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
2
during pregnancy and lactation
3 C. O. Lemleya, A. M. Meyera, T. L. Nevillea, D. M. Hallfordb, L. E. Camachoa, K. R. Maddock-
5
Carlina, T. A. Wilmothc, M. E. Wilsonc, G. A. Perryd, D. A. Redmera, L. P. Reynoldsa, J. S.
6
Catona, and K. A. Vonnahmea
RI PT
4
8
a
9
University, Fargo, ND 58108
SC
7
10
b
11
88003
12
c
13
26506
14
d
M AN U
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
TE D
Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV,
Department of Animal Sciences, South Dakota State University, Brookings, SD, 57007
15
EP
16
Address for reprint requests and other correspondence: K. A. Vonnahme, 181 Hultz Hall, Dept
18
7630 PO Box 6050, Fargo, ND 58108-6050 (e-mail: [email protected]).
AC C
17
1
ACCEPTED MANUSCRIPT Revised Version Abstract
20
Objectives were to examine effects of selenium (Se) supply and maternal nutritional plane during
21
gestation on placental size at term and maternal endocrine profiles throughout gestation and early
22
lactation. Ewe lambs (n = 84) were allocated to treatments that included Se supply of adequate
23
Se (ASe, 11.5 µg/kg BW) or high Se (HSe, 77 µg/kg BW) initiated at breeding and nutritional
24
plane of 60% (RES), 100% (CON), or 140% (EXC) of requirements beginning on day 40 of
25
gestation. At parturition, lambs were removed from their dams, and ewes were transitioned to a
26
common diet that met requirements of lactation. Blood samples were taken from a subset of ewes
27
(n = 42) throughout gestation, during parturition, and throughout lactation to determine hormone
28
concentrations. Cotyledon number was reduced (P = 0.03) in RES and EXC compared with
29
CON ewes. Placental delivery time tended (P = 0.08) to be shorter in HSe compared with ASe
30
ewes, whereas placental delivery time was longer (P = 0.02) in RES compared with CON and
31
EXC ewes. During gestation, maternal progesterone, estradiol-17β, and growth hormone were
32
increased (P < 0.05) in RES and decreased (P < 0.05) in EXC compared with CON ewes. In
33
contrast, maternal cortisol, IGF-I, prolactin, triiodothyronine, and thyroxine were decreased in
34
RES and increased in EXC compared with CON ewes during gestation. Selenium supply did not
35
alter maternal hormone profiles during gestation. During parturition and lactation, maternal
36
hormone concentrations were influenced by both Se and maternal nutritional plane. During the
37
parturient process, HSe ewes tended to have greater (P = 0.06) concentrations of estradiol-17β
38
compared to ASe ewes. Three h after parturition there was a surge of growth hormone in ASe-
39
RES ewes that was muted in HSe-RES, and not apparent in other ewes. Growth hormone area
40
under the curve during the parturient process was increased (P < 0.05) in ASe-RES vs HSe-RES
41
ewes. Ewes that were overfed during gestation had reduced (P < 0.05) estradiol-17β, but greater
AC C
EP
TE D
M AN U
SC
RI PT
19
2
ACCEPTED MANUSCRIPT Revised Version IGF-I, T3, and T4 (P < 0.05) compared to RES ewes. Even though ewes were transitioned to a
43
common diet after parturition, endocrine status continued to be impacted into lactation.
44
Moreover, it appears that gestational diet may partially impact lactational performance through
45
altered endocrine status.
46
Key words: endocrinology, placenta, pregnancy, selenium, sheep
AC C
EP
TE D
M AN U
SC
RI PT
42
3
ACCEPTED MANUSCRIPT Revised Version 47 48
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].
50
Endocrine profiles during gestation have been associated with placental nutrient transport
51
capacity in the ewe [5] and improvements in endocrine and/or metabolic profiles during
52
nutritional stress may be directly implicated in fetal growth and subsequent offspring
53
performance. In addition to altered nutrient partitioning to the uterus during late gestation,
54
endocrine profiles including steroids, prolactin, and growth hormone (GH) during gestation and
55
lactation also impact proper mammary development and lactogenesis, which are vital for
56
postnatal nutrition and development. In our experimental model, altered nutritional plane during
57
gestation, followed by realimentation during lactation, affects colostrum and milk composition
58
and yield in ewes [6,7]. Moreover, supranutritional selenium (Se) supplementation increased
59
colostrum yield and milk yield in ewes [7]. Therefore, nutritional plane and Se supply may alter
60
endocrine profiles during gestation, leading to changes in mammary development and
61
preparation for the subsequent lactation. These endocrine effects could therefore directly cause
62
alterations in postnatal growth and development irrespective of placental nutrient utilization
63
during gestation. Despite this, there is a paucity of data linking gestational nutrition with
64
gestational and lactational endocrine profiles and postpartum milk production, especially in the
65
face of supranutritional Se.
SC
M AN U
TE D
EP
AC C
66
RI PT
49
The objectives of the current experiment were to determine placental size at term and
67
maternal endocrine profiles during gestation, parturition, and lactation in under or overnourished
68
ewes, with or without supranutritional dietary Se, during gestation. We hypothesized that
69
maternal placental characteristics and endocrine profiles would be altered by nutrient restriction
4
ACCEPTED MANUSCRIPT Revised Version or overnourishment, and that these changes would persist into early lactation even when ewes
71
were fed to a common nutritional plane. Additionally, we hypothesized that feeding a high Se
72
diet during gestation would affect placental and endocrine characteristics, resulting in the
73
observed increases in fetal growth and milk production.
RI PT
70
74 2. Materials and methods
76
2.1 Animals and diets
77
SC
75
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
79
care and use for this study.
M AN U
78
Ewes were bred and managed as described in Meyer et al. [7,8]. Breeding occurred at the
81
USSES, and at this time, Se treatments [adequate Se (ASe; 3.5 µg Se per kg of BW daily) or high
82
Se (HSe; 65 µg Se per kg of BW daily)] were initiated. After transport to North Dakota State
83
University on d 36 of gestation, pregnant Rambouillet ewe lambs (n = 84; 52.1 ± 6.2 kg) were
84
individually housed. Ewes remained on their Se treatments (actual intakes: ASe, 11.5 µg Se/ kg
85
BW daily; HSe, 77.0 µg Se/ kg BW daily), and on d 40 of gestation were assigned randomly to 1
86
of 3 nutritional plane treatments supplying 60% (RES), 100% (CON), or 140% (EXC) of NRC
87
[9] recommendations for 60 kg pregnant ewe lambs during mid to late gestation (weighted ADG
88
of 140 g) except for Se. This resulted in a completely randomized design with a 2 × 3 factorial of
89
Se supply x nutritional plane treatments (ASe-RES, ASe-CON, ASe-EXC, HSe-RES, HSe-CON,
90
HSe-EXC; n = 14/treatment). At parturition 42 ewes (7/treatment) that gave birth to singleton
91
lambs were selected to be mechanically milked twice daily for 20 d [7].
92
AC C
EP
TE D
80
All diets were fed once daily in a complete pelleted form (based on wheat middlings, beet
5
ACCEPTED MANUSCRIPT Revised Version pulp, alfalfa meal, and ground corn). Three pellet formulations (adequate Se, high Se, and
94
concentrated Se pellets; described in [8]) were blended to meet Se and metabolizable energy
95
(ME) intake based on the Se treatment and nutritional plane of each ewe. The adequate Se pellet
96
contained 15.9% crude protein (CP), 2.83 Mcal/kg ME, and 0.67 mg/kg Se [dry matter (DM)
97
basis]. Selenium-enriched wheat mill run was used to replace wheat middlings and corn in the
98
basal diet to make a high Se pellet (6.13 mg/kg Se, 16.6% CP, 2.82 Mcal/kg ME; DM basis).
99
Purified seleno-methionine was added to achieve 37.1 ppm Se in the concentrated Se pellet
SC
RI PT
93
(16.2% CP, 3.01 Mcal/kg ME; DM basis). Every 14 d, body weight (BW) was measured and
101
diets were adjusted accordingly. Both Se supply and nutritional plane treatments were terminated
102
at parturition. Ewes (n = 42) that were assigned to necropsy on day 20 of lactation were
103
transitioned to receive 100% of NRC [9] requirements for early lactation, provided by the
104
adequate Se pellet fed during gestation and a lactation protein supplement pellet [7]. A 5-d
105
transition period was used to increase intake from the gestation to lactation level, and feed was
106
delivered after each milking (2 times a day).
TE D
107
M AN U
100
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
109
recorded to calculate time elapsed from time of lambing to expelling of the placenta. At 3 h post-
110
partum ewes received 1mL oxytocin (20 IU; AgriLabs, St. Joseph, MO) to facilitate collection of
111
colostrum; 34 ewes had not expelled placentas at the time of oxytocin administration. Placentas
112
were stored in a sealed bag at 4°C until processing, at which time the placenta was weighed and
113
cotyledons were cut from the placenta, counted, and weighed. The inter-cotyledonary weight was
114
considered to be the remaining weight of the placenta.
115
AC C
EP
108
Endocrine patterns were determined in a subset of singleton-bearing ewes (n = 42)
6
ACCEPTED MANUSCRIPT Revised Version throughout gestation, parturition, and lactation. Blood samples were collected for both serum and
117
plasma on days 39, 53, 67, 81, 95, 109, 123, 137, and 144 of gestation; h 0, 3, 6, 12, and 24 of
118
parturition; and days 1, 3, 7, 14, and 20 of lactation.
119
2.2 Progesterone analysis
RI PT
116
Progesterone was analyzed as previously described [10]. Briefly, a 50-µl sample of
121
maternal serum was analyzed in duplicate. Progesterone concentrations were measured by
122
chemiluminescence immunoassay using the Immulite 1000 (Siemens, Los Angeles, CA), where
123
lesser-, medium-, and greater-progesterone pools were assayed in triplicate (1.4 ± 0.06, 3.2 ±
124
0.06, and 12.0 ± 0.55 ng/mL, mean ± SEM for lesser-, medium-, and greater- pools,
125
respectively). The intraassay and interassay CV were 3.4% and 5.1%, respectively.
126
2.3 Estradiol 17β analysis
M AN U
127
SC
120
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
129
4.6%, respectively. The assay sensitivity was 0.4 pg/mL.
130
2.4 Cortisol analysis
Serum samples were analyzed for cortisol concentration as previously described [6].
EP
131
TE D
128
Briefly, serum samples (10-µL) were assayed in triplicate by the chemiluminescence
133
immunoassay (Immulite 1000, Siemens, Los Angeles, CA). Within each assay, lesser-, medium-
134
, and greater-cortisol pools were assayed in duplicate (41.4 ± 0.6, 131.5 ± 1.2, and 335.6 ± 3.0
135
ng/mL, mean ± SEM for lesser-, medium-, and greater-cortisol pools, respectively). The intra-
136
and interassay CV were 8.7% and 4.8%, respectively.
137
2.5 Growth hormone analysis
AC C
132
7
ACCEPTED MANUSCRIPT Revised Version 138
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
140
(AFP0802210) and oGH-I-5 (AFP12855B) provided by the National Hormone and Peptide
141
Program (NHPP, Torrance, CA). The intra- and interassay CV were 5.5% and 9.4%,
142
respectively.
143
2.6 IGF-I analysis
The concentration of IGF-I in plasma was determined using a commercially available
SC
144
RI PT
139
ELISA (Diagnostic Systems Laboratories Inc., Webster, TX) [13,14]. Briefly, this assay used 2
146
antibodies in a sandwich-type immunoassay. Horseradish peroxidase is linked to the secondary
147
antibody, and in the presence of tetramethylbenzidine (a substrate for horseradish peroxidase),
148
the absorbance is directly proportional to the concentration of IGF-I. The assay sensitivity was
149
10 ng/mL; the intra- and interassay CV were < 10%.
150
2.7 Prolactin analysis
TE D
151
M AN U
145
Serum prolactin [15] concentrations were determined in duplicate by double-antibody RIA using primary antisera (anti-oPRL-2, AFPC35810691R) and purified standard and
153
iodination (oPRL-I-3, AFP10789B) preparations supplied by the NHPP. The intra- and
154
interassay CV were 6.0% and 9.0%, respectively.
155
2.8 Thyroid hormone analysis
AC C
EP
152
156
Serum thyroxine (T4) and triiodothyronine (T3) concentrations were determined by the
157
chemiluminescence immunoassay using the Immulite 1000 (Siemens), utilizing components of
158
commercial kits (Siemens) as we have described [4,16]. Within each assay, T4 and T3 pools
159
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
160
± 0.02, and 3.3 ± 0.06 ng/mL, mean ± SEM for lesser-, medium-, and greater-pools for T4 and
8
ACCEPTED MANUSCRIPT Revised Version T3, respectively). Fifteen-microliter and 25-µl serum samples were assayed in duplicate for T4
162
and T3, respectively. The intraassay CV was 4.4% and 5.9% for T4 and T3, respectively, and the
163
interassay CV was 4.6% and 5.0% for T4 and T3, respectively.
164
2.9 Statistical analysis
165
RI PT
161
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
167
using repeated measures ANOVA of the MIXED procedure of SAS, and means were separated
168
using the PDIFF option of the LSMEANS statement. The model statement included: nutritional
169
plane, Se supply, time, and the respective interactions. Endocrine profiles were analyzed
170
separately by gestation, parturition and lactation. In addition, hormone data were further
171
analyzed by calculating area under the curve (AUC) using SigmaPlot 8.0 (Aspire Software
172
International, Ashburn, VA). Area under the curve data were tested using the MIXED procedure
173
of SAS. The model statement included: nutritional plane, Se supply, and nutritional plane by Se
174
supply interaction. Means were separated using the PDIFF option of the LSMEANS statement.
TE D
M AN U
SC
166
175 3. Results
177
3.1 Placental characteristics
Offspring birth weights were reported previously [8]. Briefly, there was a Se supply by
AC C
178
EP
176
179
nutritional plane interaction (P = 0.08) where lambs born to ASe-RES ewes had lower birth
180
weight compared to HSe-RES, ASe-CON, HSe-CON, and ASe-EXC. Total placental weight at
181
term averaged 364 ± 12 g and was not affected (P > 0.20) by Se supply or maternal nutritional
182
plane. There was an effect of nutritional plane (P = 0.03; Figure 1A) on cotyledonary number,
183
with RES and EXC ewes having decreased number of cotyledons compared to CON ewes.
9
ACCEPTED MANUSCRIPT Revised Version Cotyledonary weight was not different (P > 0.50) due to Se supply or nutritional plane. Placental
185
inter-cotyledonary weight was not different due to Se supply or nutritional plane (P > 0.40).
186
Time of placental delivery (Figure 1B) showed a main effect of nutritional plane (P < 0.05) and a
187
tendency for a main effect of Se supply (P = 0.08). Placental delivery time was increased in RES
188
versus CON or EXC fed ewes, while placental delivery tended to decrease in HSe versus ASe
189
ewes.
190
3.2 Endocrine profiles
SC
RI PT
184
Progesterone concentrations throughout gestation showed a nutritional plane by
192
gestational day interaction (P < 0.0001; Figure 2). At day 109, 123, 137, and 144 of gestation
193
RES ewes had increased concentrations of progesterone relative to CON. Conversely,
194
progesterone concentrations were decreased in EXC ewes compared to CON on the same days.
195
A main effect of nutritional plane was observed for progesterone AUC during gestation (P
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
2
during pregnancy and lactation
3 C. O. Lemleya, A. M. Meyera, T. L. Nevillea, D. M. Hallfordb, L. E. Camachoa, K. R. Maddock-
5
Carlina, T. A. Wilmothc, M. E. Wilsonc, G. A. Perryd, D. A. Redmera, L. P. Reynoldsa, J. S.
6
Catona, and K. A. Vonnahmea
RI PT
4
8
a
9
University, Fargo, ND 58108
SC
7
10
b
11
88003
12
c
13
26506
14
d
M AN U
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
TE D
Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV,
Department of Animal Sciences, South Dakota State University, Brookings, SD, 57007
15
EP
16
Address for reprint requests and other correspondence: K. A. Vonnahme, 181 Hultz Hall, Dept
18
7630 PO Box 6050, Fargo, ND 58108-6050 (e-mail: [email protected]).
AC C
17
1
ACCEPTED MANUSCRIPT Revised Version Abstract
20
Objectives were to examine effects of selenium (Se) supply and maternal nutritional plane during
21
gestation on placental size at term and maternal endocrine profiles throughout gestation and early
22
lactation. Ewe lambs (n = 84) were allocated to treatments that included Se supply of adequate
23
Se (ASe, 11.5 µg/kg BW) or high Se (HSe, 77 µg/kg BW) initiated at breeding and nutritional
24
plane of 60% (RES), 100% (CON), or 140% (EXC) of requirements beginning on day 40 of
25
gestation. At parturition, lambs were removed from their dams, and ewes were transitioned to a
26
common diet that met requirements of lactation. Blood samples were taken from a subset of ewes
27
(n = 42) throughout gestation, during parturition, and throughout lactation to determine hormone
28
concentrations. Cotyledon number was reduced (P = 0.03) in RES and EXC compared with
29
CON ewes. Placental delivery time tended (P = 0.08) to be shorter in HSe compared with ASe
30
ewes, whereas placental delivery time was longer (P = 0.02) in RES compared with CON and
31
EXC ewes. During gestation, maternal progesterone, estradiol-17β, and growth hormone were
32
increased (P < 0.05) in RES and decreased (P < 0.05) in EXC compared with CON ewes. In
33
contrast, maternal cortisol, IGF-I, prolactin, triiodothyronine, and thyroxine were decreased in
34
RES and increased in EXC compared with CON ewes during gestation. Selenium supply did not
35
alter maternal hormone profiles during gestation. During parturition and lactation, maternal
36
hormone concentrations were influenced by both Se and maternal nutritional plane. During the
37
parturient process, HSe ewes tended to have greater (P = 0.06) concentrations of estradiol-17β
38
compared to ASe ewes. Three h after parturition there was a surge of growth hormone in ASe-
39
RES ewes that was muted in HSe-RES, and not apparent in other ewes. Growth hormone area
40
under the curve during the parturient process was increased (P < 0.05) in ASe-RES vs HSe-RES
41
ewes. Ewes that were overfed during gestation had reduced (P < 0.05) estradiol-17β, but greater
AC C
EP
TE D
M AN U
SC
RI PT
19
2
ACCEPTED MANUSCRIPT Revised Version IGF-I, T3, and T4 (P < 0.05) compared to RES ewes. Even though ewes were transitioned to a
43
common diet after parturition, endocrine status continued to be impacted into lactation.
44
Moreover, it appears that gestational diet may partially impact lactational performance through
45
altered endocrine status.
46
Key words: endocrinology, placenta, pregnancy, selenium, sheep
AC C
EP
TE D
M AN U
SC
RI PT
42
3
ACCEPTED MANUSCRIPT Revised Version 47 48
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].
50
Endocrine profiles during gestation have been associated with placental nutrient transport
51
capacity in the ewe [5] and improvements in endocrine and/or metabolic profiles during
52
nutritional stress may be directly implicated in fetal growth and subsequent offspring
53
performance. In addition to altered nutrient partitioning to the uterus during late gestation,
54
endocrine profiles including steroids, prolactin, and growth hormone (GH) during gestation and
55
lactation also impact proper mammary development and lactogenesis, which are vital for
56
postnatal nutrition and development. In our experimental model, altered nutritional plane during
57
gestation, followed by realimentation during lactation, affects colostrum and milk composition
58
and yield in ewes [6,7]. Moreover, supranutritional selenium (Se) supplementation increased
59
colostrum yield and milk yield in ewes [7]. Therefore, nutritional plane and Se supply may alter
60
endocrine profiles during gestation, leading to changes in mammary development and
61
preparation for the subsequent lactation. These endocrine effects could therefore directly cause
62
alterations in postnatal growth and development irrespective of placental nutrient utilization
63
during gestation. Despite this, there is a paucity of data linking gestational nutrition with
64
gestational and lactational endocrine profiles and postpartum milk production, especially in the
65
face of supranutritional Se.
SC
M AN U
TE D
EP
AC C
66
RI PT
49
The objectives of the current experiment were to determine placental size at term and
67
maternal endocrine profiles during gestation, parturition, and lactation in under or overnourished
68
ewes, with or without supranutritional dietary Se, during gestation. We hypothesized that
69
maternal placental characteristics and endocrine profiles would be altered by nutrient restriction
4
ACCEPTED MANUSCRIPT Revised Version or overnourishment, and that these changes would persist into early lactation even when ewes
71
were fed to a common nutritional plane. Additionally, we hypothesized that feeding a high Se
72
diet during gestation would affect placental and endocrine characteristics, resulting in the
73
observed increases in fetal growth and milk production.
RI PT
70
74 2. Materials and methods
76
2.1 Animals and diets
77
SC
75
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
79
care and use for this study.
M AN U
78
Ewes were bred and managed as described in Meyer et al. [7,8]. Breeding occurred at the
81
USSES, and at this time, Se treatments [adequate Se (ASe; 3.5 µg Se per kg of BW daily) or high
82
Se (HSe; 65 µg Se per kg of BW daily)] were initiated. After transport to North Dakota State
83
University on d 36 of gestation, pregnant Rambouillet ewe lambs (n = 84; 52.1 ± 6.2 kg) were
84
individually housed. Ewes remained on their Se treatments (actual intakes: ASe, 11.5 µg Se/ kg
85
BW daily; HSe, 77.0 µg Se/ kg BW daily), and on d 40 of gestation were assigned randomly to 1
86
of 3 nutritional plane treatments supplying 60% (RES), 100% (CON), or 140% (EXC) of NRC
87
[9] recommendations for 60 kg pregnant ewe lambs during mid to late gestation (weighted ADG
88
of 140 g) except for Se. This resulted in a completely randomized design with a 2 × 3 factorial of
89
Se supply x nutritional plane treatments (ASe-RES, ASe-CON, ASe-EXC, HSe-RES, HSe-CON,
90
HSe-EXC; n = 14/treatment). At parturition 42 ewes (7/treatment) that gave birth to singleton
91
lambs were selected to be mechanically milked twice daily for 20 d [7].
92
AC C
EP
TE D
80
All diets were fed once daily in a complete pelleted form (based on wheat middlings, beet
5
ACCEPTED MANUSCRIPT Revised Version pulp, alfalfa meal, and ground corn). Three pellet formulations (adequate Se, high Se, and
94
concentrated Se pellets; described in [8]) were blended to meet Se and metabolizable energy
95
(ME) intake based on the Se treatment and nutritional plane of each ewe. The adequate Se pellet
96
contained 15.9% crude protein (CP), 2.83 Mcal/kg ME, and 0.67 mg/kg Se [dry matter (DM)
97
basis]. Selenium-enriched wheat mill run was used to replace wheat middlings and corn in the
98
basal diet to make a high Se pellet (6.13 mg/kg Se, 16.6% CP, 2.82 Mcal/kg ME; DM basis).
99
Purified seleno-methionine was added to achieve 37.1 ppm Se in the concentrated Se pellet
SC
RI PT
93
(16.2% CP, 3.01 Mcal/kg ME; DM basis). Every 14 d, body weight (BW) was measured and
101
diets were adjusted accordingly. Both Se supply and nutritional plane treatments were terminated
102
at parturition. Ewes (n = 42) that were assigned to necropsy on day 20 of lactation were
103
transitioned to receive 100% of NRC [9] requirements for early lactation, provided by the
104
adequate Se pellet fed during gestation and a lactation protein supplement pellet [7]. A 5-d
105
transition period was used to increase intake from the gestation to lactation level, and feed was
106
delivered after each milking (2 times a day).
TE D
107
M AN U
100
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
109
recorded to calculate time elapsed from time of lambing to expelling of the placenta. At 3 h post-
110
partum ewes received 1mL oxytocin (20 IU; AgriLabs, St. Joseph, MO) to facilitate collection of
111
colostrum; 34 ewes had not expelled placentas at the time of oxytocin administration. Placentas
112
were stored in a sealed bag at 4°C until processing, at which time the placenta was weighed and
113
cotyledons were cut from the placenta, counted, and weighed. The inter-cotyledonary weight was
114
considered to be the remaining weight of the placenta.
115
AC C
EP
108
Endocrine patterns were determined in a subset of singleton-bearing ewes (n = 42)
6
ACCEPTED MANUSCRIPT Revised Version throughout gestation, parturition, and lactation. Blood samples were collected for both serum and
117
plasma on days 39, 53, 67, 81, 95, 109, 123, 137, and 144 of gestation; h 0, 3, 6, 12, and 24 of
118
parturition; and days 1, 3, 7, 14, and 20 of lactation.
119
2.2 Progesterone analysis
RI PT
116
Progesterone was analyzed as previously described [10]. Briefly, a 50-µl sample of
121
maternal serum was analyzed in duplicate. Progesterone concentrations were measured by
122
chemiluminescence immunoassay using the Immulite 1000 (Siemens, Los Angeles, CA), where
123
lesser-, medium-, and greater-progesterone pools were assayed in triplicate (1.4 ± 0.06, 3.2 ±
124
0.06, and 12.0 ± 0.55 ng/mL, mean ± SEM for lesser-, medium-, and greater- pools,
125
respectively). The intraassay and interassay CV were 3.4% and 5.1%, respectively.
126
2.3 Estradiol 17β analysis
M AN U
127
SC
120
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
129
4.6%, respectively. The assay sensitivity was 0.4 pg/mL.
130
2.4 Cortisol analysis
Serum samples were analyzed for cortisol concentration as previously described [6].
EP
131
TE D
128
Briefly, serum samples (10-µL) were assayed in triplicate by the chemiluminescence
133
immunoassay (Immulite 1000, Siemens, Los Angeles, CA). Within each assay, lesser-, medium-
134
, and greater-cortisol pools were assayed in duplicate (41.4 ± 0.6, 131.5 ± 1.2, and 335.6 ± 3.0
135
ng/mL, mean ± SEM for lesser-, medium-, and greater-cortisol pools, respectively). The intra-
136
and interassay CV were 8.7% and 4.8%, respectively.
137
2.5 Growth hormone analysis
AC C
132
7
ACCEPTED MANUSCRIPT Revised Version 138
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
140
(AFP0802210) and oGH-I-5 (AFP12855B) provided by the National Hormone and Peptide
141
Program (NHPP, Torrance, CA). The intra- and interassay CV were 5.5% and 9.4%,
142
respectively.
143
2.6 IGF-I analysis
The concentration of IGF-I in plasma was determined using a commercially available
SC
144
RI PT
139
ELISA (Diagnostic Systems Laboratories Inc., Webster, TX) [13,14]. Briefly, this assay used 2
146
antibodies in a sandwich-type immunoassay. Horseradish peroxidase is linked to the secondary
147
antibody, and in the presence of tetramethylbenzidine (a substrate for horseradish peroxidase),
148
the absorbance is directly proportional to the concentration of IGF-I. The assay sensitivity was
149
10 ng/mL; the intra- and interassay CV were < 10%.
150
2.7 Prolactin analysis
TE D
151
M AN U
145
Serum prolactin [15] concentrations were determined in duplicate by double-antibody RIA using primary antisera (anti-oPRL-2, AFPC35810691R) and purified standard and
153
iodination (oPRL-I-3, AFP10789B) preparations supplied by the NHPP. The intra- and
154
interassay CV were 6.0% and 9.0%, respectively.
155
2.8 Thyroid hormone analysis
AC C
EP
152
156
Serum thyroxine (T4) and triiodothyronine (T3) concentrations were determined by the
157
chemiluminescence immunoassay using the Immulite 1000 (Siemens), utilizing components of
158
commercial kits (Siemens) as we have described [4,16]. Within each assay, T4 and T3 pools
159
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
160
± 0.02, and 3.3 ± 0.06 ng/mL, mean ± SEM for lesser-, medium-, and greater-pools for T4 and
8
ACCEPTED MANUSCRIPT Revised Version T3, respectively). Fifteen-microliter and 25-µl serum samples were assayed in duplicate for T4
162
and T3, respectively. The intraassay CV was 4.4% and 5.9% for T4 and T3, respectively, and the
163
interassay CV was 4.6% and 5.0% for T4 and T3, respectively.
164
2.9 Statistical analysis
165
RI PT
161
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
167
using repeated measures ANOVA of the MIXED procedure of SAS, and means were separated
168
using the PDIFF option of the LSMEANS statement. The model statement included: nutritional
169
plane, Se supply, time, and the respective interactions. Endocrine profiles were analyzed
170
separately by gestation, parturition and lactation. In addition, hormone data were further
171
analyzed by calculating area under the curve (AUC) using SigmaPlot 8.0 (Aspire Software
172
International, Ashburn, VA). Area under the curve data were tested using the MIXED procedure
173
of SAS. The model statement included: nutritional plane, Se supply, and nutritional plane by Se
174
supply interaction. Means were separated using the PDIFF option of the LSMEANS statement.
TE D
M AN U
SC
166
175 3. Results
177
3.1 Placental characteristics
Offspring birth weights were reported previously [8]. Briefly, there was a Se supply by
AC C
178
EP
176
179
nutritional plane interaction (P = 0.08) where lambs born to ASe-RES ewes had lower birth
180
weight compared to HSe-RES, ASe-CON, HSe-CON, and ASe-EXC. Total placental weight at
181
term averaged 364 ± 12 g and was not affected (P > 0.20) by Se supply or maternal nutritional
182
plane. There was an effect of nutritional plane (P = 0.03; Figure 1A) on cotyledonary number,
183
with RES and EXC ewes having decreased number of cotyledons compared to CON ewes.
9
ACCEPTED MANUSCRIPT Revised Version Cotyledonary weight was not different (P > 0.50) due to Se supply or nutritional plane. Placental
185
inter-cotyledonary weight was not different due to Se supply or nutritional plane (P > 0.40).
186
Time of placental delivery (Figure 1B) showed a main effect of nutritional plane (P < 0.05) and a
187
tendency for a main effect of Se supply (P = 0.08). Placental delivery time was increased in RES
188
versus CON or EXC fed ewes, while placental delivery tended to decrease in HSe versus ASe
189
ewes.
190
3.2 Endocrine profiles
SC
RI PT
184
Progesterone concentrations throughout gestation showed a nutritional plane by
192
gestational day interaction (P < 0.0001; Figure 2). At day 109, 123, 137, and 144 of gestation
193
RES ewes had increased concentrations of progesterone relative to CON. Conversely,
194
progesterone concentrations were decreased in EXC ewes compared to CON on the same days.
195
A main effect of nutritional plane was observed for progesterone AUC during gestation (P
