Dietary selenium and nutritional plane alter specific aspects of maternal endocrine status during pregnancy and lactation

Accepted Manuscript Dietary selenium and nutritional plane alter specific aspects of maternal endocrine status during pregnancy and lactation C.O. Lemley, A.M. Meyer, T.L. Neville, D.M. Hallford, L.E. Camacho, K.R. MaddockCarlin, T.A. Wilmoth, M.E. Wilson, G.A. Perry, D.A. Redmer, L.P. Reynolds, J.S. Caton, K.A. Vonnahme PII:

S0739-7240(13)00114-8

DOI:

10.1016/j.domaniend.2013.09.006

Reference:

DAE 6041

To appear in:

Domestic Animal Endocrinology

Received Date: 24 July 2013 Revised Date:

13 September 2013

Accepted Date: 15 September 2013

Please cite this article as: Lemley CO, Meyer AM, Neville TL, Hallford DM, Camacho LE, Maddock-Carlin KR, Wilmoth TA, Wilson ME, Perry GA, Redmer DA, Reynolds LP, Caton JS, Vonnahme KA, Dietary selenium and nutritional plane alter specific aspects of maternal endocrine status during pregnancy and lactation, Domestic Animal Endocrinology (2013), doi: 10.1016/ j.domaniend.2013.09.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Revised Version 1

Dietary selenium and nutritional plane alter specific aspects of maternal endocrine status

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during pregnancy and lactation

3 C. O. Lemleya, A. M. Meyera, T. L. Nevillea, D. M. Hallfordb, L. E. Camachoa, K. R. Maddock-

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Carlina, T. A. Wilmothc, M. E. Wilsonc, G. A. Perryd, D. A. Redmera, L. P. Reynoldsa, J. S.

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Catona, and K. A. Vonnahmea

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a

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University, Fargo, ND 58108

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Center for Nutrition and Pregnancy, Department of Animal Sciences, North Dakota State

Department of Animal and Range Sciences, New Mexico State University, Las Cruces, NM

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Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV,

Department of Animal Sciences, South Dakota State University, Brookings, SD, 57007

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Address for reprint requests and other correspondence: K. A. Vonnahme, 181 Hultz Hall, Dept

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7630 PO Box 6050, Fargo, ND 58108-6050 (e-mail: [email protected]).

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ACCEPTED MANUSCRIPT Revised Version Abstract

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Objectives were to examine effects of selenium (Se) supply and maternal nutritional plane during

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gestation on placental size at term and maternal endocrine profiles throughout gestation and early

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lactation. Ewe lambs (n = 84) were allocated to treatments that included Se supply of adequate

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Se (ASe, 11.5 µg/kg BW) or high Se (HSe, 77 µg/kg BW) initiated at breeding and nutritional

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plane of 60% (RES), 100% (CON), or 140% (EXC) of requirements beginning on day 40 of

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gestation. At parturition, lambs were removed from their dams, and ewes were transitioned to a

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common diet that met requirements of lactation. Blood samples were taken from a subset of ewes

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(n = 42) throughout gestation, during parturition, and throughout lactation to determine hormone

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concentrations. Cotyledon number was reduced (P = 0.03) in RES and EXC compared with

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CON ewes. Placental delivery time tended (P = 0.08) to be shorter in HSe compared with ASe

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ewes, whereas placental delivery time was longer (P = 0.02) in RES compared with CON and

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EXC ewes. During gestation, maternal progesterone, estradiol-17β, and growth hormone were

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increased (P < 0.05) in RES and decreased (P < 0.05) in EXC compared with CON ewes. In

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contrast, maternal cortisol, IGF-I, prolactin, triiodothyronine, and thyroxine were decreased in

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RES and increased in EXC compared with CON ewes during gestation. Selenium supply did not

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alter maternal hormone profiles during gestation. During parturition and lactation, maternal

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hormone concentrations were influenced by both Se and maternal nutritional plane. During the

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parturient process, HSe ewes tended to have greater (P = 0.06) concentrations of estradiol-17β

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compared to ASe ewes. Three h after parturition there was a surge of growth hormone in ASe-

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RES ewes that was muted in HSe-RES, and not apparent in other ewes. Growth hormone area

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under the curve during the parturient process was increased (P < 0.05) in ASe-RES vs HSe-RES

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ewes. Ewes that were overfed during gestation had reduced (P < 0.05) estradiol-17β, but greater

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ACCEPTED MANUSCRIPT Revised Version IGF-I, T3, and T4 (P < 0.05) compared to RES ewes. Even though ewes were transitioned to a

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common diet after parturition, endocrine status continued to be impacted into lactation.

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Moreover, it appears that gestational diet may partially impact lactational performance through

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altered endocrine status.

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Key words: endocrinology, placenta, pregnancy, selenium, sheep

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1. Introduction Several researchers have shown alterations in steroid, somatotropic axis and thyroid hormones in animals fed above or below maintenance requirements during gestation [1,2,3,4].

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Endocrine profiles during gestation have been associated with placental nutrient transport

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capacity in the ewe [5] and improvements in endocrine and/or metabolic profiles during

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nutritional stress may be directly implicated in fetal growth and subsequent offspring

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performance. In addition to altered nutrient partitioning to the uterus during late gestation,

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endocrine profiles including steroids, prolactin, and growth hormone (GH) during gestation and

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lactation also impact proper mammary development and lactogenesis, which are vital for

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postnatal nutrition and development. In our experimental model, altered nutritional plane during

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gestation, followed by realimentation during lactation, affects colostrum and milk composition

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and yield in ewes [6,7]. Moreover, supranutritional selenium (Se) supplementation increased

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colostrum yield and milk yield in ewes [7]. Therefore, nutritional plane and Se supply may alter

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endocrine profiles during gestation, leading to changes in mammary development and

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preparation for the subsequent lactation. These endocrine effects could therefore directly cause

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alterations in postnatal growth and development irrespective of placental nutrient utilization

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during gestation. Despite this, there is a paucity of data linking gestational nutrition with

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gestational and lactational endocrine profiles and postpartum milk production, especially in the

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face of supranutritional Se.

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The objectives of the current experiment were to determine placental size at term and

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maternal endocrine profiles during gestation, parturition, and lactation in under or overnourished

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ewes, with or without supranutritional dietary Se, during gestation. We hypothesized that

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maternal placental characteristics and endocrine profiles would be altered by nutrient restriction

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ACCEPTED MANUSCRIPT Revised Version or overnourishment, and that these changes would persist into early lactation even when ewes

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were fed to a common nutritional plane. Additionally, we hypothesized that feeding a high Se

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diet during gestation would affect placental and endocrine characteristics, resulting in the

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observed increases in fetal growth and milk production.

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74 2. Materials and methods

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2.1 Animals and diets

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Institutional Animal Care and Use Committees at North Dakota State University, Fargo, and the USDA, ARS, U.S. Sheep Experiment Station (USSES; Dubois, ID) approved animal

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care and use for this study.

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Ewes were bred and managed as described in Meyer et al. [7,8]. Breeding occurred at the

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USSES, and at this time, Se treatments [adequate Se (ASe; 3.5 µg Se per kg of BW daily) or high

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Se (HSe; 65 µg Se per kg of BW daily)] were initiated. After transport to North Dakota State

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University on d 36 of gestation, pregnant Rambouillet ewe lambs (n = 84; 52.1 ± 6.2 kg) were

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individually housed. Ewes remained on their Se treatments (actual intakes: ASe, 11.5 µg Se/ kg

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BW daily; HSe, 77.0 µg Se/ kg BW daily), and on d 40 of gestation were assigned randomly to 1

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of 3 nutritional plane treatments supplying 60% (RES), 100% (CON), or 140% (EXC) of NRC

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[9] recommendations for 60 kg pregnant ewe lambs during mid to late gestation (weighted ADG

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of 140 g) except for Se. This resulted in a completely randomized design with a 2 × 3 factorial of

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Se supply x nutritional plane treatments (ASe-RES, ASe-CON, ASe-EXC, HSe-RES, HSe-CON,

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HSe-EXC; n = 14/treatment). At parturition 42 ewes (7/treatment) that gave birth to singleton

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lambs were selected to be mechanically milked twice daily for 20 d [7].

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All diets were fed once daily in a complete pelleted form (based on wheat middlings, beet

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ACCEPTED MANUSCRIPT Revised Version pulp, alfalfa meal, and ground corn). Three pellet formulations (adequate Se, high Se, and

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concentrated Se pellets; described in [8]) were blended to meet Se and metabolizable energy

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(ME) intake based on the Se treatment and nutritional plane of each ewe. The adequate Se pellet

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contained 15.9% crude protein (CP), 2.83 Mcal/kg ME, and 0.67 mg/kg Se [dry matter (DM)

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basis]. Selenium-enriched wheat mill run was used to replace wheat middlings and corn in the

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basal diet to make a high Se pellet (6.13 mg/kg Se, 16.6% CP, 2.82 Mcal/kg ME; DM basis).

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Purified seleno-methionine was added to achieve 37.1 ppm Se in the concentrated Se pellet

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(16.2% CP, 3.01 Mcal/kg ME; DM basis). Every 14 d, body weight (BW) was measured and

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diets were adjusted accordingly. Both Se supply and nutritional plane treatments were terminated

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at parturition. Ewes (n = 42) that were assigned to necropsy on day 20 of lactation were

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transitioned to receive 100% of NRC [9] requirements for early lactation, provided by the

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adequate Se pellet fed during gestation and a lactation protein supplement pellet [7]. A 5-d

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transition period was used to increase intake from the gestation to lactation level, and feed was

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delivered after each milking (2 times a day).

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All births were attended, and lambs were removed from their dams immediately after birth. After parturition, ewes were monitored closely until placentas were expelled, and time was

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recorded to calculate time elapsed from time of lambing to expelling of the placenta. At 3 h post-

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partum ewes received 1mL oxytocin (20 IU; AgriLabs, St. Joseph, MO) to facilitate collection of

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colostrum; 34 ewes had not expelled placentas at the time of oxytocin administration. Placentas

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were stored in a sealed bag at 4°C until processing, at which time the placenta was weighed and

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cotyledons were cut from the placenta, counted, and weighed. The inter-cotyledonary weight was

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considered to be the remaining weight of the placenta.

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Endocrine patterns were determined in a subset of singleton-bearing ewes (n = 42)

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ACCEPTED MANUSCRIPT Revised Version throughout gestation, parturition, and lactation. Blood samples were collected for both serum and

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plasma on days 39, 53, 67, 81, 95, 109, 123, 137, and 144 of gestation; h 0, 3, 6, 12, and 24 of

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parturition; and days 1, 3, 7, 14, and 20 of lactation.

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2.2 Progesterone analysis

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Progesterone was analyzed as previously described [10]. Briefly, a 50-µl sample of

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maternal serum was analyzed in duplicate. Progesterone concentrations were measured by

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chemiluminescence immunoassay using the Immulite 1000 (Siemens, Los Angeles, CA), where

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lesser-, medium-, and greater-progesterone pools were assayed in triplicate (1.4 ± 0.06, 3.2 ±

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0.06, and 12.0 ± 0.55 ng/mL, mean ± SEM for lesser-, medium-, and greater- pools,

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respectively). The intraassay and interassay CV were 3.4% and 5.1%, respectively.

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2.3 Estradiol 17β analysis

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Circulating concentrations of estradiol-17β were analyzed in all serum samples by RIA using methodology described by Perry and Perry [11]. Inter and intra-assay CVs were 4.2% and

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4.6%, respectively. The assay sensitivity was 0.4 pg/mL.

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2.4 Cortisol analysis

Serum samples were analyzed for cortisol concentration as previously described [6].

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Briefly, serum samples (10-µL) were assayed in triplicate by the chemiluminescence

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immunoassay (Immulite 1000, Siemens, Los Angeles, CA). Within each assay, lesser-, medium-

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, and greater-cortisol pools were assayed in duplicate (41.4 ± 0.6, 131.5 ± 1.2, and 335.6 ± 3.0

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ng/mL, mean ± SEM for lesser-, medium-, and greater-cortisol pools, respectively). The intra-

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and interassay CV were 8.7% and 4.8%, respectively.

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2.5 Growth hormone analysis

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Serum concentrations of GH were determined in the samples using the RIA procedures described by Hoefler and Hallford [12]. The double antibody RIA used rabbit anti-oGH-3

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(AFP0802210) and oGH-I-5 (AFP12855B) provided by the National Hormone and Peptide

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Program (NHPP, Torrance, CA). The intra- and interassay CV were 5.5% and 9.4%,

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respectively.

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2.6 IGF-I analysis

The concentration of IGF-I in plasma was determined using a commercially available

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ELISA (Diagnostic Systems Laboratories Inc., Webster, TX) [13,14]. Briefly, this assay used 2

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antibodies in a sandwich-type immunoassay. Horseradish peroxidase is linked to the secondary

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antibody, and in the presence of tetramethylbenzidine (a substrate for horseradish peroxidase),

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the absorbance is directly proportional to the concentration of IGF-I. The assay sensitivity was

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10 ng/mL; the intra- and interassay CV were < 10%.

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2.7 Prolactin analysis

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Serum prolactin [15] concentrations were determined in duplicate by double-antibody RIA using primary antisera (anti-oPRL-2, AFPC35810691R) and purified standard and

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iodination (oPRL-I-3, AFP10789B) preparations supplied by the NHPP. The intra- and

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interassay CV were 6.0% and 9.0%, respectively.

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2.8 Thyroid hormone analysis

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Serum thyroxine (T4) and triiodothyronine (T3) concentrations were determined by the

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chemiluminescence immunoassay using the Immulite 1000 (Siemens), utilizing components of

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commercial kits (Siemens) as we have described [4,16]. Within each assay, T4 and T3 pools

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were assayed in duplicate (20.5 ± 0.68, 73.3 ± 1.27, and 113.6 ± 1.41 ng/mL and 0.7 ± 0.02, 1.4

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± 0.02, and 3.3 ± 0.06 ng/mL, mean ± SEM for lesser-, medium-, and greater-pools for T4 and

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ACCEPTED MANUSCRIPT Revised Version T3, respectively). Fifteen-microliter and 25-µl serum samples were assayed in duplicate for T4

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and T3, respectively. The intraassay CV was 4.4% and 5.9% for T4 and T3, respectively, and the

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interassay CV was 4.6% and 5.0% for T4 and T3, respectively.

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2.9 Statistical analysis

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Placental data were analyzed using the MIXED procedure of SAS. The model statement included: nutritional plane, Se supply, and their interaction. Endocrine profiles were analyzed

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using repeated measures ANOVA of the MIXED procedure of SAS, and means were separated

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using the PDIFF option of the LSMEANS statement. The model statement included: nutritional

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plane, Se supply, time, and the respective interactions. Endocrine profiles were analyzed

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separately by gestation, parturition and lactation. In addition, hormone data were further

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analyzed by calculating area under the curve (AUC) using SigmaPlot 8.0 (Aspire Software

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International, Ashburn, VA). Area under the curve data were tested using the MIXED procedure

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of SAS. The model statement included: nutritional plane, Se supply, and nutritional plane by Se

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supply interaction. Means were separated using the PDIFF option of the LSMEANS statement.

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175 3. Results

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3.1 Placental characteristics

Offspring birth weights were reported previously [8]. Briefly, there was a Se supply by

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nutritional plane interaction (P = 0.08) where lambs born to ASe-RES ewes had lower birth

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weight compared to HSe-RES, ASe-CON, HSe-CON, and ASe-EXC. Total placental weight at

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term averaged 364 ± 12 g and was not affected (P > 0.20) by Se supply or maternal nutritional

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plane. There was an effect of nutritional plane (P = 0.03; Figure 1A) on cotyledonary number,

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with RES and EXC ewes having decreased number of cotyledons compared to CON ewes.

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ACCEPTED MANUSCRIPT Revised Version Cotyledonary weight was not different (P > 0.50) due to Se supply or nutritional plane. Placental

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inter-cotyledonary weight was not different due to Se supply or nutritional plane (P > 0.40).

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Time of placental delivery (Figure 1B) showed a main effect of nutritional plane (P < 0.05) and a

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tendency for a main effect of Se supply (P = 0.08). Placental delivery time was increased in RES

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versus CON or EXC fed ewes, while placental delivery tended to decrease in HSe versus ASe

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ewes.

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3.2 Endocrine profiles

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Progesterone concentrations throughout gestation showed a nutritional plane by

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gestational day interaction (P < 0.0001; Figure 2). At day 109, 123, 137, and 144 of gestation

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RES ewes had increased concentrations of progesterone relative to CON. Conversely,

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progesterone concentrations were decreased in EXC ewes compared to CON on the same days.

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A main effect of nutritional plane was observed for progesterone AUC during gestation (P <

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0.0001; Figure 2), which was increased in RES and decreased in EXC compared to CON.

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Estradiol-17β concentrations throughout gestation, parturition, and lactation are

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illustrated in Figure 3A, B, and C, respectively. A nutritional plane by gestation day interaction

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was observed for estradiol-17β concentrations (P < 0.05; Figure 3A), where RES ewes had

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elevated estradiol-17β at day 109 and 137 of gestation compared to CON, while EXC ewes had

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decreased estradiol-17β at day 109, 123, 137, and 144 of gestation compared to CON ewes.

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Estradiol-17β AUC during gestation showed a main effect of nutritional plane (P < 0.005; Figure

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3A), where estradiol-17β was increased in RES and decreased in EXC compared to CON.

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Concentrations of estradiol-17β postpartum showed a main effect of hour (P < 0.0001; Figure

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3B), which decreased dramatically during the first 6 h postpartum. Postpartum estradiol-17β

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AUC showed a main effect of nutritional plane (P < 0.05; Figure 3B), which was decreased in

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ACCEPTED MANUSCRIPT Revised Version EXC versus RES and CON ewes. In addition, postpartum estradiol-17β AUC showed a trend for

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a main effect of Se supply (P = 0.06; Figure 3B), which was increased in HSe versus ASe.

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Estradiol-17β concentrations during lactation showed a nutritional plane by lactation day

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interaction (P < 0.001; Figure 3C), where RES was increased and EXC was decreased at

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lactation day 1 and 3 compared to CON. No significant differences were observed for estradiol-

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17β AUC during lactation (Figure 3C).

Cortisol concentrations during gestation showed a main effect of gestation day (P <

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0.001; Figure 4A), where cortisol decreased from day 39 to day 67 of gestation then remained

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stable. Both gestational cortisol concentration and AUC showed a main effect of nutritional

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plane (P < 0.0001; Figure 4A), which was decreased in RES and increased in EXC compared to

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CON ewes. A main effect of hour was observed for postpartum cortisol concentrations (P <

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0.0001; Figure 4B), which decreased dramatically during the first 3 h postpartum. No effects of

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lactation day, nutritional plane, Se supply or their respective interactions were observed for

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cortisol concentrations during lactation (data not shown).

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Maternal GH concentrations throughout gestation, parturition, and lactation are illustrated in Figure 5A, B, and C, respectively. A trend for a nutritional plane by gestation day interaction

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was observed for GH concentrations (P = 0.06; Figure 5A), where RES ewes had increased GH

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concentrations at day 137 of gestation compared to CON ewes, and EXC ewes had decreased

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GH concentrations at day 109 and 137 of gestation compared to CON ewes. A main effect of

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nutritional plane was observed for gestational GH AUC (P < 0.05; Figure 5A), where EXC ewes

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had decreased GH AUC compared to RES and CON ewes. A nutritional plane by Se supply by

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postpartum hour interaction was observed for GH concentrations (P < 0.01; Figure 5B), where

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ASe-RES ewes had an increase in GH concentrations 3 h postpartum compared to all other ewes.

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ACCEPTED MANUSCRIPT Revised Version A main effect of nutritional plane (P < 0.001) and Se supply (P < 0.05) were also observed for

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GH AUC (Figure 5B), where GH AUC was increased in RES and decreased in EXC compared

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to CON ewes, and postpartum GH AUC was decreased in HSe compared to ASe ewes. A

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nutritional plane by lactation day interaction was observed for GH concentrations (P < 0.05;

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Figure 5C), where RES ewes had increased GH on day 1 of lactation compared to CON ewes,

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and EXC ewes had decreased GH concentrations on day 1 and 3 of lactation compared to CON

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ewes. A main effect of nutritional plane (P < 0.05) was observed for lactation GH AUC, which

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was decreased in EXC compared to RES and CON ewes (Figure 5C).

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Maternal IGF-1 concentration during gestation, parturition, and lactation are illustrated in

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Figure 6A, B, and C, respectively. A nutritional plane by gestation day interaction was observed

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for maternal IGF-1 concentrations (P < 0.0001; Figure 6A), where IGF-1 concentrations were

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increased in EXC compared to CON ewes at gestation day 123, 137, and 144. In contrast

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maternal IGF-I was decreased in RES compared to CON ewes at gestation day 109, 123, 137,

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and 144. Gestational IGF-I AUC showed a main effect of nutritional plane (P < 0.05; Figure

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6A), where RES ewes had decreased IGF-I AUC compared to EXC and CON ewes. Postpartum

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IGF-1 concentrations and AUC showed a main effect of nutritional plane (P < 0.0001), where

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IGF-1 was decreased in RES compared to CON ewes and increased in EXC compared to CON

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ewes. The main effect of postpartum h (P < 0.001) on IGF-1 concentrations showed a steady

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increase from 0 to 12 h postpartum and a decline back to baseline at 24 h postpartum. Maternal

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IGF-1 concentrations during lactation showed a nutritional plane by lactation day interaction (P

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< 0.0001; Figure 6C), where IGF-1 concentrations decreased in EXC and CON ewes during the

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first 7 days of lactation, while RES ewes remained low throughout lactation. The IGF-1 AUC for

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lactation was decreased in RES and increased in EXC compared to CON ewes (P < 0.001;

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Figure 6C). Maternal prolactin concentrations for gestation, parturition, and lactation are illustrated in

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Figure 7A, B, and C, respectively. The nutritional plane by gestation day interaction (P < 0.0001)

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for prolactin revealed an increase in EXC compared to CON ewes at day 123, 137, and 144 of

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gestation, while RES were decreased at day 144 of gestation compared to CON ewes (Figure

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7A). Gestational prolactin AUC revealed a main effect of nutritional plane (P < 0.0001), where

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EXC was increased compared to RES and CON ewes (Figure 7A). Postpartum prolactin

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concentrations decreased in all groups from 0 to 24 h (P < 0.0001; Figure 7B). A tendency (P <

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0.08) for decreased prolactin concentrations was observed for RES compared to EXC and CON

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ewes; however, no significant effect of nutritional plane (P = 0.18) was observed for postpartum

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prolactin AUC (Figure 7B). A main effect of nutritional plane (P < 0.005) and lactation day (P <

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0.01) was observed for prolactin (Figure 7C). Prolactin concentrations and AUC were increased

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in EXC compared to RES and CON ewes. In addition, prolactin concentrations decreased the

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first 7 days of lactation.

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Figure 8A, B, and C, respectively. Gestational T3 concentrations and AUC showed a main effect

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of nutritional plane (P ≤ 0.001; Figure 8A), where T3 was increased in EXC ewes versus RES

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and CON ewes. Postpartum T3 concentrations and AUC showed a main effect of nutritional

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plane (P < 0.05; Figure 8B), where RES ewes had decreased T3 compared to CON and EXC

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ewes. Lactation T3 concentrations showed a nutritional plane by day interaction (P < 0.005;

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Figure 8C), where EXC ewes had increased T3 on days 1, 3, and 7 of lactation compared to

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CON ewes. In addition, RES ewes had decreased T3 on day 1 of lactation compared to CON

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ewes. T3 AUC showed a main effect of nutritional plane (P < 0.01), where EXC ewes were

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increased compared to CON and RES ewes. Moreover, T3 AUC showed a main effect of Se

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supply (P < 0.05), where HSe ewes were decreased compared to ASe ewes. Maternal T4 concentrations during gestation, parturition, and lactation are illustrated in

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Figure 9A, B, and C, respectively. Gestational T4 concentrations showed a nutritional plane by

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day interaction (P < 0.001; Figure 9A), where T4 concentrations were decreased in RES ewes

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versus CON ewes at day 81, 95, 109, 123, and 137. In addition, T4 concentrations were

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increased in EXC ewes versus CON at day 137 and 144 of gestation. Chronic AUC T4

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concentrations throughout gestation (Figure 9A insert) were decreased in RES versus CON and

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EXC ewes. Postpartum T4 concentrations and AUC showed a main effect of nutritional plane (P

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< 0.0001; Figure 9B), where RES ewes had decreased T4 concentrations, while EXC ewes had

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increased T4 concentrations compared to CON ewes. Lactation T4 concentrations and AUC

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showed a main effect of nutritional plane (P < 0.001; Figure 9C), where EXC ewes had increased

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T4 compared to CON and RES ewes.

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4. Discussion

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In the current study, placental weight at term was not altered by nutritional plane or Se supply. Thus, the previously observed reduction in birth weight due to undernutrition and the

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rescue in birth weight due to supranutritional Se supply [8] were not due to alterations in

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placental weight at term. Even though cotyledonary number was reduced, total cotyledonary

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weight was similar between treatment groups. Placental delivery time was increased in RES

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ewes compared to CON and EXC ewes. A subset of animals (n = 34) received i.m. oxytocin

297

prior to placenta delivery (oxytocin was administered 3 h postpartum to facilitate colostrum

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ACCEPTED MANUSCRIPT Revised Version collection; [7]); however, the RES ewes still retained placentas on average 3 h after oxytocin

299

administration vs. approximately 1.5 to 1 hour after oxytocin administration for the CON and

300

EXC ewes, respectively. Microscopic examination of placental delivery has been previously

301

characterized in the ewe [17]. In general the rupture of fetal epithelial cells, rather than sole

302

separation of the interlocking microvilli, aids in placental delivery time [17], therefore nutrient

303

restriction during gestation may delay this rupture, resulting in delayed placental delivery time.

304

Additionally, supranutritional Se tended to reduce time to placenta delivery, which suggests that

305

high Se may accelerate rupture of fetal epithelial cells. Previous data in dairy cows suggest that

306

Se supplementation, with or without vitamin E, may reduce retained placentas in dairy cows,

307

although this Se supplementation is more effective in animals with low dietary Se (reviewed by

308

[18]).

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During the last third of gestation, both progesterone and estradiol-17β concentrations

310

were increased in nutrient restricted ewes and decreased in overnourished ewes compared to

311

controls. Our laboratory has previously reported this observation in a similar experiment [4].

312

Moreover, similar findings have been reported in gestating overnourished adolescent ewe lambs,

313

which had decreased progesterone concentrations from mid- to late gestation compared to

314

control fed dams [19]. Supplementing exogenous progesterone to overnourished adolescent ewe

315

lambs during the first third of gestation partially rescued fetal growth compared to control fed

316

ewe lambs [20]. In the Wallace et al. [20] study, fetal placental mass remained similar across

317

treatment groups; therefore, placental nutrient transfer capacity may have been altered via

318

progesterone supplementation during the time of maximal placental growth. Altered peripheral

319

steroid concentrations during a lower or higher nutritional plane may be due to differences in

320

hepatic steroid clearance, placental steroid production, and/or a combination of both metabolic

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plane [21], supporting the hypothesis of differences in steroid clearance with differences in

323

intake. Interestingly, during mid-gestation overnourished ewes showed no discernible differences

324

in placental steroidogenic acute regulatory protein expression or steroidogenic biosynthetic

325

enzyme expression in a previous study [22]. However, during late gestation placental mRNA

326

expression of P450 side chain cleavage was reduced in overnourished ewes with lower

327

peripheral progesterone concentrations [22]. Therefore, depending on the stage of pregnancy,

328

both hepatic steroid clearance and placental steroid synthesis capacity may directly alter serum

329

concentrations.

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In addition to the altered steroid profiles during gestation, these effects remain evident in estradiol-17β concentrations during the first few days of lactation. Estradiol-17β concentrations

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during the transition period from late pregnancy to early lactation have been implicated in fatty

333

acid mobilization [23]. Moreover, increased estradiol-17β during early lactation may have

334

partially altered milk nutrient composition, whereas RES ewes had elevated milk butterfat

335

concentrations versus EXC ewes [7].

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Average area under the curve for GH concentrations was decreased in EXC versus CON

337

and RES ewes. In contrast, IGF-I concentrations were increased in EXC versus RES, with CON

338

ewes being intermediate. Nutritional plane has been previously related to the somatotropic axis

339

in both cattle and sheep [2]. In addition, nutrient restriction has been associated with reduced

340

somatotropic negative feedback as a result of decreased hypothalamic somatostatin secretion,

341

increased GH pulse frequency, and decreased hepatic GH receptors, which results in decreased

342

circulating IGF-1 concentrations [2]. The positive association between nutritional plane and

343

hepatic GH receptor expression and/or binding affinity may explain the increased IGF-1

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concentrations in relation in overnourished adolescent ewe lambs with decreased GH

345

concentrations compared to control fed ewes.

346

Moreover, these responses in the somatotropic axis remain apparent during the first few days of lactation, even though ewes were placed on a common nutritional plane following

348

parturition. Ewes were transitioned to this common diet over 5 d, however, thus intakes were less

349

for RES and CON ewes than EXC during this period. Previous data from this study showed main

350

effects of both Se supply and nutritional plane on milk yield, where HSe ewes had greater milk

351

yield compared to ASe ewes, and RES ewes had decreased milk yield compared to CON and

352

EXC ewes during the first 20 days of lactation [7]. We did not observe any interactions with Se

353

supply or any main effects of Se supply on steroid, GH, IGF-1, or prolactin concentrations

354

during the first 20 days of lactation. However, GH concentrations during lactation were

355

decreased in EXC ewes compared to CON and RES ewes, while IGF-1 concentrations increased

356

from RES to CON to EXC ewes during the first 20 days of lactation. Moreover, concentrations

357

of prolactin during early lactation were increased in EXC versus CON and RES ewes. Pearson

358

correlation coefficients of total milk yield over the 20-day lactation [7] to average AUC for GH

359

concentrations revealed a positive association in CON ewes (P < 0.05; Figure 10) and a negative

360

association in EXC ewes (P < 0.05; Figure 10). Ewes with the lowest total milk yield, RES ewes,

361

showed no association of milk yield with AUC for GH, IGF-1 or prolactin concentrations during

362

lactation (P > 0.50; data not shown). Thus overnutrition during gestation may make ewes less

363

sensitive to GH during lactation, or may even predispose ewes to become resistant to GH during

364

lactation.

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Triiodothyroine concentrations were increased in EXC ewes versus CON and RES ewes during gestation, while thyroxine concentrations were decreased in RES ewes compared to CON

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368

and T4 in overnourished adolescent ewe lambs during gestation. Previous data in rodents showed

369

a relationship between thyroid hormone administrations and increased placental growth [24],

370

although in the current study, placental weight was not altered by nutritional plane or Se supply.

371

Previous reports in ewes showed that neither maternal IGF-I, T3, or T4 concentrations are altered

372

by Se supply during gestation [25]. In addition, Se is a component of multiple deiodinases [26],

373

which highlights the role of adequate Se intake on thyroid hormone output. However, thyroid

374

hormone metabolism may only be altered by restricting maternal Se intake, while the current

375

study found no differences in comparing dams with adequate Se versus supranutritional Se

376

intake throughout gestation. Maternal intake is a major determinant of thyroid status; however,

377

as evidenced by both EXC and RES intake during gestation altering both T3 and T4 profiles

378

throughout gestation, parturition, and lactation compared to CON-fed dams. Reduced lactational

379

T3 in ewes that were previously fed the HSe diet during gestation may suggest that ewes adapted

380

to greater Se intake, resulting in decreased deiodinase production when the supranutritional Se

381

was removed from the diet.

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In summary, increasing or decreasing maternal intake during gestation altered endocrine

383

profiles not only throughout gestation, but also into parturition and lactation, even though dams

384

were provided a similar nutritional plane following lambing. Although supranutritional Se has

385

altered fetal growth in this model, Se had a much lesser effect on maternal endocrine profiles

386

than plane of nutrition. Additionally, nutritional plane from mid- to late-gestation altered

387

concentrations of GH, IGF-1, and prolactin during early lactation and these were associated with

388

total milk yield when separated amongst nutritional plane treatment groups.

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ACCEPTED MANUSCRIPT Revised Version ACKNOWLEDGEMENTS

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Partially supported by USDA-NRI grants No. 2003-35206-13621 and 2005-35206-15281, from

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the USDA-CSREES, LEAP grant to KAV from National Science Foundation Grant # HRD-

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0811239 to the NDSU Advance FORWARD program, and by NIH Grant HL 64141. Authors

394

would like to thank employees of the Animal Nutrition and Physiology Center and Ruminant

395

Nutrition, Physiology and Muscle Biology Laboratories for their contributions to this project.

396

Appreciation is expressed to Dr. Parlow and the National Hormone and Peptide Program for

397

providing reagents used in the growth hormone and prolactin radioimmunoassays. Current

398

address for A. M. Meyer, Division of Animal Science, University of Missouri, Columbia, MO

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65211. Current address for C.O. Lemley, Department of Animal and Dairy Sciences, Mississippi

400

State University, Mississippi State, MS 39762.

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REFERENCES

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[1] Breier BH, Gluckman PD, Bass JJ. The somatrotrophic axis in young steers: influence of nutritional status and oestradiol-17β on hepatic high and low-affinity somatotrophic

404

binding sites. J Endocrinol 1988;116:169-77.

407 408 409

Anim Endocrinol 1999;17:209-18.

[3] Renaville R, Hammadi M, Portetelle D. Role of the somatotropic axis in the mammalian metabolism. Dom Anim Endocrinol 2002;23:351-60.

SC

406

[2] Breier BH. Regulation of protein and energy metabolism by the somatotropic axis. Dom

[4] Vonnahme KA, Neville TL, Perry GA, Redmer DA, Reynolds LP, Caton JS. Maternal

M AN U

405

RI PT

403

410

dietary intake alters organ mass and endocrine and metabolic profiles in pregnant ewe

411

lambs. Anim Reprod Sci 2013; accepted .

413 414

[5] Fowden AL, Ward JW, Wooding FPB, Forhead AJ, Constancia M. Programming placental nutrient transport capacity. J Phys 2006;572:5-15.

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412

[6] Swanson TJ, Hammer CJ, Luther JS, Carlson DB, Taylor JB, Redmer DA, Neville TL, Reed JJ, Reynolds LP, Caton JS, Vonnahme KA. Effects of gestational plane of nutrition and

416

selenium supplementation on mammary development and colostrum quality in pregnant

417

ewe lambs. J Anim Sci 2008;86:2415-23. [7] Meyer AM, Reed JJ, Neville TL, Thorson JF, Maddock-Carlin KR, Taylor JB, Reynolds LP,

AC C

418

EP

415

419

Redmer DA, Luther JS, Hammer CJ, Vonnahme KA, Caton JS. Nutritional plane and

420

selenium supply during gestation affect yield and nutrient composition of colostrum and

421

milk in primiparous ewes. J Anim Sci 2011;89:1627-39.

422 423

[8] Meyer AM, Reed JJ, Neville TL, Taylor JB, Hammer CJ, Reynolds LP, Redmer DA, Vonnahme KA, Caton JS. Effects of plane of nutrition and selenium supply during

20

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gestation on ewe and neonatal offspring performance, body composition, and serum

425

selenium. J Anim Sci 2010;88:1786-1800. [9] NRC. Nutrient Requirements of Sheep. 6th rev. ed. Natl. Acad. Press, Washington, 1985.

427

[10] Galbreath CW, Scholljegerdes EJ, Lardy GP, Odde KG, Wilson ME, Schroeder JW,

RI PT

426

428

Vonnahme KA. Effect of feeding flax or linseed meal on progesterone clearance rate in

429

ovariectomized ewes. Dom Anim Endocrinol 2008;35:164-9.

[11] Perry GA, Perry BL. Effects of standing estrus and supplemental estradiol on changes in

SC

430

uterine pH during a fixed-time artificial insemination protocol. J Anim Sci 2008;86:

432

2928-35.

433

M AN U

431

[12] Hoefler WC, Hallford DM. Influence of suckling status and type of birth on serum hormone

434

profiles and return to estrus in early-postpartum, spring-lambing ewes. Theriogenol

435

1987; 27:887–95.

438 439 440

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437

[13] Costine BA, Inskeep EK, Wilson ME. Growth hormone at breeding modifies conceptus development and postnatal growth in sheep. J Anim Sci 2005;83:810–5. [14] Koch JM, Wilmoth TA, Wilson ME. Periconceptional growth hormone treatment alters fetal growth and development in lambs. J Anim Sci 2010;88:1619-25.

EP

436

[15] Spoon RA, Hallford DM. Growth response, endocrine profiles and reproductive performance of fine-wool ewe lambs treated with ovine prolactin before breeding.

442

Theriogenology 1989;32:45–53.

443

AC C

441

[16] O'Neil MR, Lardy GP, Wilson ME, Lemley CO, Reynolds LP, Caton JS, Vonnahme KA.

444

Estradiol-17beta and linseed meal interact to alter visceral organ mass and hormone

445

concentrations from ovariectomized ewes. Domest Anim Endocrinol 2009; 37:148-58.

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447 448 449 450

[17] Perry JS, Heap RB, Ackland N. The ultrastructure of the sheep placenta around the time of parturition. J Anat 1975;120:561-70. [18] Spears JW, Weiss WP. Role of antioxidants and trace elements in health and immunity of transition dairy cows. Vet J 2008;176:70-6.

RI PT

446

[19] Wallace JM, Da Silva P, Aitken RP, Cruichshank MA. Maternal endocrine status in relation to pregnancy outcome in rapidly growing adolescent sheep. J Endocrinol 1997;155:359-

452

68.

SC

451

[20] Wallace JM, Bourke DA, Da Silva P, Aitken RP. Influence of progesterone supplementation

454

during the first third of pregnancy on fetal and placental growth in overnourished

455

adolescent ewes. Reprod 2003;126:481-7.

456

M AN U

453

[21] Meyer AM, Reed JJ, Neville TL, Taylor JB, Reynolds LP, Redmer DA, Vonnahme KA, Caton JS. Effects of nutritional plane and selenium supply during gestation on visceral

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organ mass and indices of intestinal growth and vascularity in primiparous ewes at

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parturition and during early lactation. J Anim Sci 2012;90:2733-49.

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TE D

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[22] Lea RG, Wooding P, Stewart I, Hannah LT, Morton S, Wallace K, Aitken RP, Milne JS, Regnault TR, Anthony RV, Wallace JM. The expression of ovine placental lactogen,

462

StAR and progesterone-associated steroidogenic enzymes in placentae of overnourished

463

growing adolescent ewes. Reprod 2007;133:785-96.

465 466 467

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[23] Bell AW. Regulation of organic nutrient metabolism during transition from late pregnancy to early lactation. J Anim Sci 1995;73:2804-19. [24] Spencer GSG, Robinson GM. Stimulation of placental, fetal and neonatal growth by thyroxine administration to pregnant rats. J Endocrinol 1993;139:275-9.

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[25] Ward MA, Neville TL, Reed JJ, Taylor JB, Hallford DM, Soto-Navarro SA, Vonnahme KA, Redmer DA, Reynolds LP, Caton JS. Effects of selenium supply and dietary

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restriction on maternal and fetal metabolic hormones in pregnant ewe lambs. J Anim Sci

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2008;86:1254-62.

EP

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[26] Beckett GJ, Arthur JR. Selenium and endocrine systems. J Endocrinol 2005;184:455-65.

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ACCEPTED MANUSCRIPT Revised Version FIGURE LEGENDS

474

Figure 1. Average cotyledon number (A) at term showed a main effect of maternal nutritional

475

plane (P = 0.03). Average time of placental delivery time (B) showed a main effect of nutritional

476

plane (P < 0.05) and a tendency for a main effect of Se supply (P < 0.10). Means with different

477

letters represent significant treatment differences (P < 0.05). Values are means ± SE.

478

Figure 2. Progesterone (P4) concentrations throughout gestation showed a nutritional plane by

479

gestational day interaction (P < 0.0001). Average progesterone AUC (bar graph insert) was

480

different (P < 0.0001) across nutritional plane treatments. *Represents difference (P < 0.05)

481

from CON within the same time point. Means with different letters represent significant

482

treatment differences (P < 0.05). Values are means ± SE.

483

Figure 3. Estradiol-17β concentrations during gestation (A) showed a nutritional plane by

484

gestation day interaction (P < 0.05), while average AUC for Estradiol-17β during gestation (bar

485

graph insert) showed a main effect of nutritional plane (P < 0.005). Estradiol-17β concentrations

486

during parturition (B) showed a main effect of time (P < 0.0001), while postpartum estradiol-17β

487

AUC showed a main effect of nutritional plane (P < 0.05) and a trend (P = 0.06) for a main

488

effect of Se supply. Estradiol-17β concentrations during lactation (C) showed a nutritional plane

489

by lactation day interaction (P < 0.001), while no significant differences were observed for

490

estradiol-17β AUC during lactation. *Represents difference (P < 0.05) from CON within the

491

same time point. Means with different letters represent significant treatment differences (P <

492

0.05). Values are means ± SE.

493

Figure 4. Cortisol concentrations during gestation (A) showed a main effect of gestation day (P

494

< 0.0001) and a main effect of nutritional plane (P < 0.0001), while cortisol AUC showed a main

495

effect of nutritional plane (P < 0.0001). Cortisol concentrations during parturition (B) showed

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ACCEPTED MANUSCRIPT Revised Version main effect of time (P < 0.0001). Means with different letters represent significant treatment

497

differences (P < 0.05). Values are means ± SE.

498

Figure 5. Concentrations of GH during gestation (A) showed a trend for a nutritional plane by

499

gestation day interaction (P = 0.06), while a main effect of nutritional plane (P < 0.05) was

500

observed for gestational GH AUC. Concentrations of GH during parturition (B) showed a

501

nutritional plane by Se supply by postpartum hour interaction (P < 0.01); while a main effect of

502

nutritional plane (P < 0.001) and Se supply (P < 0.05) were observed for GH AUC.

503

Concentrations of GH during lactation (C) showed a nutritional plane by lactation day interaction

504

(P < 0.05), while a main effect of nutritional plane (P < 0.05) was observed for lactation GH

505

AUC. *Represents difference (P < 0.05) from CON within the same time point. Means with

506

different letters represent significant treatment differences (P < 0.05). Values are means ± SE.

507

Figure 6. Concentrations of IGF-1 during gestation (A) showed a nutritional plane by gestation

508

day interaction (P < 0.0001), while gestational IGF-1 AUC showed a main effect of nutritional

509

plane (P < 0.05). Concentrations of IGF-1 during parturition (B) showed a main effect of

510

nutritional plane (P < 0.0001) and a main effect of time (P < 0.001). Concentrations of IGF-1

511

during lactation (C) showed a nutritional plane by lactation day interaction (P < 0.0001), while

512

the IGF-1 AUC showed a main effect of nutritional plane (P < 0.001). Means with different

513

letters represent significant treatment differences (P < 0.05). Values are means ± SE.

514

Figure 7. Prolactin concentrations during gestation (A) showed a nutritional plane by gestation

515

day interaction (P < 0.0001), while gestational prolactin AUC revealed a main effect of

516

nutritional plane (P < 0.0001). Prolactin concentrations during parturition (B) showed a main

517

effect of time (P < 0.0001) and a tendency for nutritional plane (P < 0.08). Prolactin

518

concentrations during lactation (C) showed a main effect of nutritional plane (P < 0.005) and a

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ACCEPTED MANUSCRIPT Revised Version main effect of lactation day (P < 0.01). *Represents difference (P < 0.05) from CON within the

520

same time point. Means with different letters represent significant treatment differences (P <

521

0.05). Values are means ± SE.

522

Figure 8. Concentrations of T3 during gestation (A) showed a main effect of nutritional plane (P

523

< 0.001). Concentrations of T3 during parturition (B) showed a main effect of nutritional plane

524

(P < 0.05). Concentrations of T3 during lactation (C) showed a nutritional plane by day

525

interaction (P < 0.005), while average T3 AUC showed a main effect of nutritional plane (P <

526

0.01) and a main effect of Se supply (P < 0.05). *Represents difference (P < 0.05) from CON

527

within the same time point. Means with different letters represent significant treatment

528

differences (P < 0.05). Values are means ± SE.

529

Figure 9. Concentrations of T4 during gestation (A) showed a nutritional plane by day

530

interaction (P < 0.001), while T4 AUC during gestation showed a main effect of nutritional plane

531

(P < 0.01). Concentrations of T4 during parturition (B) showed a main effect of nutritional plane

532

(P < 0.0001). Concentrations of T4 during lactation (C) showed a main effect of nutritional plane

533

(P < 0.001). *Represents difference (P < 0.05) from CON within the same time point. Means

534

with different letters represent significant treatment differences (P < 0.05). Values are means ±

535

SE.

536

Figure 10. Pearson correlations for total milk yield over the first 20 days of lactation (milk yield

537

data taken from Meyer et al., 2011) versus AUC for GH concentrations during the first 20 days

538

of lactation. Average AUC for GH concentrations revealed a positive association in CON ewes

539

(P < 0.05) and a negative association in EXC ewes (P < 0.05). Ewes with the lowest total milk

540

yield, RES ewes, showed no association of milk yield with AUC for GH (P > 0.50; data not

541

shown).

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Figure 1.

120

Nut, P = 0.03 a b

b

80

60

40

SC

Coteyledonary Number

A 100

20

CON

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B

500

Nut, P = 0.02

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Placental drop, min

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Figure 2.

1400

Nut*Day, P = 0.0001

b

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RES CON EXC

c

800 600 400

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P4 AUC

* *

200 0

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20

CON

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Nut, P < 0.0001

a

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140

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Figure 3.

Nut, P < 0.005

1200

Nut*Day, P < 0.05

b

1000

600

RES CON

400

EXC

c

800

200

*

0

RES

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CON

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* 10

SEM

*

*

*

0 20

40

60

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100

120

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Hrs; P < 0.0001

a

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Nut, P < 0.05;

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Estradiol (pg/mL)

Estradiol, AUC

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15

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30

a

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Figure 4.

Cortisol (ng/mL)

Cortisol AUC

Nut, P < 0.0001; Day, P < 0.0001

40

RES CON EXC

30

2000

a

Nut, P < 0.0001 b

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c 1000

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AC C

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Figure 5.

1200

GH (ng/mL)

GH, AUC

12

10

Nut, P < 0.05

1000

a

800

Nut*Day, P = 0.06

a RES CON EXC

b

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SEM

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Figure 6.

400

Nut; P = 0.02 a a

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b 2

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500

IGF, AUC*1000

RES CON EXC

5

a

400

b

SEM

3

cd

0

d

d

c

1

c

c

200

a b

2

b

300

Nut; P < 0.001

4

AC C

IGF-1 (ng/mL)

Nut*Day; P < 0.0001

EP

Postpartum (hours)

C 600

RES

CON

EXC

c d

100 0

5

10

Lactation (day)

15

SC

600

IGF-1 AUC*10,000

A

20

30

ACCEPTED MANUSCRIPT

Figure 7. 200 4000

150

Nut*Day, P < 0.0001

Nut, P < 0.0001 a

RES CON EXC

3000

b

2000

b

a

1000

b

0

RES CON EXC

100

RI PT

PRL AUC

Prolactin (ng/mL)

A

c c

SEM

50

*

*

d d

d

d

20

40

60

80

100

120

140

160

Hrs, P < 0.0001 Nut, P < 0.08

Nut, P = 0.18

400

500

300 200 100

400

0

RES

CON EXC

300 SEM

200

RES CON EXC

100 0

5

10

15

TE D

Prolactin (ng/mL)

600

PRL, AUC

B

M AN U

Gestation (day)

20

25

Postpartum (hours) 250 Nut, P = 0.005 Day, P = 0.01

Nut; P < 0.001

EP

RES CON EXC

a

b

1500

b

1000 500

150

AC C

Prolactin (ng/mL)

200

2000

PRL AUC

C

100

50

0

RES

CON

EXC

SEM

0 0

5

10

Lactation (day)

15

SC

0

20

ACCEPTED MANUSCRIPT

Figure 8.

120

Nut, P < 0.001 Day, P < 0.0001 RES CON EXC

T3 (ng/mL)

2.0

Nut, P = 0.001

100

T3 AUC

2.5

a

b

b

80 60 40 20 0

RI PT

A

RES CON EXC

1.5

1.0 SEM

0.0 20

40

60

80

100

120

140

160

4

Nut, P < 0.05 a b

2.0

RES CON EXC

3

1.5

a

1.0 0.5 0.0

RES

CON EXC

2

TE D

T3 (ng/mL)

2.5

Nut, P < 0.05 Hrs, P < 0.0001

T3 AUC

B

M AN U

Gestation (day)

SEM 1

0

5

10

15

20

25

3.0 RES CON EXC

AC C

T3 (ng/mL)

2.5

25

T3 AUC

Nut*Day; P < 0.005

EP

Postpartum (hours)

C

2.0

*

1.5

*

1.0

20

Nut, P < 0.01

b

a

b

Se, P < 0.05

a

b

15 10

5 0

RES CON EXC

ASe HSe

*

*

SEM

0.5

0.0 0

5

10

Lactation (day)

15

SC

0.5

20

ACCEPTED MANUSCRIPT

Figure 9.

100

RES CON EXC

T4 AUC

8000

Nut, P < 0.01

b

b

a

6000 4000 2000 0

RES CON EXC

80

* * *

SEM

*

*

20

40

60

80

*

*

40

100

120

140

160

140

T4 (ng/mL)

120

Nut, P < 0.0001 Hrs, P < 0.0001

100

T4, AUC

B 160

RES CON EXC

120

80

Nut, P < 0.001 b c

60 40 20 0

RES

TE D

80

SEM

0

5

a

CON EXC

100

60

M AN U

Gestation (day)

10

15

20

25

EP

Postpartum (hours)

1500

Nut, P < 0.001 RES CON EXC

1000

AC C

100

T4 AUC

C 120

SEM

80

60

Nut, P < 0.001 b

b

RES

CON

a

500

0

EXC

40 0

5

10

Lactation (day)

15

SC

60

T4 (ng/mL)

RI PT

Nut*Day, P = 0.05

T4 (ng/mL)

A 120

20

ACCEPTED MANUSCRIPT

Figure 10.

RI PT

30000

25000

SC

20000

15000

CON ewes r2 = 0.57; P < 0.05 EXC ewes r2 = -0.56; P < 0.05

5000 50

100

150

200

M AN U

10000

EP

TE D

GH AUC for 20 day Lactation

AC C

Total Milk Yeild for 20 day Lactation

35000

250

300