Impact of manganese amino acid complex on tissue-specific trace mineral distribution and corpus luteum function in gilts

Abstract

Functional corpora lutea (CL) are required for pregnancy establishment and gestational maintenance in swine, and CL function is susceptible to environmental influences. Manganese (Mn) could be critical in regulating CL function since it is a component of the antioxidant enzyme Mn superoxide dismutase (MnSOD) as well as enzymes involved in cholesterol and steroid hormone synthesis. We hypothesized that a more bioavailable dietary Mn source would increase Mn content in the CL thereby influencing luteal function during the mid-luteal phase of the estrous cycle. Postpubertal gilts (n = 32) were assigned to one of four gestation diets. The control diet (CON) met or exceeded National Research Council (2012) requirements and was formulated to contain 20 parts per million (ppm) of added Mn in the form of Mn sulfate. Three additional diets included 20 (treatment [TRT]1), 40 (TRT2), or 60 (TRT3) ppm of added Mn from a Mn–amino acid complex (Availa-Mn; Zinpro Corporation) instead of Mn sulfate. Dietary treatment began at estrus synchronization onset and continued through 12 days post estrus (dpe) of the ensuing estrous cycle. Blood samples were collected at estrus onset, which was assigned as 0 dpe, as well as 4, 8, and 12 dpe. Gilts were euthanized and tissues were collected at 12 dpe. Serum progesterone (P₄) increased (P < 0.01) from 0 to 12 dpe but was unaffected by dietary treatment (P = 0.15) and there was no effect of the interaction between day and treatment (P = 0.85). Luteal Mn content increased (P ≤ 0.05) by 19%, 21%, and 24% in gilts fed TRT1, TRT2, and TRT3, respectively, compared to CON. Luteal P₄ concentrations decreased (P = 0.03) 25%, 26%, and 32% in gilts fed TRT1, TRT2, and TRT3, respectively, compared to CON. Relative to CON gilts, CL calcium content decreased (P = 0.02) by 36%, 24%, and 34% for TRT1, TRT2, and TRT3 gilts, respectively. Collectively, these data support the hypothesis that feeding a more bioavailable Mn source increases Mn accumulation in CL tissue. If and how this influences CL function may be related to altered luteal P₄ concentrations.

Introduction

The essential element manganese (Mn) is a cofactor for a wide range of metalloenzymes and is accumulated and utilized by almost all forms of life (Culotta et al., 2005). It is also involved in skeletal system development, energy metabolism, nervous system function, immune system function, and reproductive hormone function (Santamaria, 2008). Mn is a cofactor for enzymes involved in synthesis of cholesterol, the precursor for production of steroid hormones essential for proper reproductive function. It is also a component of the antioxidant enzyme Mn superoxide dismutase (MnSOD), the principal mitochondrial antioxidant enzyme (Johnson and Giulivi, 2005; Abreu and Cabelli, 2010). MnSOD protects cells from oxidative damage caused by reactive oxygen species (ROS). Normally there is an appropriate balance between ROS and antioxidants, but oxidative stress occurs when ROS production exceeds the scavenging ability of antioxidants (Agarwal et al., 2005; Wang et al., 2017). Oxidative stress results in DNA damage, lipid peroxidation, and protein damage, leading to disruptions in gene expression and cell signaling pathways (Allen and Tresini, 2000; Rizzo et al., 2012; Wang et al., 2017).

In the ovary, ROS function as critical modulators during the inflammatory reactions involved in follicular rupture; thus, ROS depletion by antioxidant enzymes during the follicular phase can hinder ovulation (Shkolnik et al., 2011; Rizzo et al., 2012). Following ovulation, the ruptured follicle transforms into the corpus luteum (CL), a temporary endocrine structure essential for the establishment and maintenance of pregnancy in pigs. Ovarian ROS are formed as a by-product of steroid hormone synthesis and electron transport associated with steroidogenesis is a primary site of free radical generation (González-Fernández et al., 2005; Rizzo et al., 2012; Wang et al., 2017). During the luteal phase, the CL produces large amounts of progesterone (P₄) which is accompanied by ROS production (Carlson et al., 1995; Wang et al., 2017). Peak luteal P₄ production occurs during the mid-luteal phase of the estrous cycle at approximately 12 days post estrus (dpe) (Stabenfeldt et al., 1969; Henricks et al., 1972; Anderson, 2009). MnSOD is the first enzyme to act on superoxide anions generated by the electron transport chain and MnSOD activity is enhanced during CL regression to protect luteal cells from oxidative damage caused by inflammation (Kodaman and Behrman, 2001; Kamiński et al., 2012). Since the CL is critical for proper reproductive function, understanding if dietary supplementation with Mn influences CL function is warranted.

The aggregate of research evaluating the effects of Mn on reproductive function is more than 50 yr old, but rapid genetic progress over the past decade has resulted in a sow herd with substantially greater fecundity. Thus, it is unclear if the current requirement estimates for Mn (National Research Council, 2012) allow for optimum reproductive performance in the modern highly prolific pig. In fact, the ability of trace mineral supplementation to alter CL function is an area of research that remains relatively unexplored in gilts, particularly as it relates to dietary Mn bioavailability. This study investigated the hypothesis that providing a more bioavailable (Liu et al., 2014) source of Mn would increase CL accumulation, consequently influencing luteal function by increasing P₄ production during the mid-luteal phase of the estrous cycle.

Materials and Methods

Animals and experimental design

All animal procedures were reviewed and approved by the Iowa State University (ISU) Institutional Animal Care and Use Committee. Thirty-two postpubertal gilts were used for this experiment. Animals were individually housed at the ISU Swine Nutrition Farm. All animals were acclimated to their pens for a minimum of 3 d with individual access to feed and water in thermal neutral conditions (18 ± 0.5 °C).

Gilts were allocated into dietary treatments by body weight (154 ± 8 kg). Diets were formulated based on National Research Council (2012) gilt requirements for less than 90 d gestation (Table 1) and Mn was included in a vitamin and mineral premix (Table 2). The control diet (CON) was formulated to contain 20 parts per million (ppm) of added Mn in the form of Mn sulfate. Three additional diets included 20 (treatment [TRT]1), 40 (TRT2), or 60 (TRT3) ppm of added Mn from a Mn amino acid complex (Availa-Mn; Zinpro Corporation) in place of Mn sulfate. Diets were mixed at the ISU Swine Nutrition Farm and samples from each diet were submitted to Midwest Laboratories (Omaha, NE) for proximate and trace mineral analysis (Table 3). All animals were limit-fed 2.7 kg at 0800 h daily throughout the trial.

Table 1.

Composition of gestation diets

Ingredient	Dietary inclusion (%)
Corn, yellow dent	62.81
DDGS1	20.00
Soybean meal (47% CP)	9.23
Premix containing vitamin and minerals2	2.50
Soybean oil	2.00
Limestone	1.55
Monocalcium phosphate	1.12
Salt	0.50
L-Lysine HCl	0.17
L-Threonine	0.03
Quantum Blue 5G Phytase	0.005
Choline3	0.08
Calculated chemical composition, as fed
Crude protein	15.06
Calcium	0.88
Phosphorous (total)	0.65
ATTD4 Phosphorous	0.32
SID5 Lysine	0.60
SID Methionine	0.24
SID Threonine	0.46

¹Corn distillers dried grains with solubles.

²Components of vitamin and mineral premix found in Table 2.

³Choline (choline chloride), 500 mg/kg.

⁴Apparent total tract digestibility.

⁵Standardized ileal digestible.

Table 2.

Vitamin and mineral premix contributions¹ to final diets

Component	Treatment2
	CON	TRT1	TRT2	TRT3
Manganese (Availa-Mn), ppm	—	20	40	60
Manganese (Mn sulfate), ppm	20	—	—	—
Zinc3, ppm	100	100	100	100
Copper4, ppm	15	15	15	15
Iron (Fe sulfate), ppm	100	100	100	100
Iodine (KI), ppm	0.50	0.50	0.50	0.50
Selenium (sodium selenite), ppm	0.30	0.30	0.30	0.30
Choline, ppm	500	500	500	500
Vitamin A, IU/kg	14,330	14,330	14,330	14,330
Vitamin D, IU/kg	2,205	2,205	2,205	2,205
Vitamin E, IU/kg	53	53	53	53
Vitamin K, mg/kg	4.0	4.0	4.0	4.0
Biotin, mg/kg	0.20	0.20	0.20	0.20
Folacin, mg/kg	2.2	2.2	2.2	2.2
Niacin, mg/kg	154	154	154	154
Pantothenic acid, mg/kg	33	33	33	33
Riboflavin, mg/kg	8.0	8.0	8.0	8.0
Thiamin, mg/kg	2.2	2.2	2.2	2.2
Vitamin B6, mg/kg	2.2	2.2	2.2	2.2
Vitamin B12, µg/kg	40	40	40	40

¹Vitamin and mineral premix included at rate of 2.5%.

2CON, control; TRT, treatment.

³Zinc = 50 ppm Availa-Zn, 50 ppm Zn sulfate.

⁴Copper = 10 ppm Availa-Cu, 5 ppm Cu sulfate.

Table 3.

Proximate and trace mineral analysis of total dietary treatments

	Treatment1
	CON	TRT1	TRT2	TRT3
Moisture, %	13.27	12.83	13.21	12.85
Dry matter, %	86.73	87.18	86.79	87.16
Protein (crude), %	14.6	14.6	14.9	15.2
Fat (crude), %	4.97	5.18	5.07	5.13
Fiber (acid detergent), %	4.8	5.0	5.0	5.2
Ash, %	5.12	4.64	4.40	4.41
Calcium, %	1.03	0.89	0.87	0.91
Copper, ppm	12.5	23.9	19.9	13.2
Iron, ppm	355	350	402	310
Magnesium, %	0.17	0.18	0.18	0.17
Manganese, ppm	44.7	37.1	67.9	85.2
Phosphorus, %	0.75	0.73	0.75	0.71
Potassium, %	0.67	0.67	0.69	0.67
Sodium, %	0.23	0.21	0.23	0.24
Sulfur, %	0.25	0.25	0.25	0.25
Zinc, ppm	113	113	154	118

¹CON, 20 ppm Mn sulfate; TRT1, 20 ppm Availa-Mn; TRT2, 40 ppm Availa-Mn; TRT3, 60 ppm Availa-Mn.

Gilts were estrus synchronized using altrenogest (Matrix; Merck Animal Health) as top dressing (15.0 mg altrenogest per day delivered per os) for 15 d. Administration of dietary treatments began at estrus synchronization (approximately 20 days before estrus [dbe]) and continued throughout the ensuing estrous cycle. Beginning 4 d post-altrenogest withdrawal, animals were observed for behavioral signs of standing estrus twice daily using direct boar exposure. Characteristics of estrus such as a swollen vulva, reddening of the vulva, and vulva discharge were recorded. Animals were classified as being in estrus when they would stand for back pressure, and time of estrus detection was assigned as 0 dpe.

Sample collection and processing

Blood samples for serum isolation were collected by jugular venipuncture at approximately 20 dbe, and 0, 4, 8, and 12 dpe before feeding. Serum tubes were kept at room temperature for 15 to 30 min to allow for appropriate clotting before separation of serum. Samples were processed by centrifugation at 4 °C for 15 min at 1,500 × g, followed by aspiration of serum for storage at −80 °C until further analysis. At 12 dpe animals were humanely euthanized using captive bolt penetration followed by exsanguination. Following euthanasia, ovaries were extracted, weighed, and the number of CL on each ovary were counted. CL on one ovary were measured by digital calipers and the ovary was fixed in 4% paraformaldehyde for 24 h and then transferred to 70% ethanol for storage. The remaining ovary had each CL excised, weighed, measured in three dimensions using digital calipers, flash frozen in liquid nitrogen, and stored at −80 °C. Additionally, uterus and heart weights were collected prior to tissue sampling and preservation. Liver tissue and left ventricle heart tissue were collected, flash frozen in liquid nitrogen, and stored at −80 °C.

Trace mineral analysis

Serum samples were submitted to the ISU Veterinary Diagnostic Laboratory (ISU-VDL) for trace mineral analysis of calcium, copper, iron, potassium, magnesium, manganese, molybdenum, phosphorus, selenium, and zinc using inductively coupled plasma-mass spectrometry (ICP-MS; Analytik Jena, Woburn, MA) in CRI mode with hydrogen as the skimmer gas. Briefly, samples were diluted in 1% nitric acid, mixed, and analyzed by ICP-MS. For quality control, Bismuth (Bi), Scandium (Sc), Indium (In), Lithium (Li), Yttrium (Y), and Terbium (Tb) were used as internal standards for the ICP-MS. Tissue samples (heart, liver, CL) were submitted to the ISU-VDL for trace mineral analysis of cadmium, calcium, chromium, cobalt, copper, iron, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, sodium, and zinc using the established standard operating procedure (SOP) of the diagnostic laboratory as previously described. Minerals with concentrations below detection limits have been omitted from results tables and are acknowledged in table footnotes. Results are reported as ppm or parts per billion (ppb) on a wet weight basis. The ISU-VDL utilizes references from “Mineral Levels in Animal Health” (Puls, 1994) when reporting results.

P₄ production and release

To assess CL function, P₄ was quantified in both circulating serum and within CL tissue. P₄ concentration in serum was determined using a P₄ enzyme-linked immunosorbent assay (ELISA) (EIA-1561) obtained from DRG International (Springfield, NJ) according to the manufacturer’s protocol. Serum from 0 and 4 dpe was run undiluted while serum from 8 and 12 dpe was diluted 1:2 and 1:3, respectively. Samples were analyzed in duplicate and the inter- and intra-assay coefficients of variation for serum P₄ were 3.80% and 3.96%, respectively.

Additionally, four CL from each animal were powdered with a mortar and pestle on dry ice and 100 mg of powdered tissue per animal was weighed into a new tube. One milliliter of 5% trichloroacetic acid was added and the tissue was homogenized (Blitek et al., 2016). Supernatant, containing the CL extract, was collected after centrifugation at 10,000 × g at 4 °C for 5 min and placed into a new tube. Luteal extracts were stored at −80 °C until P₄ concentrations were analyzed using DRG P₄ ELISA (EIA-1561) following extract dilution of 1:100. This approach has been used previously to quantify porcine P₄ (Rak-Mardyła et al., 2014; Rak et al., 2015). Samples were analyzed in duplicate and the intra-assay coefficient of variation for luteal P₄ was 4.01%.

Evaluation of superoxide dismutase activity

To assess the effects of a more bioavailable Mn source on total superoxide dismutase (SOD) activity, SOD activity was quantified using the Superoxide Dismutase Assay Kit (Item No. 706002) from Cayman Chemical (Ann Arbor, MI) according to the manufacturer’s protocol in serum collected at 20 dbe and 12 dpe. In addition, to assess CL SOD activity, cell lysates from CL tissue collected at 12 dpe were also evaluated. Samples were analyzed in duplicate and the intra-assay coefficient of variation for SOD activity in serum at 20 dbe, 12 dpe, and luteal SOD activity at 12 dpe was 4.08%, 4.77%, and 3.79%, respectively.

Evaluation of a more bioavailable Mn source on immune cell abundance and markers of inflammation

To determine the effect of Mn supplementation on immune cell profiles, whole blood samples collected at 12 dpe into plasma (ethylenediaminetetraacetic acid [EDTA]) tubes were submitted to the ISU-VDL for complete blood count (CBC) analysis using the established SOP of the diagnostic laboratory. Further, to determine if markers of inflammation were altered, tumor necrosis factor-alpha (TNFα) and lipopolysaccharide-binding protein (LBP) were evaluated in serum and plasma, respectively, collected at 12 dpe. Concentration of TNFα was determined using the Porcine TNFα Quantikine ELISA Kit (PTA00) obtained from R&D Systems (Minneapolis, MN). Concentration of LBP was determined using the LBP ELISA Kit (HK503) obtained from Hycult Biotech (Uden, The Netherlands) per manufacturer’s recommendations. Samples were analyzed in duplicate and the intra-assay coefficients of variation for TNFα and LBP were 4.03% and 3.25%, respectively.

Statistical analysis

Effects of treatment, day, when appropriate, and the interaction between treatment and day, when appropriate were assessed as a completely randomized design in SAS 9.4 (Cary, NC) utilizing a PROC MIXED analysis procedure. Data are represented as least squares means and considered significant if P ≤ 0.05 or to indicate a tendency for biological meaning if 0.05 < P < 0.10. Comparisons between individual treatments are represented as differences of least squares means using the probability of differences function. Orthogonal contrasts were used to determine the linear or quadratic effect of Availa-Mn concentration in the diet (TRT1, TRT2, and TRT3) on trace mineral concentrations in serum and tissues.

Results

Effect of Mn source and concentration on trace mineral concentration in serum and tissues

CL number (18.6 ± 1.3), weight (0.49 ± 0.09 g), diameter (9.2 ± 0.7 mm), and volume (663 ± 125 mm³) were not affected by dietary treatment (P ≥ 0.65). Similarly, no effect of dietary treatment was observed on ovary (10.55 ± 1.45 g), uterus (895.25 ± 159.81 g), or heart (609.34 ± 50.09 g) weight (P ≥ 0.60; data not shown).

In serum collected at 12 dpe there was no overall effect of dietary treatment (P ≥ 0.40) and Mn concentration ranged from 2.57 to 2.75 ppb (Table 4), although a tendency for a quadratic relationship was observed between serum iron concentration and dietary Availa-Mn concentration (P = 0.09). In left ventricle heart tissue, there was no overall effect of dietary treatment (P > 0.10) and Mn concentration ranged from 0.263 to 0.275 ppm (Table 5). However, a linear decrease in magnesium (P = 0.08) and molybdenum (P = 0.01) concentration was observed as dietary Availa-Mn increased. Trace mineral concentration in liver tissue collected at 12 dpe was altered by dietary treatment (Table 6). Specifically, a tendency for a decrease in calcium in TRT1 compared to CON was observed (P = 0.07) along with a linear decrease in calcium as dietary Availa-Mn increased (P = 0.05). Additionally, sodium concentration was decreased in TRT2 and TRT3 compared to TRT1 (P < 0.05), but no difference was observed between CON and TRT1 (P = 0.25). Mn concentration ranged from 2.38 to 2.5 ppm but was not affected by dietary treatment (P = 0.81). Trace mineral concentration in luteal tissue collected at 12 dpe was altered by dietary treatment and Mn concentrations ranged from 0.60 to 0.75 ppm (Table 7). Mn concentration increased (P = 0.05) and calcium concentration decreased (P = 0.02) in Availa-Mn treatments compared to CON, although no differences were observed when comparing TRT1 with TRT2 and TRT3 (P ≥ 0.33).

Table 4.

Trace mineral concentration 12 dpe in serum from gilts fed a control diet (CON) or diets supplemented with Availa-Mn

	Treatment1					P-value
Minerals	CON	TRT1	TRT2	TRT3	SEM2	Source3	Treatment4	Linear	Quadratic
Calcium, ppm	103.6	102.4	100.4	98.6	3.2	0.79	0.70	0.45	0.99
Copper, ppm	1.79	1.71	1.75	1.55	0.12	0.65	0.49	0.37	0.45
Iron, ppm	1.86	1.91	1.38	1.90	0.27	0.90	0.44	0.97	0.09
Magnesium, ppm	18.7	18.3	17.9	17.6	0.7	0.65	0.71	0.53	0.99
Manganese, ppb	2.63	2.75	2.57	2.75	0.30	0.75	0.96	1.00	0.65
Molybdenum, ppb	3.63	3.88	3.50	3.38	0.27	0.52	0.61	0.25	0.73
Phosphorus, ppm	40.2	39.2	38.5	37.1	1.3	0.59	0.43	0.33	0.86
Potassium, ppm	146.1	143.3	142.5	143.1	4.1	0.64	0.93	0.98	0.90
Selenium, ppb	287.8	277.4	262.1	247.0	19.2	0.71	0.47	0.29	0.99
Zinc, ppm	0.900	0.875	0.838	0.838	0.053	0.74	0.80	0.65	0.79

¹CON, 20 ppm Mn sulfate; TRT1, 20 ppm Availa-Mn; TRT2, 40 ppm Availa-Mn; TRT3, 60 ppm Availa-Mn.

²Each mean represents eight individually fed gilts.

³Source P-value reflects differences between CON and TRT1.

⁴Treatment P-value reflects the main effect of treatment.

Table 5.

Trace mineral concentration in heart tissue from gilts fed a control diet (CON) or diets supplemented with Availa-Mn

	Treatment1					P-value
Minerals2	CON	TRT1	TRT2	TRT3	SEM3	Source4	Treatment5	Linear	Quadratic
Calcium, ppm	72.5	84.1	75.8	76.6	8.3	0.33	0.79	0.54	0.66
Chromium, ppm	0.031	0.034	0.032	0.033	0.004	0.50	0.91	0.90	0.80
Cobalt, ppm	0.002	0.001	0.002	0.002	0.0004	0.18	0.40	0.26	0.28
Iron, ppm	43.8	42.3	42.6	44.1	1.7	0.55	0.85	0.49	0.81
Magnesium, ppm	179.4	187.0	178.3	179.8	2.9	0.07	0.14	0.08	0.15
Manganese, ppm	0.263	0.263	0.275	0.275	0.020	1.00	0.94	0.67	0.80
Molybdenum, ppm	0.033	0.036	0.028	0.024	0.004	0.55	0.11	0.01	0.63
Phosphorus, ppm	1,591	1,648	1,586	1,603	24	0.11	0.27	0.18	0.18
Potassium, ppm	2,747	2,816	2,762	2,740	44	0.28	0.61	0.27	0.78
Selenium, ppm	0.318	0.324	0.315	0.321	0.011	0.69	0.94	0.84	0.47
Sodium, ppm	1,073	994.4	1,070	968.0	49.1	0.27	0.33	0.68	0.12
Zinc, ppm	12.8	13.3	12.6	12.9	0.2	0.13	0.27	0.24	0.12

¹CON, 20 ppm Mn sulfate; TRT1, 20 ppm Availa-Mn; TRT2, 40 ppm Availa-Mn; TRT3, 60 ppm Availa-Mn.

²Cadmium and copper omitted from results due to detection limits of cadmium and a mechanical error in detection of copper.

³Each mean represents eight individually fed gilts.

⁴Source P-value reflects differences between CON and TRT1.

⁵Treatment P-value reflects the main effect of treatment.

Table 6.

Trace mineral concentration in liver tissue from gilts fed a control diet (CON) or diets supplemented with Availa-Mn

	Treatment1					P-value
Minerals	CON	TRT1	TRT2	TRT3	SEM2	Source3	Treatment4	Linear	Quadratic
Cadmium, ppm	0.022a	0.028b	0.023ab	0.027ab	0.002	0.03	0.07	0.71	0.09
Calcium, ppm	134.1b	98.9ab	66.8a	62.9a	13.2	0.07	< 0.01	0.05	0.36
Chromium, ppm	0.026	0.022	0.021	0.022	0.002	0.29	0.49	0.85	0.71
Cobalt, ppm	0.016	0.019	0.017	0.019	0.001	0.06	0.13	0.94	0.16
Copper, ppm	16.0	12.1	11.0	13.1	1.9	0.17	0.32	0.68	0.44
Iron, ppm	301.0	300.1	281.1	333.5	25.6	0.98	0.55	0.33	0.23
Magnesium, ppm	193.5	191.5	192.3	186.8	3.2	0.66	0.47	0.35	0.48
Manganese, ppm	2.48	2.38	2.39	2.50	0.11	0.53	0.81	0.46	0.73
Molybdenum, ppm	1.21	1.19	1.21	1.23	0.04	0.74	0.89	0.49	0.99
Phosphorus, ppm	3,209	3,163	3,181	3,121	55	0.56	0.72	0.62	0.60
Potassium, ppm	3,102	3,063	3,134	3,042	53	0.61	0.62	0.79	0.25
Selenium, ppm	0.821	0.806	0.809	0.788	0.022	0.64	0.76	0.57	0.68
Sodium, ppm	1,242a	1,134a	782.9b	776.1b	65.4	0.25	<0.01	<0.01	0.06
Zinc, ppm	61.5	61.4	65.4	61.6	4.7	0.99	0.92	0.97	0.55

¹CON, 20 ppm Mn sulfate; TRT1, 20 ppm Availa-Mn; TRT2, 40 ppm Availa-Mn; TRT3, 60 ppm Availa-Mn.

²Each mean represents eight individually fed gilts.

³Source P-value reflects differences between CON and TRT1.

⁴Treatment P-value reflects the main effect of treatment.

^abValues within a row with differing superscripts denote differences (P ≤ 0.05) between individual treatments.

Table 7.

Trace mineral concentration in luteal tissue from gilts fed a control diet (CON) or diets supplemented with Availa-Mn

	Treatment1					P-value
Minerals2	CON	TRT1	TRT2	TRT3	SEM3	Source4	Treatment5	Linear	Quadratic
Calcium, ppm	201.5b	129.3a	153.1ab	132.6a	17.2	< 0.01	0.02	0.83	0.12
Chromium, ppm	0.109	0.123	0.151	0.153	0.021	0.63	0.38	0.34	0.62
Cobalt, ppm	0.009a	0.017ab	0.018ab	0.027b	0.005	0.24	0.08	0.19	0.49
Copper, ppm	1.63	1.50	1.75	1.63	0.18	0.63	0.81	0.63	0.40
Iron, ppm	33.5	42.5	40.5	53.0	5.6	0.27	0.13	0.26	0.36
Magnesium, ppm	127.3	121.5	120.4	114.3	3.9	0.31	0.16	0.21	0.61
Manganese, ppm	0.600a	0.713b	0.725b	0.750b	0.039	0.05	0.05	0.54	0.91
Molybdenum, ppm	0.029	0.038	0.034	0.043	0.005	0.21	0.26	0.52	0.35
Phosphorus, ppm	1,979	1,991	1,964	1,819	86	0.93	0.46	0.19	0.59
Potassium, ppm	2,700	2,691	2,686	2,511	91	0.95	0.41	0.18	0.46
Selenium, ppm	0.465	0.463	0.488	0.441	0.018	0.92	0.35	0.38	0.09
Sodium, ppm	1,472	1,248	1,452	1,453	87	0.08	0.24	0.12	0.37
Zinc, ppm	8.25	8.38	8.38	7.63	0.31	0.78	0.27	0.14	0.38

¹CON, 20 ppm Mn sulfate; TRT1, 20 ppm Availa-Mn; TRT2, 40 ppm Availa-Mn; TRT3, 60 ppm Availa-Mn.

²Cadmium results omitted from table due to detection limits.

³Each mean represents eight individually fed gilts.

⁴Source P-value reflects differences between CON and TRT1.

⁵Treatment P-value reflects the main effect of treatment.

^abValues within a row with differing superscripts denote differences (P ≤ 0.05) between individual treatments.

Source of Mn decreased luteal concentration of P₄ at 12 dpe but did not affect serum P₄

Circulating P₄ increased from 0 to 12 dpe as expected in a normal estrous cycle (P < 0.01; Figure 1), but there was no overall effect of treatment on circulating P₄ (P = 0.15). There was also no interaction between treatment and day (P = 0.85). Interestingly, in CL extracts, P₄ concentrations decreased 25%, 26%, and 32% in gilts fed TRT1, TRT2, and TRT3, respectively, compared to CON (P = 0.03; Figure 2). However, evaluation of serum concentration of P₄ at only 12 dpe was not different between treatments (P = 0.50).

Figure 1.

Serum P₄ concentrations in gilts. P₄ was measured in serum of gilts supplemented with 20 ppm Mn sulfate (CON), 20 ppm Availa-Mn (TRT1), 40 ppm Availa-Mn (TRT2), or 60 ppm Availa-Mn (TRT3).

Figure 2.

Effect of dietary treatment on luteal P₄ concentrations in gilts. Gilts were fed 20 ppm Mn sulfate (CON), 20 ppm Availa-Mn (TRT1), 40 ppm Availa-Mn (TRT2), or 60 ppm Availa-Mn (TRT3). P₄ concentrations decreased (P < 0.05) 25%, 26%, and 32% in gilts fed TRT1, TRT2, and TRT3, respectively, compared to CON. Bars with differing superscripts denote significant differences between treatments (P < 0.05).

Effect of Mn source on SOD activity, immune cell abundance, and markers of inflammation

SOD activity was analyzed in serum at 20 dbe (prior to implementation of dietary treatments) and at 12 dpe. There was no effect of treatment at either time point (P ≥ 0.30) and no effect of the interaction between treatment and day (P = 0.80), although serum SOD activity was increased (P < 0.01) at 12 dpe (10.96 ± 3.72 units/mL) compared to 20 dbe (8.60 ± 3.20 units/mL). SOD activity was also analyzed in cell lysates from CL tissue collected at 12 dpe and there was no effect of treatment on luteal SOD activity (11.13 ± 1.95 units/mL; P = 0.77). CBC analysis of whole blood (EDTA) was performed to evaluate the effects of Mn on circulating immune cell populations and blood parameters at 12 dpe. No effect of dietary treatment was observed on any parameters measured (P ≥ 0.10; Table 8). The effect of Mn supplementation on markers of inflammation (TNFα and LBP) in circulation was examined. At 12 dpe, no effect of treatment was observed on TNFα concentration (35.42 ± 1.66; P = 0.65), while LBP concentration increased 66% in TRT2 (P = 0.01) and 50% in TRT3 (P < 0.06) compared to CON (Figure 3).

Table 8.

CBC 12 dpe from gilts fed a control (CON) diet or diets supplemented with Availa-Mn

	Treatment1
Parameters2	CON	TRT1	TRT2	TRT3	SEM	P-value
White blood cells, ×103/µL	18.8	17.6	17.3	17.2	0.8	0.46
Red blood cells, ×106/µL	8.10	8.06	8.20	8.29	0.21	0.85
Hemoglobin, g/dL	15.0	15.3	15.4	15.6	0.3	0.68
Hematocrit, %	45.7	46.2	46.1	46.6	0.9	0.92
MCV, fL	56.7	57.5	56.2	56.3	1.0	0.79
MCH, pg	18.6	19.1	18.8	18.8	0.4	0.86
MCHC, g/dL	32.8	33.2	33.3	33.4	0.2	0.14
RDW, %	15.9	15.9	15.8	15.5	0.4	0.84
Platelets, ×103/µL	250.6	241.8	254.5	234.4	26.3	0.94
MPV, fL	9.36	9.43	8.56	9.14	0.48	0.53
Neutrophils, ×103/µL	5.07	4.51	4.66	4.52	0.43	0.77
Lymphocytes, ×103/µL	12.0	11.5	11.1	11.2	0.6	0.73
Monocytes, ×103/µL	0.834	0.818	0.784	0.778	0.066	0.91
Eosinophils, ×103/µL	0.593	0.490	0.524	0.440	0.066	0.40
Basophils, ×103/µL	0.166	0.143	0.098	0.110	0.027	0.27
LUC, ×103/µL	0.140	0.120	0.141	0.145	0.024	0.86

¹CON, 20 ppm Mn sulfate; TRT1, 20 ppm Availa-Mn; TRT2, 40 ppm Availa-Mn; TRT3, 60 ppm Availa-Mn.

²MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red cell distribution width; MPV, mean platelet volume; LUC, large unstained cells.

Figure 3.

Effect of dietary treatment on LBP concentration in gilts. The concentration of LBP in plasma was quantified at 12 dpe in gilts fed 20 ppm Mn sulfate (CON), 20 ppm Availa-Mn (TRT1), 40 ppm Availa-Mn (TRT2), or 60 ppm Availa-Mn (TRT3). Concentration of LBP increased 66% in TRT2 (P = 0.01) and 50% in TRT3 (P = 0.06) compared to CON. Bars with differing superscripts denote significant differences between treatments (P ≤ 0.05).

Discussion

Mn is an essential trace mineral required for a variety of metabolic functions including enzyme activation, immune system function, and reproductive hormone function (Santamaria, 2008). It is also a cofactor for enzymes including MnSOD and enzymes involved in cholesterol synthesis (Curran, 1954; Benedict et al., 1965; Goering, 2003). During the luteal phase, the CL produces P₄ using cholesterol as a precursor and P₄ levels peak during the mid-luteal phase of the estrous cycle (around 12 dpe). Previous investigations on the effects of Mn on reproductive function and tissue Mn concentration date back to the 1950s and 1960s, leaving researchers to rely on data that are not reflective of the current swine genetics. This led us to conduct this study to evaluate the effects of supplementing gilt diets with various concentrations of a more bioavailable source of Mn on tissue trace mineral distribution and CL function. We hypothesized that the more bioavailable Mn would accumulate in the CL, influencing luteal function by increasing P₄ production during the mid-luteal phase of the estrous cycle.

Across multiple species, absorption of Mn takes place in the gastrointestinal tract, primarily in the duodenum and jejunum (Miller et al., 1972; Abrams et al., 1976; Ji et al., 2006), but some absorption can occur in the ileum (Bai et al., 2008). After absorption, Mn is transported to mitochondria-enriched tissues and organs for storage until it is needed for enzyme activation (Deng et al., 2013). Mn can be toxic in cases of overexposure; therefore, for Mn to reach its target it must be guided down a designated trafficking pathway by homeostatic factors such as cell surface and intracellular Mn transporters and Mn chaperones while avoiding detoxification factors designed to eliminate Mn from the cell (Culotta et al., 2005). True absorption of Mn is difficult to assess but tissue and blood concentrations of Mn are thought to remain stable regardless of dietary intake (Britton and Cotzias, 1966; Hansen et al., 2006a). This suggests Mn absorption or retention is dependent on Mn concentrations in the body, so absorption will not increase when dietary Mn rises above the amount of Mn needed for normal function (Tuormaa, 1996). Results from this study partially agree with this observation, with Mn concentrations remaining stable in the heart, liver, and serum, across dietary treatments. Conversely, we observed increased Mn concentrations in CL of gilts in Availa-Mn treatments compared to CL of gilts receiving the CON diet containing Mn sulfate. The increased luteal Mn observed in TRT1 gilts compared to CON gilts can be explained as an effect of dietary Mn source, given that these diets contained the same concentration of Mn. On the other hand, the reason behind the increase in luteal Mn in TRT2 and TRT3 gilts compared to CON is more complicated to explain, as both source and concentration of Mn differ in TRT2 and TRT3 diets compared to CON.

Although some literature on Mn content in the CL exists, recent work is scant. A study in ewes injected intravenously with radiolabeled ⁵⁴Mn chloride reported that Mn uptake was greatest in the Graafian follicles and CL compared to other ovarian components (Hidiroglou, 1975). This observation corroborates our findings, supporting the notion of tissue-specific absorption and accumulation of Mn. Preferential Mn uptake by the CL may be explained by the fact that the CL contains abundant mitochondria, where the highest concentration of Mn is found among different cellular components (Maynard and Cotzias, 1955). Additionally, the same study in ewes reported that luteal Mn uptake increased from the 4th to the 11th day of the estrous cycle when luteal P₄ production was greatest (Hidiroglou, 1975). This suggests Mn may be involved in luteal metabolism or activity.

Intestinal Mn absorption is impacted by several factors including developmental stage, dietary constituents, concentration of other trace minerals in the diet, and source of the mineral (Aschner and Aschner, 2005; Williams et al., 2012). Inorganic forms (sulfates, chlorides, carbonates, oxides) can combine with digesta components in the intestine forming insoluble complexes, resulting in excretion. Organic forms (e.g., metal amino acid complexes, metal amino acid chelates) are protected from these interactions, absorbed through the intestinal mucosa, and circulated to target tissues (Acda and Chae, 2002).

It has been previously reported that Mn absorption is inhibited by a high load of dietary cations such as calcium and cobalt, suggesting these cations compete for a common site of transfer (Dupuis et al., 1992). Results from our study may also demonstrate an interaction between Mn and calcium, with calcium concentration in the tissues being altered by the source or concentration of dietary Mn. This effect was observed in the liver and CL where calcium concentration was decreased in the Availa-Mn treatments compared to CON. Conclusions from this observation should consider that the CON diet contained a higher concentration of calcium than the Availa-Mn diets, which may influence these results. The relationship between Mn and calcium has been examined in other studies, with Hidiroglou et al. reporting that Mn deficiency appears to alter utilization of other minerals, particularly calcium (Hidiroglou et al., 1978). In ewes fed a low Mn diet (8 ppm) or a Mn-supplemented diet (60 ppm), the calcium content of kidney and muscle tissue was lower in ewes fed supplemental Mn. In day-old lambs born to Mn-supplemented ewes, calcium levels were higher in the heart and kidney (Hidiroglou et al., 1978). Previous studies in cattle have reported relationships between fertility and the calcium–phosphorous–manganese complex. Dairy cattle were able to maintain fertility when fed diets with less than 40 ppm of Mn when the calcium and phosphorus content of the diet were adequate and balanced (Hignett, 1960; Hidiroglou, 1979). However, fertility was depressed when dietary Mn was less than 40 ppm and the calcium to phosphorus ratio was high (Hignett, 1960; Hidiroglou, 1979). Additionally, fertility was restored when the diet contained high calcium to phosphorus ratios and when dietary Mn was high (over 100 ppm). Hignett (1960) also proposed that the influence of the calcium–phosphorus–manganese complex on bovine fertility suggests these minerals affect the utilization of the Mn required for the enzyme systems essential to the establishment and maintenance of pregnancy. Although our study did not evaluate reproductive performance parameters, we did observe a relationship between Mn and calcium in regard to tissue mineral concentration.

Mn is a cofactor for several enzymes involved in cholesterol synthesis including mevalonate kinase and farnesyl pyrophosphate synthase (Curran, 1954; Benedict et al., 1965; Goering, 2003). Cholesterol is a precursor for synthesis of steroid hormones including progestogens, androgens, and estrogens, all essential to proper reproductive function. Our discovery that increasing luteal Mn content was associated with decreased luteal P₄ concentrations countered our hypothesis. However, while luteal P₄ was decreased, circulating P₄ at 12 dpe was not different between treatments. Previous studies administering Mn via the diet reported no changes in serum cholesterol or P₄ in broilers or cattle, and there was no difference in P₄ in sheep (Hidiroglou and Shearer, 1976; Hansen et al., 2006b; Xie et al., 2014). The observation that differences in luteal concentration of P₄ due to treatment were unexpected, particularly given that serum P₄ remained unchanged between treatment groups. The consistency between treatments with respect to serum P₄ concentrations suggests the biological consequence for target tissues of luteal P₄ is unlikely to be different between groups but we cannot explicitly conclude if this response is positive or negative.

Trace minerals are essential for health and immunity as they are components of SOD enzymes, glutathione reductase, glutathione peroxidase, thioredoxin reductase, and catalase (Yatoo et al., 2013). These enzymes act as antioxidants and prevent oxidative stress induced by conditions such as environmental stress, production stress, and immune activation stress. Animals used in our study were presumably stress-free, which may explain why no changes were observed in SOD activity, circulating immune cells or TNFα. Conversely, LBP concentrations were increased in TRT2 and TRT3 compared to CON. This is possibly an effect of Mn concentration in the diet rather than Mn source, since TRT2 and TRT3 contained 40 and 60 ppm of supplemental Mn while CON and TRT1 contained only 20 ppm of supplemental Mn. Previous studies using trace mineral supplements (Availa-Zn; Zinpro Corporation) have also reported increases in circulating LBP which corroborates results from our study (Sanz Fernandez et al., 2014), although it is unclear if source or concentration of the mineral is responsible for the observed changes. This presents the question of whether circulating LBP is increased in response to a bacterial infection, or if circulating LBP is increased as a protective measure to better respond to potential bacterial challenges. If the latter, utilization of a stress model, such as heat stress, with known impacts in specific tissues, including compromising gut barrier integrity, would enable further evaluation of the putative value of Mn-induced increases of circulating LBP. A study in rats reported that lipopolysaccharide (LPS) injection at day 15 of pregnancy caused an increase in MnSOD mRNA in the CL and a decrease in serum P₄ levels, which led to the conclusion that MnSOD is induced by inflammatory cytokines (Sugino et al., 1998). While the precise mechanism of luteal SOD accumulation in response to heat stress remains unresolved, increased circulating LPS has been repeatedly demonstrated in heat-stressed pigs (Pearce et al., 2013; Ross et al., 2017) and has negative impacts on the female reproductive system (Bidne et al., 2018).

Conclusion

The genetic advancement of the U.S. sow herd has enabled unprecedented fecundity and opportunities to maximize the number of quality pigs weaned over a sow’s lifetime. However, understanding how specific dietary components, such as trace minerals and their bioavailability, influence the function of specific reproductive tissues is relatively unexplored. In this study, dietary supplementation with a more bioavailable Mn demonstrated accumulation in the CL during the mid-luteal phase of the estrous cycle in gilts. While luteal P₄ concentrations were reduced at 12 dpe, concomitant serum P₄ differences were not observed, suggesting the functional outcome of Mn source may be minimal, although a more thorough investigation into the molecular response of the CL as the result of Mn source is warranted. Additionally, the ability of higher dietary levels of Availa-Mn to elevate circulating LBP warrants further investigation of these diets to mitigate the deleterious results on production in environments or conditions which may elevate bacterial exposure.

Glossary

Abbreviations

CBC

complete blood count

CL

corpora lutea

dbe

days before estrus

dpe

days post estrus

EDTA

ethylenediaminetetraacetic acid

LBP

lipopolysaccharide-binding protein

LPS

lipopolysaccharide

Mn

manganese

MnSOD

manganese superoxide dismutase

P₄

progesterone

ROS

reactive oxygen species

SOD

superoxide dismutase

SOP

standard operating procedure

TNFα

tumor necrosis factor-alpha

Conflict of interest statement

J.M.S., Z.E.K., B.M.G., A.F.K., L.H.B., and J.W.R. report no conflict of interest and Z.J.R., W.P.S., M.E.W., and C.R. are employed by the Zinpro Corporation.