of cows in mid
321
estrous cycle might not have influenced the overall outcome of the PG+G program since
322
percentage of cows with functional CL at time of PGF2α of Ovsynch and P/AI following
323
timed-AI was similar between treatments.
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PG+G might be a good substitution for Presynch-14 or -12 [13,14]. These programs
325
use the same weekdays for injections as Ovsynch but take longer to execute. Additionally,
326
these protocols are commonly used in combination with AI after estrus detection following
327
the PGF2α administration of the pre-synchronization and subsequent timed-AI for cows that
328
were not detected in estrus [33-36]. Fertility for these cows following estrus after PGF2α of
329
Presynch has been reported to be similar [33-35] or reduced [36] compared to cows that
330
only received timed-AI. On the other hand, the use of the PG+G program for first and
331
subsequent inseminations with the addition to detection of estrus between PG+G pre-
332
synchrony and first GnRH of Ovsynch may be a good strategy for farms that want to limit
333
the number of days in the week they are handling cows. PG+G increased the percent of
334
cows that ovulated during the 5 d prior to the 1st GnRH of Ovsynch. These cows would
335
likely be in early stages of follicle development (pre-deviation) at time of PG+G and may
336
have normal fertility following AI after estrus detection [37].
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5. Conclusion
Results from the present experiment indicate that administering both PGF2α and
340
GnRH on the same day, 7 d prior to the start of Ovsynch (PG+G) may offer a reasonable
341
alternative to more complex fertility programs to enhance P/AI for first and subsequent
342
services. The protocol PG+G appeared to provide sufficient ovulatory response to the first
343
GnRH of Ovsynch and concentrations of progesterone at time of PGF2α of Ovsynch. The
344
increase in the percent of cows in the PG+G treatment that ovulated during the 5 d prior to
345
the 1st GnRH of Ovsynch is a concern and need to be further investigated.
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Acknowledgments 15
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A special thanks to Ken and Kerry Nobis and all the employees of Nobis Dairy
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Farms (St. Johns, MI) for allowing us the use of their cows and facilities and providing
350
Fertagyl and Lutalyse used in this project. J.P. Martins was supported by The National
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Council for Scientific and Technological Development (CNPq - Brazil) fellowship –
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Science without Borders program, and M.R. Yousuf was supported by PAK-US S&T coop.
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program Phase IV.
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Table 1: Percentage of cows with functional corpus luteum (CL) at beginning of treatment, and effect of treatment (G6G vs. PG+G) on pre-synchronization outcomes. PG+G
P-value
Cows with functional CL at beginning of treatments1, % (n/n)
56 (65/117)
66 (79/119)
0.11
Cows with complete luteolysis to the PGF2α of the pre-synchronization2, % (n/n)
89 (58/65)
Ovulation to the GnRH of the pre-synchronization, % (n/n)
71 (56/79)
0.01
80 (93/116)
58 (70/120)
0.002
Cows with functional CL at beginning of treatments1, % (n/n)
95 (62/65)
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G6G (Control)
54 (43/79)
<0.001
Cows without functional CL at beginning of treatments1, % (n/n)
61 (31/51)
65 (26/40)
0.51
16.1 ± 0.4
16.2 ± 0.5
0.89
28 (26/93)
16 (11/70)
0.14
69 (80/116)
45 (54/120)
<0.001
Double ovulation to the GnRH of the presynchronization (%)
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Cows with new estrous cycle following presynchronization4, % (n/n) 1
Cows with functional CL = Serum progesterone (P4) ≥ 1.00 ng/mL. Only cows with functional CL were used in this analysis. Complete luteolysis was consider when cows with functional CL at d of PGF2α injection had serum P4 < 0.5 ng/mL 2 d later. 3 Only cows with single ovulations used on calculations. 4
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Cows with a new estrous cycle = (1) cows with a functional CL at beginning of treatment that had complete luteal regression and ovulation for the PGF2α and GnRH presynchronization injections, respectively, or (2) cows without functional CL at beginning of treatment that ovulated after the GnRH injection of the pre-synchronization
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Table 2: Effect of treatment (G6G vs. PG+G) on luteal and follicular functions at first GnRH of Ovsynch and response to first GnRH of Ovsynch.
Serum P4 at the 1st GnRH of the Ovsynch, ng/mL ± SEM Ovulation to the 1st GnRH of Ovsynch, % (n/n) Size of the ovulatory follicle of the 1st GnRH of the Ovsynch2, mm ± SEM Double ovulation to the 1st GnRH of Ovsynch, % (n/n)
P-values
91 (104/115)
76 (90/119)
0.03
2.22 ± 0.18 67 (78/116) 14.3 ± 0.3
2.14 ± 0.18
0.78
68 (82/120)
0.86
15.7 ± 0.3
0.002
22 (17/78)
23 (19/82)
0.79
Cows with functional corpus luteum (CL) at the 1st GnRH of Ovsynch were defined as having at least one of the following: (1) ovulation to the pre-synchrony GnRH, (2) no complete luteolysis after the pre-synchrony PGF2α and serum progesterone (P4) at time of 1st GnRH of Ovsynch ≥ 0.5 ng/mL, or (3) ovulation between the 5 d period before the 1st GnRH of Ovsynch and serum P4 at time of 1st GnRH of Ovsynch ≥ 0.5 ng/mL. 1
2
Only cows with single ovulation.
AC C
EP
TE D
373
M AN U
366 367 368 369 370 371 372
PG+G
RI PT
Cows with functional CL at the first GnRH of the Ovsynch 1, % (n/n)
G6G (Control)
SC
364 365
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Table 3: Effect of treatment (G6G vs. PG+G) on luteal function at PGF2α of Ovsynch and response to PGF2α of Ovsynch. G6G PG+G P-values (Control) Cows with functional CL at the PGF2α of Ovsynch 1, % (n/n) Serum P4 at the PGF2α of Ovsynch, ng/mL ± SEM Cows with complete luteolysis after the PGF2α of Ovsynch2, % (n/n) 1
5.75 ± 0.28 96 (103/107)
93 (111/120)
0.67
4.64 ± 0.24
0.002
97 (108/111)
0.68
SC
Cows with functional corpus luteum (CL) at PGF2α of Ovsynch were defined as having ovulation to at least one GnRH treatment or during the 5 d period before the 1st GnRH of Ovsynch and no CL regression prior to PGF2α of Ovsynch. 2
EP
TE D
M AN U
Only cows with functional CL were used in this analysis. Complete luteolysis was consider when cows with functional CL at d of PGF2α injection had serum progesterone (P4) < 0.5 ng/mL 2 d later.
AC C
376 377 378 379 380 381 382 383
94 (108/115)
RI PT
374 375
19
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Fig. 1: Schematic illustration of treatments, blood collections (B) and ultrasonography examinations (US). PG= PGF2α; US= Ultrasonography examination of ovaries; B=Blood sample collection
SC
RI PT
384 385 386
387
AC C
EP
TE D
M AN U
388
20
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389 390 391 392
Fig. 2: Effect of treatment (G6G vs. PG+G) on serum concentrations of progesterone (ng/mL) at first GnRH of Ovsynch and at PGF2α of Ovsynch for first and second or greater service cows. Interaction treatment and service at first GnRH of Ovsynch: P = 0.22 and at PGF2α of Ovsynch: P = 0.03.
8
G6G (control)
6.19 6 4
2.68
2.35
n=55
n=57
2 n=55
First GnRH of Ovsynch 393
G6G (control)
6 4
n=60
AC C
0
PG+G
P = 0.46
5.35
4.93
n=60
n=62
P = 0.29
1.79 2
PGF2α of Ovsynch
TE D
8
n=58
Second or greater service cows
1.94
EP
Serum progesterone (ng/mL)
10
SC
4.32
P = 0.50
0
n=62
First GnRH of Ovsynch
394
P < 0.001
PG+G
RI PT
First service cows
M AN U
Serum progesterone (ng/mL)
10
PGF2α of Ovsynch
21
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Fig. 3: Effect of treatments (G6G vs. PG+G) on pregnancies per AI (P/AI) at 35 and 49 d postAI. Interaction service by treatment was not significant for 35 d post-AI (P = 0.82) and for 49 d post-AI (P = 0.76). 100 G6G (control) PG+G 90 80 P = 0.27
P/AI (%)
70
P = 0.33
60 50
57
54
50
47
SC
40 30
n=110
n=120
0 35 d post-AI 398
n=109
n=118
49 d post-AI
AC C
EP
TE D
399
M AN U
20 10
RI PT
395 396 397
22
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[4]
[5]
[6]
[7]
[8]
[9]
[10] [11] [12]
[13]
[14]
RI PT
SC
[3]
M AN U
[2]
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TE D
[1]
EP
401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444
References
AC C
400
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[19]
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TE D
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[15]
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[32]
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[31]
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[30]
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[29]
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Highlights A new pre-synchronization strategy was tested for Ovsynch.
Fertility was similar in PG+G compared to G6G.
RI PT
PG and GnRH were administered simultaneously one week prior to Ovsynch.
AC C
EP
TE D
M AN U
SC
This strategy may be more logistically feasible for producers to utilize.