Extending the duration of treatment with progesterone and equine chorionic gonadotropin improves fertility in suckled beef cows with low body condition score subjected to timed artificial insemination

Accepted Manuscript Extending the duration of treatment with progesterone and treatment with eCG improves fertility in suckled beef cows with low body condition score subjected to timed artificial insemination M.G. Bilbao, N. Massara, S. Ramos, L.O. Zapata, M.F. Farcey, J. Pesoa, E. Turic, M.I. Vázquez, J.A. Bartolome PII:

S0093-691X(16)00064-9

DOI:

10.1016/j.theriogenology.2016.02.003

Reference:

THE 13504

To appear in:

Theriogenology

Received Date: 6 September 2015 Revised Date:

2 February 2016

Accepted Date: 3 February 2016

Please cite this article as: Bilbao M, Massara N, Ramos S, Zapata L, Farcey M, Pesoa J, Turic E, Vázquez M, Bartolome J, Extending the duration of treatment with progesterone and treatment with eCG improves fertility in suckled beef cows with low body condition score subjected to timed artificial insemination, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.02.003. 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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Extending the duration of treatment with progesterone and treatment with eCG improves fertility in suckled beef cows with low body condition score subjected to timed artificial insemination Bilbao MGa, Massara Nb, Ramos Sa, Zapata LOa, Farcey MFa, Pesoa Jc, Turic Ec, Vázquez MId, Bartolome JAa* a

Facultad de Ciencias Veterinarias, Universidad Nacional de La Pampa, Argentina Actividad Privada, Lomas de Zamora, Buenos Aires, Argentina c Biogénesis Bagó SA, Garin, Buenos Aires, Argentina d Facultad de Agronomía y Veterinaria, Universidad Nacional de Rio Cuarto, Argentina * Corresponding author. E-mail address: [email protected]

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Abstract

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The objective of this study was to evaluate the effect of an extended progesterone treatment on follicular development and fertility in postpartum, suckled beef cows subjected to timed-AI (TAI). In Experiment 1, cows (n=24) with body condition score (BCS) ≥ 4.5 received either a 2 g progesterone intravaginal device on Day -23 or a 0.558 g progesterone intravaginal device on Day -9. Then, all cows received 2 mg of estradiol benzoate on Day -9, removal of the device, 1 mg estradiol cypionate, and PGF2α on Day -2 and TAI on Day 0. Metabolic status was assessed between Days -9 and -2. Ovarian structures and plasma progesterone were determined weekly from Day -23 to -9, daily from Day -9 to 0, and weekly until Day 28. In Experiment 2, cows (n = 302) with BCS ≥ 4.5 received identical treatment to cows in Experiment 1, but on Day -2 cows received 400 IU of two different commercial preparations of eCG. Ovarian structures were determined on Days -23 and -9 on a subset of cows (n = 40). Pregnancy was determined 39 days after TAI. In Experiment 3, multiparous cows (n = 244) with BCS < 5.0 received identical treatment as cows in Experiment 1 initiated on Day -18, and on Day -2 cows received 400 IU of eCG or no treatment. Ovarian structures were determined in a subset of cows (n = 31) on Days -3, -2, -1, 0, 1, and on Day 10. Pregnancy was determined 39 days after TAI. The results indicated that in Experiment 1, plasma progesterone was higher in treated than non-treated (control cows) during the first 14 days (P = 0.0001). The extended progesterone treatment increased the size of the largest follicle between Days -23 and Day -5 (Group by Day, P = 0.04), and tended to increase the size of the dominant follicle from Day -5 to Day -1 (Group by Day, P = 0.06). There was no effect of metabolic status or interaction between metabolic status and day on follicular growth. In Experiment 2, extended progesterone treatment tended to increase the size of the largest follicle between Day -23 and -9 (P = 0.06). There was no effect of Group, eCG, BCS and parity on pregnancy per AI. In Experiment 3, extended progesterone treatment combined with eCG increased the size of the dominant follicle (P = 0.01). Both extended progesterone treatment (P = 0.02) and eCG (P = 0.03) increased pregnancy per AI. In conclusion, an extended progesterone treatment stimulated follicular growth post partum and improved fertility only in cows with low BCS.

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Key words: timed insemination, progesterone, eCG, beef cow, body condition 1. Introduction Protocols for induction of estrus and synchronization of ovulation have facilitated the use of AI in large and free-ranged beef herds where detection of estrus is difficult to implement [1-3]. Delayed interval from calving to first ovulation (postpartum anestrus), and reduced fertility to first estrus (short luteal phase post-ovulation) are the two main problems that limit the use of AI in suckled beef cows [4]. Postpartum anestrus seems to be caused by the inhibitory effect of estrogen on LH release in postpartum nursing beef cows [5]. Treatment with norgestomet for

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9 days in combination with estradiol enhanced LH pulsatility [6], increased the number LH receptors in the dominant follicle [7], and improved follicular development that consequently produced more estradiol [8] which enhanced the response to GnRH after norgestomet removal [9]. Progesterone treatment during nine [10] or seven [11] days, in combination with GnRH [12] or estradiol [13], has been shown to induce estrus in postpartum cows. In addition, inclusion of progesterone in estrous synchronization protocols has been shown to improve fertility to first estrus [14] and induced ovulation in noncyclic cows [15,16].

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The understanding of follicular dynamics during the estrous cycle has allowed for developing different estrous synchronization protocols in cows and heifers. Different protocols combining estradiol, progesterone and PGF2α [17,18], GnRH and PGF2α [19], or GnRH, progesterone and PGF2α [20,21] provide acceptable pregnancy rates in beef cows. Furthermore, protocols including GnRH improved conception rate by reducing the interval from GnRH to PGF2α to five days [22,23]. However, in cows with low body condition score (BCS) or in anestrus, the addition of eCG at the end of progesterone treatment can increase pregnancy rates to AI [24,25].

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Postpartum nutritional status affects the growth of the dominant follicle, ovulation and fertility either by a direct effect on hypothalamic GnRH, pituitary gonadotropins, or indirectly through the somatotrophic axis [26]. Low levels of glucose and elevated levels of β-hydroxybutyrate (BHB) in blood are associated with reduced energy intake, anaerobic metabolism, and those levels have been associated with early or late conception in suckled beef cows [27]. Glucose availability affected the level of insulin-like growth factor 1 (IGF-1) in anestrous suckled beef cows [28], and IGF-1 levels have been associated with the length of anestrus [29]. Therefore, BCS, circulating levels of BHB, glucose, and IGF-1 could determine postpartum metabolic status, and provide a better understanding of the effect of exogenous progesterone treatment on ovarian response and fertility.

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Our hypothesis was that a combination of an extended progesterone treatment and eCG will stimulate follicular growth and increase fertility in postpartum beef cows. The objective of Experiment 1 was to evaluate the effect of an extended progesterone treatment and metabolic status on follicular growth during early postpartum in suckled beef cows. The objective of Experiment 2 and 3 was to evaluate the effect of an extended progesterone treatment and eCG on fertility in suckled beef cows with high and low BCS subjected to a protocol of synchronization of ovulation and timed-AI (TAI). 2. Materials and methods

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2.1. Study populations

Suckled Polled Hereford cows located northeast of La Pampa province, Argentina, were used in Experiment 1 (breeding season 2012-2013). Cows were grazing oat pastures during the morning and grazing deferred corn during the rest of the day. Suckled Angus cows from a commercial herd located in the South of San Luis Province, Argentina were used in both Experiment 2 (breeding season 2012-2013) and Experiment 3 (breeding season 2013-2014). During the breeding season cows grazed native pastures within semi-arid woodlands, the Calden woodlands of Prosopis caldenia Burk. During the breeding season of 2012-2013 annual total rainfall was 860 mm, and during the breeding season of 2013-2014 it was only 515 mm. All procedures were performed with the approval of the Committee of Ethics in Biological Science Research (School of Veterinary Sciences, University of La Pampa, Argentina, Resolution 247/11) and according to the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching [30].

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2.2. Experiment 1

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Suckled Polled Hereford cows (n = 24) with BCS ≥ 4.5 (1 = emaciated to 9 = obese; [31]) were randomized to receive either a 2 g progesterone intravaginal device (2 g of progesterone in a solid matrix of silicone polymers over a plastic spine, patent pending) on Day -23 (Treatment group, n = 12, BCS = 6.04 ± 0.68; days postpartum = 35.5 ± 1.1) or a 0.558 g progesterone intravaginal device (Cronipress monodosis®, Biogénesis Bagó, Garin, Argentina) on Day -9 (Control group, n = 12, BCS = 5.95 ± 0.54, days postpartum= 35.4 ± 0.9). Cows in both groups received 2 mg of estradiol benzoate (2 mL, i.m., Bioestrogen®, Biogénesis Bagó) on Day -9. On Day -2 the intravaginal progesterone device was removed and cows received 25 mg of dinoprost tromethamine (5 mL, i.m., Lutalyse® Sterile Solution, Zoetis, Florham Park, NJ, USA) and 1 mg of estradiol cypionate (1 mL, i.m., Cipiosyn®, Syntex, Luis Guillón, Argentina). Cows were TAI 48 h later (Day 0) using one bull with proven fertility (Figure 1).

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Blood samples were collected on Days - 9, - 6 and - 2 by venipuncture of the median coccygeal vein or artery and whole blood was used to determine glucose and BHB levels. On Days -9 and -2, samples were centrifuged at 1100 X g for 20 min and serum was stored at -20 ºC until assayed for IGF-1. Blood samples were also collected by venipuncture of the coccygeal vein or artery into evacuated tubes containing EDTA (Vacutainer®; BD, Franklin Lakes, NJ, USA) on Days -23, -16, -9, daily until Day 0, and on Days 7, 14 and 28, and were immediately placed on ice. Samples were centrifuged at 1100 X g for 20 min, and plasma was stored at -20 ºC until assayed for progesterone.

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Concentrations of glucose and BHB in whole blood were determined using a hand-held glucose/ketone meter system (FreeStyleOptium Blood Glucose Monitoring System, Abbott Diabetes Care, Alameda, California, USA) according to the manufacturer's operating guidelines. Quality control reagents were run on a daily basis before any samples were analyzed [32].

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Concentrations of IGF-1 were determined at the Laboratory of Nuclear Techniques, Veterinary Faculty, Montevideo, Uruguay using a commercial radioimmunoassay (RIA) kit (IGF1-133 RIACT Cis Bio International, GIF-SUR-YVETTE CEDEX, France) [33]. The kit contained two monoclonal antibodies against two different antigenic sites of the IGF-1 molecule; one was coated on the solid phase, and the other radiolabeled with iodine-125. All samples were measured in one assay. The sensitivity of the assay was 0.38 ng/mL. The intra-assay coefficients of variation (CVs) for control 1 (35 ng/mL) and control 2 (458 ng/mL) were 14.1% and 13.4%, respectively. Plasma progesterone concentrations were determined at the Laboratory of Nuclear Techniques, Veterinary Faculty, Montevideo, Uruguay using a direct, solid-phase RIA (Siemens Healthcare Diagnostics Inc. Los Angeles, CA, USA). The RIA had a sensitivity of 0.083 ng/mL. The intra-assay CVs for low (0.92 ng/mL), medium (2.89 ng/mL) and high (9.9 ng/mL) controls were 12.1%, 7.5% and 5.9%, respectively.

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Transrectal ultrasonography (5 MHz transrectal linear transducer, HS-101V, Honda Electronics, Japan) of the ovaries was conducted on Day -23, -9, daily from Day -9 to -1, and on Day 7 to confirm ovulation by the presence of a corpus luteum on the ovaries [34]. Pregnancy was evaluated by transrectal ultrasonography of the uterus 28 and 43 days after AI [35]. Baseline comparisons between groups for days post partum, BCS, BHB, IGF-1, and glucose concentrations were done using ANOVA in the SAS System® (SAS Inst. Inc., Cary, NC, USA). Cows were classified in low metabolic status when BHB tended to increase and glucose and IGF-1 tended to decrease between Days -9 and -2. The effect of treatment and metabolic

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status on size of the largest follicle between Days -23 and -9, between Day -9 and -5 and preovulatory follicle between Day -5 and -1, and plasma progesterone were determined using ANOVA for repeated measures in the Proc Mixed of SAS. The effect of treatment, metabolic status, and their interaction on pregnancy per AI was analyzed using the backward elimination procedure [36] in Proc Logistic of SAS. All results for variables with continuous distribution are presented as means ± SEM. Significant effects were declared at P ≤ 0.05, and tendencies declared at P ≤ 0.10.

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Primiparous and multiparous suckled Angus cows (n = 302) between 40 to 60 days post partum with BCS ≥ 4.5 (1 = emaciated to 9 = obese; [31) received at random identical treatments to cows in Experiment 1 (Treatment group, n=151 and Control group n=151), but on Day -2 received 0.150 mg D-cloprostenol (2 mL, i.m., Enzaprost®, Biogénesis Bagó) instead of dinoprost tromethamine. Also on Day -2, cows were assigned at random to receive 400 IU of equine chorionic gonadotropin (eCG) of two different commercial preparations (2 mL, i.m., Novormon®, Syntex, n = 149 or 2mL, i.m., Ecegon®, Biogénesis Bagó, n=153). Cows were TAI 48 h later (Day 0) with one bull with proven fertility. The proportion of cows treated with either Novormon® or Ecegon® was 49.0% (74/151) and 51.0% (77/151) in the Treatment group and 49.7% (75/151) and 50.3% (76/151) in the Control group (P = 0.90). The proportion of primiparous and multiparous cows were 37.7% (57/151) and 62.3% (94/151) in the Treatment group and 34.4% (52/151) and 65.6% (99/151) in the Control group (P = 0.54). Ultrasonography of the ovaries [34] was conducted on Days -23 and -9 on a subset of cows (n = 40). Pregnancy was evaluated 39 days after AI by transrectal palpation of the uterus [35].

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Size of the largest follicle between Day -23 and -9 was analyzed using the repeated measures procedure with Proc Mixed of SAS. The effect of group (Treatment vs. Control) on pregnancy per AI adjusted by eCG type (Novormon® or Ecegon®), parity (primiparous vs. multiparous), and interactions was analyzed using the backward elimination procedure [36] in Proc Logistic of SAS. All results for variables with continuous distribution are presented as means ± SEM. Significant effects were declared at P ≤ 0.05 and tendencies declared at P ≤ 0.10.

Multiparous suckled Angus cows (n = 244) with 40 to 60 days postpartum and BCS < 5.0 (1 = emaciated to 9 = obese; [31]) received an identical treatment to cows in Experiment 2 (Treatment group, n = 127, Control group, n = 117) but this was initiated on Day -18. Also on Day -2, instead of receiving two different commercial preparations of eCG, cows were assigned to receive either 400 IU of eCG (2 mL, i.m., Novormon®, Syntex, n = 124) or no treatment (n = 120). Cows were TAI 48 h later (Day 0) with one bull with proven fertility. The proportion of cows treated with eCG or control was 50.4% (64/127) and 49.6% (63/127) in the Treatment group and 48.7% (57/117) and 51.3% (60/117) in the Control group (P = 0.88).

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Ovaries were examined by ultrasonography in a subset of cows (n = 31) on Days -3, -2, -1, 0,and 1 to evaluate follicular growth, and on Day 10 to confirm ovulation by the presence of a corpus luteum [34]. Pregnancy was determined 39 days after AI by transrectal palpation of the uterus [35]. The effect of treatment on size of the dominant follicle on Days -3, -2, -1, 0, and 1 was analyzed using the repeated measures procedure with Proc Mixed of SAS. The effect of Group (Treatment vs. Control) on pregnancy per AI adjusted by eCG treatment (400 IU eCG vs. Control) and interactions were analyzed using the backward elimination procedure [36] in Proc

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Logistic of SAS. All results for variables with continuous distribution are presented as means ± SEM. Significant effects were declared at P ≤ 0.05 and tendencies declared at P ≤ 0.10. 3. Results 3.1. Experiment 1

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One intravaginal device was not present at Day -9 in the Treatment group and two devices, one in each treatment, were not present at Day -2 (these cows were included in the analysis). There was no difference in days post partum (P= 0.95) and BCS (P= 0.74) for cows in the Control and Treatment groups.

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There was no difference in glucose concentrations between cows in the Control and Treatment groups (P = 0.18), but there was an interaction between Group and Day (P = 0.0005). Blood concentrations of glucose were different (P = 0.01) on Day -9 (Treatment group 58.9 ± 3.01 mg/dL and Control group 47.0 ± 2.07 mg/dL), but not different on Day -6 (Treatment group 47.6 ± 1.93 mg/dL and Control group 51.4 ± 1.52 mg/dL) and on Day -2 (Treatment group 55.4 ± 3.39 mg/dL and Control group 52.2 ± 1.84 mg/dL). There was no difference in BHB concentrations between cows in the Control and Treatment groups (P = 0.79) and no interaction between Group and Day (P= 0.33), but there was an effect of Day (P = 0.0007). Concentrations of BHB were higher on Day -2 (0.40 ± 0.02 mmol/L) compared to Day -9 (0.27 ± 0.02 mmol/L, P = 0.003) and Day -6 (0.32 ± 0.03 mmol/L, P = 0.02). There was no difference in IGF-1 concentrations between cows in the Control and Treatment groups (P = 0.61) and no interaction between Group and Day (P = 0.15), but IGF-1 concentrations were different (P = 0.002) between Day -9 (95.3 ± 10.6 ng/mL) and Day -2 (45.7 ± 10.9 ng/mL).

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There was an effect of Group (P = 0.04), Day (P = 0.0001), and an interaction between Group and Day (P = 0.001) on plasma progesterone concentrations (Figure 2). There was an effect of Day (P = 0.0001), Group (P = 0.04) and Group by Day (P = 0.04) on size of the largest follicle between Days -23 and -5. There was no difference on Day -23, but the largest follicle was greater (P = 0.04) on Day -9 and from Day -9 to -5 (P = 0.04) for cows in the Treatment group (Figure 3A). There was no effect of metabolic status (P = 0.28) or interaction between metabolic status and day (P = 0.49) on the size of the largest follicle between Days -23 and -9. There was no effect of metabolic status (P = 0.29) or interaction between metabolic status and Day (P = 0.95) on the size of the largest follicle between Days -9 and -5. The extended progesterone treatment tended (Group by Day P = 0.06) to increase the size of the dominant preovulatory follicle from Day -5 to Day -1 (Figure 3B). The dominant preovulatory follicle was larger (P = 0.04) on Day -3 for cows in the Treatment than in the Control group. There was no effect of metabolic status (P = 0.93) or interaction between metabolic status and day (P = 0.20) on the size of the dominant preovulatory follicle from Day -5 to Day -1.

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There was no difference in the presence of a CL on Day 7 (Treatment group 9/12 and Control 10/12, P = 0.61) and on Day 14 (Treatment group 10/12 and Control group 11/12, P = 0.53). There was no difference (P = 0.97) in pregnancy per AI on Days 28 (Treatment group 50%, 6/12 and Control group 50%, 6/12), and no difference on Day 43 (Treatment group 41.7%, 5/12 and Control group 41.7%, 5/12). There was no effect of metabolic status (P = 0.97) or interaction between Group and metabolic status (P = 0.97) on pregnancy per AI on Days 28 and 43. 3.2. Experiment 2 Body condition score was greater in cows in the Treatment group compared to the Control group (5.06 ± 0.69 vs 4.96 ± 0.68; P = 0.01). At Day -9, the intravaginal progesterone device

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was not present in six cows in the Treatment group (3.9 %), and on Day -2 it was not present in four cows (2.6 %) in the Treatment group, and three cows (1.9 %) in the Control group. In the subset of cows evaluated on Days -23 and -9 (n = 40), BCS was 5.30 ± 0.84 for cows in the Treatment group and 4.97 ± 0.85 for the cows in the Control group (P = 0.18). Treatment tended (P = 0.06) to increase the size of the largest follicle between Day -23 and -9 (Figure 4).

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There was no effect of Group (Treatment or Control), treatment with eCG (Novormon® or Ecegon®) and parity (primiparous or multiparous) on pregnancy per AI (Table 1). 3.3. Experiment 3

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Body condition score for cows in the Treatment group was 3.88 ± 0.56 and for cows in the Control group was 3.85 ± 0.64 (P = 0.68). At Day -9, the intravaginal progesterone device was not present in seven cows in the Treatment group (5.5 %), and on Day -2 it was not present in four cows (3.1 %) in the Treatment group and three cows (2.6 %) in the Control group.

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There was an interaction between progesterone treatment and eCG treatment (P = 0.01) on the size of the dominant preovulatory follicle. Cows receiving eCG and an extended progesterone treatment had a larger dominant preovulatory follicle (Figure 5). Ovulation rate was 1/8 for Control-Control, 2/8 for Control-eCG, 1/8 for Treatment-Control and 4/7 for Treatment-eCG (P = 0.41). Both an extended treatment with progesterone (P = 0.02) and treatment with eCG (P = 0.03) increased pregnancy per AI in suckled beef cows with low BCS and the interaction was not significant (Table 2). 4. Discussion

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The intravaginal device containing 2 g of progesterone applied around 35 days post partum was able to maintain progesterone levels for 21 days over 1.9 ng/mL (Experiment 1), and increased the size of the largest follicle by the time the synchronization of ovulation protocol was initiated (Experiment 1 and 2). It also increased the size of the dominant preovulatory follicle, but failed to increase pregnancy per AI in cows with high BCS (Experiment 1 and 2). However, in cows with low BCS, extended progesterone treatment and eCG increased growth of the preovulatory follicle and pregnancy per AI (Experiment 3). The extended progesterone treatment applied in this study increased circulating concentrations of progesterone during the first 14 days (Days -23 to -9) of the treatment protocol compared to the non-treated control cows, but concentrations of progesterone were less during the period in which synchronization of follicular waves was expected (Days -9 to -2). No differences in concentrations of progesterone in plasma were subsequently found after the synchronized estrus and ovulation (Days -2 to 28). The initial increase in progesterone due to the intravaginal device was expected since the majority of cows at 34 to 36 days post partum would not have yet ovulated. Then, since the progesterone intravaginal device blocked ovulation in cows of the Treatment group between Days -23 and -9, some cows in the Control group ovulated and had higher plasma progesterone concentrations once they received the intravaginal device on Day -9. Therefore, despite the fact that extended progesterone treatment tended to increase follicular growth, progesterone was similar between cows in the Treatment and Control groups after ovulation and TAI.

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Intravaginal progesterone devices have previously been used in cows in order to control the estrous cycle [37]. In the present study, the aim of administering progesterone early post

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partum was to stimulate follicular development in order to improve the response to a protocol of induction and synchronization of ovulation for TAI. The 2 g progesterone intravaginal device applied around 35 days post partum in suckled beef cows increased the size of the largest follicle before the initiation of the synchronization of ovulation protocol (Experiments 1 and 2), and also increased the diameter of the preovulatory follicle (Experiments 1 and 3). Progesterone has a negative feedback effect on LH secretion [38, 39], and low concentrations of progesterone result in increased LH pulse frequency [15,40, 41]. It has also been proposed that progesterone treatment may reduce the negative feedback effect of estradiol by reducing the number of estrogen receptors in the hypothalamus and the negative feedback effect of estradiol on the release of GnRH, thereby allowing an increased frequency of LH release [4]. Administration of norgestomet for 9 days around 23 days post partum in suckled beef cows increased the weight of the follicle and the number of LH receptors in the largest follicle [7]. The inclusion of a 1.38 g intravaginal progesterone device in a Co-Synch protocol in anestrous cows increased the rate of follicular growth in the last two days of treatment [42]. In contrast, a treatment with 0.5 mg of melengestrol acetate for 14 days did not affect the size of the largest follicle in anestrous cows, and these authors proposed that the ability of the dominant follicle to respond to LH pulses may be affected in anestrous cows [43].

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Experiments in the present study were conducted in range beef cows of commercial herds where BCS depends largely on climate conditions during the calving season. This is the reason for the different BCS for cows in the different experiments. Extended progesterone treatment increased pregnancy rate in cows with low BCS (Experiment 3), but not in cows with high BCS (Experiments 1 and 2). In addition, the effect of treatment on size of the dominant preovulatory follicle was more evident in Experiment 3 compared to Experiment 1. In agreement with results of the this study, administration of norgestomet during the early postpartum period increased the number of cows showing estrus, and reduced the incidence of short luteal phases [44,45,46]. In addition, administration of melengestrol acetate for 14 days and treatment with PGF2α 17 days after melengestrol removal increased the number of cows exhibiting estrus, and also increased conception and calving rates in postpartum suckled beef cows [14]. In contrast, Giles et al. [47] showed a benefit of extending the progesterone from 5 to 14 days in cows with high BCS. However, these authors used a protocol that included GnRH to synchronize the follicular wave. In Brahman heifers, Cavalieri et al. [48], did not report differences in expression of estrus and fertility in cows treated with a norgestomet implant for either 11 or 17 days combined with an acute progesterone treatment for 24 h three days before implant removal, and insemination at detected estrus.

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In the present study, glucose and BHB were determined in Experiment 1 in order to ensure comparability of the groups and evaluate a potential effect of metabolic status on follicular growth. In addition, we classified cows in low or high metabolic status when BHB tended to increase and glucose and IGF-1 tended to decrease during the study. Blood levels of these metabolites were under normal ranges, and did not affect follicular growth or pregnancy rates. This is probably due to the fact that cows in Experiment 1 had high BCS. Circulating BHB, together with insulin, glucose, NEFA (non-esterified fatty acids) and IGF-1, are considered to be indicators of energy balance in cows. Holstein Friesian first lactation dairy cows with delayed ovulation post partum had lower IGF-1 and higher BHB concentrations [49]. This metabolic profile was also observed by Huszenicza et al. [50] who observed that cows which ovulated within 35 days post partum had higher IGF-I, higher glucose, and lower NEFA and BHB concentrations. Mulliniks et al. [27] found that BHB concentrations were reduced in early

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conception cows compared with late conception, indicating that BHB concentrations may have delayed conception date. In suckled beef cows, ovulation and pregnancy rate after estrous synchronization treatment were related to mobilization of energy stores before treatment [51]. In early lactation cows, the hormonal environment created by lactation may potentially affect the capacity of ovarian cells to respond to gonadotropins. Low blood glucose could potentially compromise a variety of essential metabolic processes in ovarian cells, including oocytes that depend on glucose for energy [52]. Although glucose has been suggested as one of the most important metabolic substrates required for proper function of reproductive processes in beef cows [53], other research did not show any changes in glucose values in pregnant and non-pregnant suckled beef cows 7 weeks before AI [51]. Increased circulating IGF-I levels are associated with improved nutritional status in suckled beef heifers [54]. Increased diameter, growth rate, and persistence of the dominant follicle were associated with increased concentrations of LH, estradiol, and IGF-I during the transition from nutritionallyinduced anovulation to resumption of ovulatory cycles [55]. It has been proposed that one mechanism by which reduced body energy reserves can decrease LH release may involve a decrease in IGF-1 secretion [56].

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Administration of eCG in protocols for synchronization of ovulation and TAI have been extensively used in Bos indicus [57] and Bos taurus [58] cows. This approach improved follicular development, especially in cows with low BCS, or cows in postpartum anestrus. The inclusion of 400 IU of eCG increased the size of the dominant follicle, ovulation and pregnancy rate in postpartum anestrus Bos indicus cows subjected to a protocol for TAI that used estradiol for follicular wave synchronization, and progesterone and GnRH for induction of ovulation [25]. In the present study, eCG administration and extended progesterone treatment increased pregnancy rate independently, but increase of the size of the dominant follicle was observed only with the interaction of both treatments (Experiment 3). In agreement with the results of the present study, administration of 400 IU of eCG at the time of removal of the intravaginal progesterone device in anestrous suckled Hereford cows between 60 and 75 days post partum increased ovulation rate, but the effect on the size of the dominant follicle was not significant [59]. In contrast, treatment with either 300 or 400 IU of eCG increased the size of the dominant follicle at TAI in suckled Bos taurus beef cows subjected to a TAI protocol combining progesterone and estradiol [60]. Since eCG is obtained from a pool of different pregnant mares, eCG products and batches probably can vary in purity and potency. In Experiment 2, we compared two commercial eCG compounds which did not show differences in pregnancy per AI in cows with high BCS.

402 403 404

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5. Conclusion

In conclusion, the results of this study showed that it is possible to maintain high plasma progesterone concentrations for at least 21 days using a 2 g intravaginal progesterone device, and this stimulated follicular growth in postpartum beef cows. Extended progesterone treatment did not increase fertility in cows with high BCS, but in cows with poor BCS, the combination of extended progesterone and eCG treatment increased growth of preovulatory follicle and pregnancy per AI.

6. Acknowledgements

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The authors thank “Cabaña El Pampa” and “San Miguel” beef farms and their employees for assistance during the study and the PICT 2011-0238, Prestamo BID, Secyt-UNLPam research grant for financial support. We are grateful of Drs. Mike F. Smith and Louis F. Archbald for critical reading and English editing. 7. References

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[29] Roberts AJ, Klindt J, Jenkins TG. Effects of varying energy intake and sire breed on duration of postpartum anestrus, insulin like growth factor-1, and growth hormone in mature crossbred cows. J Anim Sci 2005;83:1705-14.

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[33] Adrien ML, Mattiauda D a, Artegoitia V, Carriquiry M, Motta G, Bentancur O, et al. Nutritional regulation of body condition score at the initiation of the transition period in primiparous and multiparous dairy cows under grazing conditions: milk production, resumption of post-partum ovarian cyclicity and metabolic parameters. Animal 2012;6:292-9. [34] Pierson RA, Ginther OJ. Ultrasonography of the bovine ovary. Theriogenology 1984;21:495-504. [35] Zemjanis R. Diagnostic and therapeutic techniques in animal reproduction. Baltimore:Williams &Wilkins; 1962.

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[39] Adams GP, Matteri RL, Kastelic JP, Ko JC, Ginther OJ. Association between surges of follicle-stimulating hormone and the emergence of follicular waves in heifers. J Reprod Fertil 1992;94:177-88. [40] Kinder JE, Kojima FN, Bergfeld EG, Wehrman ME, Fike KE. Progestin and estrogen regulation of pulsatile LH release and development of persistent ovarian follicles in cattle. J Anim Sci 1996;74:1424-40.

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[47] Giles RL, Ahola JK, Whittier JC, French JT, Repenning PE, Kruse SG, et al. Administration of a GnRH analog on day 9 of a 14-day controlled internal drug release insert with timed artificial insemination in lactating beef cows. J Anim Sci 2013;91:1866-73. [48] Cavalieri J, Kinder JE, De’ath G, Fitzpatrick LA. Effects of short-term treatment with progesterone superimposed on 11 or 17 days of norgestomet treatment on the interval to oestrus and fertility in Bos indicus heifers. Anim Reprod Sci 1998;51:169-83.

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[49] Taylor V, Beever D, Bryant M, Wathes D. Metabolic profiles and progesterone cycles in first lactation dairy cows. Theriogenology 2003;59:1661-77.

[50] Huszenicza GY, Kulcsar M, Nikolic JA, Schmidt J, Korodi P, Katai L, et al. Plasma leptin concentration and its interrelation with some blood metabolites, metabolic hormones and the resumption of cyclic ovarian function in postpartum dairy cows supplemented with Monensin or inert fat in feed. Fertil. High Prod. Dairy Cow Proc. an Int. Symp., 2001, p. 405–9. [51] Grimard B, Humblot P, Mialot JP, Jeanguyot N, Sauvant D, Thibier M. Absence of response to oestrus induction and synchronization treatment is related to lipid mobilization in suckled beef cows. Reprod Nutr Dev 1997;37:129-40.

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[59] Núñez-Olivera R, de Castro T, García-Pintos C, Bó G, Piaggio J, Menchaca A. Ovulatory response and luteal function after eCG administration at the end of a progesterone and estradiol based treatment in postpartum anestrous beef cattle. Anim Reprod Sci 2014;146:111-16.

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[60] Pessoa GA, Martini AP, Carloto GW, Rodrigues MC, Claro Júnior I, Baruselli PS, et al. Different doses of equine chorionic gonadotropin on ovarian follicular growth and pregnancy rate of suckled Bos taurus beef cows subjected to timed artificial insemination protocol. Theriogenology 2015 (Epub ahead of print).

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ACCEPTED MANUSCRIPT PGF2a , 1 mg EC

2 mg EB

TAI

0.558 g progesterone A

48 hours

2 g Progesterone Day -23

-9

-2

0

PGF2a, 1 mg EC 400 UI eCG

2 mg EB

TAI

0.558 g progesterone 2 g Progesterone Day -23

48 hours

-9

-2

0.558 g progesterone 2 g Progesterone Day - 18

748

TE D

747

EP

746

AC C

745

-2

0

Figure 1. Treatment groups for Experiment 1 (A), Experiment 2 (B) and Experiment 3 (C). On Day -23 (Experiments 1 and 2) and on Day -18 (Experiment 3) cows were randomly assigned to receive either a 2 g of progesterone device or no treatment (control). Then, on Day -9 cows in the Control group received a 0.558 g progesterone device. On Day -2 cows received 25 mg of dinoprost tromethamine on Experiment 1 and 0.150 mg D-cloprostenol on Experiments 2 and 3. EB= estradiol benzoate, EC= estradiol cypionate, eCG= equine chorionic gonadotropin; TAI= timed-AI.

743 744

48 hours

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736 737 738 739 740 741 742

-9

TAI

SC

735

0

PGF2a, 1 mg EC ± 400 UI eCG

2 mg EB

C

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B

15

ACCEPTED MANUSCRIPT 9

*

7

*

*

* *

6 5

**

*

*

4 3 2 1 0 -23

-16

-9

-8

-7

-6

-5

-4

-3

-2

Control

750 751 752 753

759 760 761 762 763

TE D

758

EP

757

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756

7

14

28

Figure 2. Plasma progesterone concentrations in cows treated with a 2 g progesterone device for 21 days (Treatment group) or a 0.558 progesterone device for 7 days (Group P = 0.04, Day P = 0.0001, Group by Day P = 0.001). *P = 0.01 and **P = 0.06 for the effect of Group on plasma progesterone concentration (Experiment 1).

754 755

0

Treatment

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749

-1

SC

Experimental Day

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Plasma Progesterone (ng/mL)

8

16

ACCEPTED MANUSCRIPT A *

*

*

*

10 5 0 -23

-9

-8

-7

-6

Experimental Day Treatment

5 0 -5

M AN U

10

-4

-3 -2 Experimental Day Control Treatment

-1

Figure 3. A. Size of the largest follicle on Experimental Day -23, -9 and between -9 and -5 in lactating beef cows for Treatment and Control groups (Group by Day P = 0.04). *P = 0.04 for the effect of group on size of the largest follicle. B. Size of the dominant preovulatory follicle (Group by Day P = 0.06). *P = 0.04 and **P = 0.11 for the effect of Group on size of the dominant preovulatory follicle (Experiment 1).

EP

771

**

*

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766 767 768 769 770

15

TE D

Preovulatory Follicle (mm)

B

-5

SC

Control

764

765

*

15

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Largest Follicle (mm)

20

17

ACCEPTED MANUSCRIPT 14

12 11 10 9 8 7 6 -23

-9

Control

SC

Experimental day Treatment

772

Figure 4. Size of the largest follicle (mm) on Days -23 and -9 for a subset of cows in the Treatment (n=20) and Control groups (n=20; Group by Day P = 0.06; Experiment 2).

M AN U

773 774

16

12

8 6 4 2 0

AC C

-3

Control-Control

776 777 778 779 780 781 782

*

**

0

1

TE D

10

EP

Preovulatory follicle (mm)

14

775

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Largest follicle (mm)

13

-2

-1 Days from PGF2αα administration

Control-eCG

Treatment-Control

Treatment-eCG

Figure 5. Size of dominant preovulatory follicle in cows in Experiment 3 from Days -3 to 1 of treatment in cows treated with either a 2 g progesterone device for 18 days or a 0.558 g progesterone device for 7 days and treated or not with eCG at the time of device removal (Group by eCG treatment by Day P = 0.006). *P = 0.02 and **P = 0.01 for the effect Group by eCG treatment on the size of the dominant preovulatory follicle (Experiment 3).

18

ACCEPTED MANUSCRIPT

783 Table 1. Pregnancy per AI, adjusted odds ratio (AOR), 95 % confidence interval (CI), and P value for cows in both groups adjusted for eCG type and parity (Experiment 2). Explanatory Variable

Pregnancy per AI % N 57.6 51.6

87/151 78/151

Reference 0.82

57.7 51.6

86/149 79/153

Reference 0.78

49.5 57.5

54/109 111/193

Reference 1.47

M AN U

Pregnancy per AI % N

AOR

95 % CI

16/117 36/127

Reference 1.52

Reference 1.08-2.15

16/120 36/124

Reference 1.57

Reference 1.11-2.22

TE D

13.6 28.3

EP

13.3 29.0

AC C

793

Reference 0.89-2.43

Table 2. Pregnancy per AI, adjusted odds ratio (AOR), 95 % confidence interval (CI), and P value for cows in both groups adjusted for eCG treatment (Experiment 3).

Group Control Treatment eCG Control 400 UI eCG

792

Reference 0.49-1.23

0.11

Explanatory Variable

791

Reference 0.51-1.31

0.26

787

790

P value 0.77

786

788 789

95 % CI

RI PT

Group Control Treatment eCG type Novormon Ecegon Parity Primiparous Multiparous

AOR

SC

784 785

P value 0.02

0.03

ACCEPTED MANUSCRIPT Revised Extending the duration of treatment with progesterone and treatment with eCG improves fertility in suckled beef cows with low body condition score subjected to timed artificial insemination

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Highlights Artificial insemination in suckled beef cows is troublesome due to difficulties to implement estrus detection and the high incidence of postpartum anestrus and short luteal phases Protocols for timed insemination avoid estrus detection, allow for timed-AI, but not always resolve the problem of postpartum anestrus and short luteal phases

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This study evaluated the effect of a long term progesterone treatment combined with a protocol of synchronization of ovulation, eCG and timed-AI in suckled beef cows with good or poor body condition score

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The results indicated that long term progesterone treatment stimulates follicular growth and both extended progesterone treatment and eCG enhances conception rates in suckled beef cows with poor body condition score