Examination of the role of the rumen in zinc metabolism

Abstract

Three experiments were conducted to characterize Zn appearance and disappearance in the rumen, rumen epithelial Zn importers and exporters, the directionality of Zn movement, and the effect of supplemental Zn on rumen epithelial structure. In experiments 1 and 2, ruminally fistulated steers (n = 4) weighing 736 ± 23.3 kg were used in a crossover experiment with 2 dietary Zn concentrations (ZINC; 0 [CON] or 120 mg supplemental Zn/kg DM [Zn120], from ZnSO₄) in a 45% forage, 55% concentrate diet (experiment 1) or a 92.5% concentrate and 7.5% forage diet (experiment 2) to assess Zn appearance and disappearance in the rumen using the washed reticulo-rumen technique (WRR). Experiment 3 used 20 individually housed Hampshire crossbred wether lambs (35.1 ± 4.57 kg) fed for 60-d to evaluate the impact of dietary grain and Zn concentrations on ruminal epithelium Zn concentration, Zn transporters, and proteins integral to epithelial integrity. This experiment was a 2 × 2 factorial, with 2 diet types (DIET; 45% forage and 55% concentrate [HF] or 7.5% forage and 92.5% concentrate [HG]), and 2 supplemental Zn treatments (ZINC; 0 mg [CON] or 120 mg [ZN] supplemental Zn/kg diet DM) from ZnSO_4. In experiments 1 and 2, ZINC did not affect the rate of disappearance (k) or plateau (B) digestive model parameters for buffer Zn (P ≥ 0.34) but ruminal Zn disappearance was numerically greater in experiment 2. In experiment 3, there was a DIET × ZINC effect (P = 0.05) where HG + ZN had the greatest rumen epithelial Zn, and HF + ZN had the least. There was an interaction between DIET and ZINC on papillae length (P = 0.05), where papillae length was shorter in HF than HG, and within HG, ZN animals had shorter papillae than CON (P = 0.01). A DIET × ZINC effect was noted for ZnT1 RNAscope score (P = 0.01) within the stratum spinosum layer where HG + CON wethers had greater mean ZnT1 RNAscope score than other treatments (P ≤ 0.006). Ruminal Claudin-7 protein expression was lesser in HF (P = 0.01) and tended to be lesser in ZN (P = 0.09). Overall, these experiments highlight the dynamic nature of ruminal Zn metabolism and are supporting evidence for the relationship between ruminal health and dietary grain and Zn concentrations.

The ruminal epithelia are involved in Zn metabolism, and Zn is absorbed into the rumen epithelia. Overall, the findings better characterize the dynamic nature of ruminal Zn metabolism and its importance for rumen health under differing nutritional conditions.

Introduction

In addition to the imperative roles of zinc (Zn) for the growth and reproduction of beef cattle, Zn is a vital component for epithelial integrity of the gastrointestinal tract (Sturniolo et al., 2002; Zhang and Guo, 2009; Opgenorth et al., 2021). Current recommendations indicate (NASEM, 2016) that beef cattle diets should contain 30 mg Zn/kg diet dry matter (DM); however, provision of Zn above NASEM (2016) recommendations can result in improved growth and feed efficiency in cattle (90 to 180 mg supplemental Zn/kg diet DM; Genther-Schroeder et al., 2016; Messersmith and Hansen, 2021; Messersmith et al., 2022). Yet high dietary Zn concentrations, from ZnSO₄ (~ 50 µg Zn/mL added in vitro; up to 120 mg Zn/kg added in vivo, ZnSO₄) may decrease overall DM and fiber digestion (Eryavuz and Dehority, 2009; Genther and Hansen, 2015).

In the feedlot, diets with high proportions of grain relative to forage are common due to the advantages in energy content and improvements in efficiency and growth performance (Loerch, 1990; Owens, 2010). High-grain diets are rich in rapidly fermentable carbohydrates and result in greater, more rapid production of short-chain fatty acids (SCFA) compared to a high-forage diet. Ruminal gross morphology has been shown to adapt to increasing concentrations of dietary grain, by increasing the size and surface area of the papillae, resulting in increased absorptive capacity for SCFA (Odongo et al., 2006). Increased supply of highly fermentable carbohydrates, like concentrations found in feedlot diets, can result in rapid production and accumulation of SCFA in the rumen (Gressley et al., 2011), creating a ruminal environment with low pH and shifts in microbial populations potentially leading to altered barrier function, cellular stress (Bondzio et al., 2011) inflammation (Ametaj et al., 2009) and decreased rates for SCFA absorption (Schwaiger et al., 2013). Zinc supplementation has been shown to improve intestinal barrier function and intestinal architecture characteristics in cattle (Horst et al., 2020; Opgenorth et al., 2021). However, the role of Zn related to functional characteristics of the ruminal epithelium remains poorly understood. Likewise, the role of the rumen in trace mineral (TM) metabolism remains largely unknown.

While ruminal Zn uptake by the ruminal epithelium has been shown to occur (Arora et al., 1969; Wright et al., 2008), prior work suggests there is no appreciable basolateral release of Zn across the ruminal epithelium (Wright et al., 2008). There is also evidence that the ruminal epithelium may release small amounts of Zn into the lumen (Weston and Kastelic, 1967) helping to partially explain the greater ruminal Zn concentration than would be predicted with dietary supply alone. Despite these findings, TM transporters are poorly characterized in ruminants, and virtually unknown within the rumen epithelium.

Therefore, the objectives of the experiments described herein were to evaluate Zn appearance and disappearance in the rumen, characterize Zn importers and exporters found within the rumen epithelium determine the directionality of Zn movement, and evaluate the effect of supplemental Zn on ruminal epithelial structure. Our hypothesis was that the ruminal epithelium will play a significant role in Zn flux, and more Zn will be absorbed in response to a grain-based diet. Additionally, it was hypothesized that Zn supplementation will positively impact ruminal tight junction (TJ) protein expression.

Materials and Methods

All animal procedures and protocols were approved by the Iowa State University Animal Care and Use Committee (IACUC Log #: 9-14-7858-B). A series of 3 experiments were conducted to evaluate the role of the rumen in Zn metabolism, and the impact of supplemental Zn on rumen epithelium structures.

Experiment 1

Four individually housed ruminally fistulated steers weighing 736 ± 23.3 kg were used in crossover experiment consisting of 2 dietary Zn concentrations (ZINC) in a 45% forage:55% concentrate diet (0 (CON) or 120 mg supplemental Zn/kg DM (Zn120), from ZnSO₄; Table 1) to assess Zn appearance and disappearance in the rumen using the washed reticulo-rumen technique (WRR). Two ruminal buffers with differing Zn concentrations were incubated independently in the WRR (0 or 0.14 mM Zn, from ZnSO₄; Table 2), and the selection of 0.14 mM Zn was based on the ruminal Zn concentration for steers receiving 120 mg Zn/kg dietary DM. Feed intake was measured daily and TMR samples were collected weekly. This experiment took place from August through mid-September in Ames, IA, with an average daily air temperature of 23.2 °C. In experiment 1, the CON and Zn120 diets were analyzed and contained 18 and 99 mg Zn/kg DM, respectively. Each period consisted of 13-d of diet adaptation where pH was continuously monitored (15 min intervals) using indwelling pH boluses (Well Cow Limited, Roslin, UK).

Table 1.

Diet ingredient composition and nutrient content for experiments 1 and 2

	Experiment 11	Experiment 22
Ingredient	% of diet DM
Cracked corn	18	62
Corn silage	50	15
Beet pulp	20	5
Soybean meal	10.05	16.55
Limestone	1	1
Urea	0.5
Salt	0.31	0.31
Vitamin A premix3	0.11	0.11
Trace mineral premix4	0.016	0.016
Rumensin905	0.0135	0.0135
Nutrient analyses
CP6, %	13.03	14.58
aNDF6, %	25.71	12.59
Fat (EE)6, %	2.61	2.85
Ash6, %	5.25	3.64
NEm7, Mcal/kg	2.04	1.87
NEg7, Mcal/kg	1.39	1.24
Cu8, mg/kg	16.1	12.9
Fe8, mg/kg	169	88
Zn8, mg/kg	18	23

¹Experiment 1 Zn treatments: supplemental Zn (0 mg [CON] or 120 mg [Zn120; analyzed as 99 mg Zn/kg] supplemental Zn/kg diet DM) from ZnSO_4.

²Experiment 2 Zn treatments: supplemental Zn (0 mg [CON] or 120 mg [Zn120 analyzed as 115 mg Zn/kg] supplemental Zn/kg diet DM) from ZnSO_4.

³Vitamin A premix contained 4,400,000 IU vitamin A/kg.

⁴Provided per kg of diet: 20 mg Mn, 10 mg Cu, 0.5 mg I, 0.1 mg Co, and 0.1 mg Se, either 0 mg Zn for CON, or 120 mg Zn for Zn120 treatment diets (all inorganic sources).

⁵Provided at 27 g/907 kg of diet (Elanco Animal Health, Greenfield, IN).

⁶Proximate analyses completed by Dairyland Laboratories (Arcadia, WI).

⁷Calculated based on diet nutrient analyses.

⁸Cr and Zn analyses were completed via ICP-OES (Pogge and Hansen, 2013).

Table 2.

Wash and incubation buffer composition for experiments 1 and 2

		Incubation buffer
Chemical, mmol/L	Wash buffer	CON Buffer	ZN Buffer
Calcium chloride		2.0	2.0
Magnesium chloride		2.0	2.0
Sodium chloride	105	5.0	5.0
Potassium chloride		5.0	5.0
Sodium acetate	10	30.0	30.0
Potassium acetate		35.0	35.0
Sodium propionate	20	35.0	35.0
Sodium butyrate		8.0	8.0
Butyric acid		7.0	7.0
Mannitol		5.0	5.0
Sodium bicarbonate	25	25.0	25.0
Zinc sulfate			0.14
Chromium EDTA		2.0	2.0
Analyzed composition
Cr1, mg/L		55	63
Zn1, mg/L		0.09	5.5
Osmolality2, osm	310	304	307
pH	6.24	6.23	6.23

¹Cr and Zn analyses were completed via ICP-OES (Pogge and Hansen, 2013).

²Osmolality was measured using a freezing point osmometer.

On day 14 (wash day 1), the temporarily isolated WRR was employed using each of 2 treatment buffers (15 L) containing 0 or 10 mg Zn/L (140 μM Zn) from ZnSO₄ in a randomly selected order per steer. The WRR was performed as detailed in previous work (Zhang, Albornoz, et al., 2013; Zhang, Aschenbach, et al., 2013). Briefly, ruminal contents were evacuated and stored in a covered container. The reticulo-rumen was washed using warm tap water, and a pre-heated washing buffer solution (38 °C, Table 2). The reticulo-rumen was isolated using devices designed to occlude the esophagus and reticulo-omasal orifice. Saliva was continually aspirated using a vacuum pump and collected into a 10-L carboy. Fifteen liters of an incubation buffer containing SCFA, chromium-EDTA (Cr), and the Zn treatments were incubated in the rumen for 65 min. The osmolality of the experimental buffer (before infusion with no preservative) was measured in duplicate via a freezing point osmometer (Osmette XL, Precision Systems, Natick, MA). One hundred percent CO₂ was continuously infused into the buffer to facilitate mixing. Samples of the incubation buffer were taken prior to insertion into the rumen, and at 5, 20, 35, 50, and 65 min after infusion. After the initial buffer incubation period, the reticulo-rumen was washed twice using a pre-heated washing buffer solution, and the second experimental buffer was incubated. Disappearance rates were adjusted for ruminal water flux using Cr concentration (Zhang, Aschenbach, et al., 2013). On day 16 (wash day 2), the WRR technique was completed again using both the CON and ZN buffers. To effectively characterize Zn disappearance from ruminal buffers, we created a model with 2 parameters, a coefficient/rate (k) and a plateau of disappearance (B).

Experiment 2

After the completion of experiment 1, the same 4 steers were transitioned over 4 wk to a diet of 92.5% concentrate and 7.5% forage, representing a common finishing cattle diet. For experiment 2, these steers were again used in a crossover experiment consisting of 2 dietary Zn concentrations (ZINC; 0 [CON] or 120 mg supplemental Zn/kg DM [Zn120], from ZnSO₄; Table 1). In experiment 2, the CON and Zn120 diets analyzed Zn concentrations of 23 and 115 mg/kg DM, respectively. The steers were on the final diet for 2 wk prior to the beginning of experiment 2. Feed intake was measured daily and TMR samples were collected weekly. This experiment took place from October through mid-November in Ames, IA, with an average daily air temperature of 11.7 °C. The WRR was completed as described in experiment 1 on days 14 and 16 of each period, and as in experiment 1, 2 ruminal buffers with differing Zn concentrations were incubated independently during the WRR (0 or 0.14 mM Zn, from ZnSO₄). Disappearance of Zn from ruminal buffers was again characterized using a model with two parameters, a coefficient/rate (k) and a plateau of disappearance (B).

Experiment 3

Twenty individually housed Hampshire crossbred wether lambs (35.1 ± 4.57 kg initial BW) were utilized in a 60-d experiment to evaluate the impact of dietary grain inclusion and dietary Zn concentration on ruminal epithelial Zn concentration, Zn transporters, and proteins integral to epithelial integrity. This experiment was conducted as a 2 × 2 factorial, with 2 diet types (DIET; 45% forage and 55% concentrate [HF] or 7.5% forage and 92.5% concentrate [HG]), and 2 concentrations of supplemental Zn (ZINC; 0 mg [CON] or 120 mg [ZN] supplemental Zn/kg diet DM) from ZnSO₄ (Table 3)_. The HF diets, HF-CON and HF-ZN, were analyzed and contained 17 and 108 mg Zn/kg DM, respectively. The HG diets, HG-CON and HG-ZN, were analyzed and contained 25 and 104 mg Zn/kg DM. Wethers were weighed on days 0, 28, and 56 (the final day of the performance period) of the experiment. Feed intake was measured daily and TMR samples were collected weekly. This experiment took place from June through mid-August in Ames, IA, with an average daily air temperature of 23.9 °C.

Table 3.

Diet ingredient and nutrient composition for Experiment 3

	DIET
	HF		HG
	ZINC
	CON	ZN	CON	ZN
Ingredients	% of diet DM
Cracked corn	15.5	15.5	67	67
Corn silage	50	50	10	10
Beet pulp	20	20	5	5
Soybean meal	11.95	11.92	15.05	15.02
Urea	0.6	0.6	0.5	0.5
Limestone	0.5	0.5	1.5	1.5
Dicalcium phosphate	0.5	0.5
Ammonium chloride	0.5	0.5	0.5	0.5
Salt	0.31	0.31	0.31	0.31
Vitamin ADE premix3	0.1	0.1	0.1	0.1
Trace mineral premix4	0.022	0.056	0.022	0.056
Bovatec5	0.015	0.015	0.015	0.015
Analyzed composition, DM
CP6, %	13.63		15.84
aNDF6, %	31.29		11.58
Fat (EE)6, %	2.18		2.59
Ash6, %	5.10		4.13
NEm7, Mcal/kg	1.75		2.07
NEg7, Mcal/kg	1.13		1.40
Cu8, mg/kg	9.5	10.0	8.9	9.7
Fe8, mg/kg	170	152	85	91
Zn8, mg/kg	17	107	25	123

¹Dietary forage concentration: 45% forage and 55% grain (HF) or 90% concentrate and 10% forage (HG).

²Zn treatments: supplemental Zn (0 mg [CON] or 120 mg [ZN] supplemental Zn/kg diet DM) from ZnSO_4.

³Vitamin ADE premix provided 900,000 IU Vitamin A, 225,000 IU vitamin D, and 180 IU vitamin E per kg of premix.

⁴Provided per kg of diet: 25 mg Cu, 6 mg Cu, 0.6 mg I, 0.22 mg Se, 0.2 mg Cu, and either 0 mg (CON) or 120 mg (ZN) for treatment diets (all inorganic sources).

⁵Provided lasalocid at 27 mg/908 kg diet DM, Zoetis, New York, NY).

⁶Proximate analyses completed by Dairyland Laboratories (Arcadia, WI).

⁷Calculated based on diet nutrient analyses.

⁸Cr and Zn analyses were completed via ICP-OES (Pogge and Hansen, 2013).

On days 60 to 62, 4 to 8 wethers representing each treatment combination were euthanized per day, prior to feeding (approximately 24 h after their last feeding). Wethers were transported to the Iowa State University Veterinary School, and following blood collection, wethers were euthanized using an intravenous dose of pentobarbital sodium by a licensed veterinarian. Following euthanasia, caudal ventral ruminal tissue samples were collected and thoroughly rinsed with deionized water. The muscularis and serosal layers were stripped, and the epithelia were immediately frozen in liquid nitrogen. Additional samples were placed on ice and frozen at −20 °C for Zn analysis, or placed in 10% buffered formalin in preparation for histology and in situ hybridization. A 5 cm³ section of liver from the right posterior lobe was also collected and frozen at −20 °C for Zn analysis.

Analytical procedures

For all experiments, diet samples were dried in a forced-air oven at 70 °C for 48 h, ground to pass through a 2-mm screen, and composited by period (experiments 1 and 2) or month (experiment 3) on an equal dry weight basis prior to analysis. Diet samples were digested using trace metal grade nitric acid prior to mineral analysis (CEMS MARSXpress, Matthews, NC) as described by Richter et al. (2012). Samples collected from the WRR procedure were acidified with trace metal-grade nitric acid prior to Cr and Zn analysis. Chromium and Zn analyses were completed via ICP-OES using methods previously described (Pogge and Hansen, 2013).

In experiment 3, ruminal epithelia and liver samples for Zn analysis were dried in a forced-air oven at 70 °C for 72 h and digested using trace metal grade nitric acid prior to mineral analysis (CEMS MARSXpress, Matthews, NC) for subsequent TM analysis via ICP-OES. Blood samples collected at euthanasia (collected in tubes containing K₂EDTA for TM analysis) were spun in a centrifuge at 1,200 × g for 10 min at 4 °C and plasma was removed and stored at −80 °C until analysis. Plasma samples were analyzed for Zn using methods previously described (Genther and Hansen, 2014).

A single section of the ruminal epithelium was analyzed for protein expression. Proteins were extracted from ruminal epithelial tissues using methods detailed by Hansen et al. (2009) using a modified radioimmunoprecipitation assay buffer (Nam et al., 2013). Western blotting was performed as described by Hansen et al. (2008). Briefly, before electrophoresis, samples were heated at 70 °C for 10 min and then electrophoresis was performed using precast NuPage Novex 4% to 12 % Bis–Tris gels and the Novex X-Cell Surelock Mini-Cell electrophoresis system (Invitrogen Corp., Carlsbad, CA, USA). Magic Mark XP Western Protein Standards (Invitrogen Corp.) were used for molecular weight determination. Approximately 60 µg total protein was loaded into each well. Proteins were separated under reducing and denaturing conditions and transferred onto a polyvinylidene diflouride membrane. Membranes were probed for 2 h with antibodies purchased from Invitrogen Corp. Antibodies shown to be successful in sheep tissue for occludin, claudin-1, -4, -7, ZO-1, and β-actin (Stumpff et al., 2011) were utilized. Primary antibody dilutions were as follows: occludin (1:1,000), claudin-1 (1:2,000), claudin-4 (1:1,000), claudin -7 (1:2,000), ZO-1 (1:1,000), and β-actin (1:10,000). A pooled sample containing equal amounts of protein from all 20 experimental samples was included on each blot. β-actin lacked utility as an internal control due to significant treatment differences in expression. Thus, protein expression was expressed as a relative ratio compared with the pooled sample. Blots for claudin-1, claudin-7, and ZO-1 were incubated with secondary goat, anti-rabbit HRP conjugated antibody (1:10,000). Blots for claudin-4, occludin, and β-actin utilized were incubated with secondary goat, anti-mouse HRP-conjugated antibody (1:5,000 for occludin and 1:10,000 for claudin-4 and β-actin). Protein bands were detected using a chemiluminescent detection kit as described by the manufacturer (ECL, Amersham Phamacia Biotech, City, State). The density of the protein bands was quantified using ChemiImager 5500 (Alpha Innotech, San Leandro, CA) and Alpha Ease FC (v. 2.03; Alpha Innotech).

Samples reserved for in situ hybridization and histology were embedded in paraffin and de-paraffinized at the Iowa State University Veterinary School Histology laboratory. In situ hybridization was completed following the manufacturer’s instructions (RNAScope 2.0 Assay, ACDBio, Washington, DC). A negative control probe (DapB, Bacillus subtillus strain SMY methylglyoxal synthase (mgsA) gene; ACDBio), and a positive control probe (Oa-SOD1, Ovis aries SOD1, accession No. XM_004012626.1) were included. Experimental probes were ZIP4 (Ovis aries-SLC39A4, accession No. XM_012184237.1), ZIP5 (Ovis aries-SLC39A5, accession No. XM_012176069.1), and ZnT1 (Ovis aries-SLC30A1, accession No. XM_012189987.1). Ten images per animal were collected for each transporter of interest using a Leica microscope (Leica Microsystems Inc., Buffalo Grove, IL) with a connected camera (Q Imaging, Surrey, BC), and QCapture Pro software (version 6.0.0.412, Q Imaging). Each ruminal epithelial layer (strata basal, spinosum, granulosum, and corneum) in situ hybridization image was scored using the system adapted from the manufacturer, as follows: 0 = no staining; 1 = 0 to 1 stained spot per cell, not all cells stained; 2 = 1 to 2 stained spots per cell; 3 = 3 to 4 stained spots per cell; 4 = 5 to 6 stained spots per cell; 5 = 7 + stained spots per cell.

Ruminal epithelial sections for histology were dehydrated, embedded in paraffin, sectioned, and stained with hemotoxylin-eosin stain at the Iowa State University Veterinary School Histology Laboratory. Ruminal epithelial samples were evaluated for papillae width, epithelial layer width, keratinized layer width, and non-keratinized layer width. Ten images per animal were collected were collected using a Leica microscope (Leica Microsystems Inc.) with a connected camera (Q Imaging) and QCapture Pro software (version 6.0.0.412, Q Imaging), and images were measured using Image-Pro Plus software (version 7.0.0.591, Media Cybernetics, Rockville, MD).

Statistical analyses

Experiments 1 and 2 data were analyzed by ANOVA as a crossover design using the MIXED procedure of SAS 9.4 (SAS Institute Inc., Cary, NC). The model included the fixed effects of ZINC, period, wash day and buffer order, and random effect of steer. DM intake, as well as mean, minimum, and maximum rumen pH data, were analyzed as repeated measures (day as the repeated effect) using the MIXED procedure of SAS 9.4. The model included the fixed effects of ZINC, period, day and their interactions, and the random effect of the steer. Data from Exp. 3 were analyzed as a completely randomized design, using the MIXED procedure of SAS, with fixed effects of DIET, ZINC, DIET × ZINC, and random effect of lamb nested within treatment. Lamb was the experimental unit (n = 5 per treatment). In situ hybridization data for ZnT1 was analyzed using a normal approximation in the GLIMMIX procedure of SAS, with the fixed effects of DIET, ZINC, DIET × ZINC, and the random effect of the image. Low expression of ZIP4 and ZIP5 prevented statistical analysis. Mean differences were compared using the PDIFF statement. Data are reported as LS means, with significance declared at P ≤ 0.05, with tendencies set at P ≤ 0.10.

Results

Experiment 1

There was no difference in DMI (Table 4; P = 0.99) or mean or minimum ruminal pH (P ≥ 0.13). Zn120 tended to have greater maximum ruminal pH (P = 0.06) mainly driven by a ZINC × day interaction (P = 0.06) where Zn120 had greater maximum ruminal pH from day 11 through day 16. There was also no difference in saliva weight (P = 0.55), collected during the WRR between dietary treatments, although Zn120 had greater rumen digesta weight (P = 0.01). Dietary Zn concentration did not affect the rate of disappearance (k) or plateau (B) digestive model parameters for buffer Zn (Table 5; P ≥ 0.34) and the majority of ruminal Zn disappeared in the first 20 min of incubation (Figure 1). The control buffer could not be modeled due to variable, and very low Zn concentrations.

Table 4.

Effect of dietary Zn concentration on rumen pH, dry matter intake, digesta weight, and saliva weight in steers consuming a 45:55 (experiment 1) or a 10:90 forage:concentrate diet (experiment 2)¹

	ZINC1
	CON	Zn120	SEM	P-value
Forage experiment 1
Ruminal pH
Mean2	6.15	6.45	0.161	0.13
Minimum3	5.36	5.63	0.276	0.31
Maximum4	6.70	7.22	0.257	0.06
DMI5, kg/d	13.6	13.6	0.60	0.99
Digesta wt, kg	71.1	73.5	3.37	0.01
Saliva wt, kg	7.0	6.4	0.94	0.55
Concentrate experiment 2
Ruminal pH
Mean6	6.18	5.93	0.245	0.31
Minimum7	5.31	5.57	0.362	0.61
Maximum8	6.72	6.36	0.333	0.35
DMI, kg	14.7	15.0	0.31	0.10
Digesta wt, kg	71.7	71.9	5.09	0.93
Saliva wt, kg	6.9	6.9	1.01	0.96

¹ZINC: supplemental Zn (0 mg [CON] or 120 mg [Zn120] supplemental Zn/kg diet DM) from ZnSO_4.

²ZINC × Day: P = 0.78.

³ZINC × Day: P = 0.14.

⁴ZINC × Day: P = 0.06, on day 11 Zn is higher through day 16.

⁵ZINC × Day: P = 0.24.

⁶ZINC × Day: P = 0.85.

⁷ZINC × Day: P = 0.97.

⁸ZINC × Day: P = 0.94.

Table 5.

Experiment 1 and 2—dietary Zn effect on percent Zn remaining from ruminal contents of steers fed either 45:55 (experiment 1) or a 10:90 forage:concentrate diet (experiment 2)¹

	ZINC2
	CON	Zn120	SEM	P-value
Forage experiment 1
k (rate constant)	0.111	0.070	0.0216	0.34
B, % (plateau)	55.3	60.6	4.99	0.52
Concentrate experiment 2
k (rate constant)	0.104	0.105	0.0124	0.93
B, % (plateau)	47.0	47.2	1.52	0.94

¹Model parameters, coefficient/rate (k), and a plateau (B) of disappearance were developed to more effectively characterize Zn remaining in ruminal contents.

²ZINC: supplemental Zn (0 mg [CON] or 120 mg [Zn120] supplemental Zn/kg diet DM) from ZnSO₄.

Figure 1.

Effect of dietary Zn concentration on ruminal Zn as a percent of initial Zn concentration (raw data, average +/− SD) over time in steers receiving a 45:55 forage:concentrate diet in experiment 1. Zn treatments: supplemental Zn (0 mg [CON] or 120 mg [Zn120] supplemental Zn/kg diet DM) from ZnSO₄.

Experiment 2

There was no difference in mean, maximum, or minimum ruminal pH, digesta weight, or saliva weight (Table 4; P ≥ 0.31). There was a trend for Zn120 to have greater DMI than CON (P = 0.10). There was no effect of ZINC on the k or B digestive model parameters in the Zn buffer (Table 5; P ≥ 0.93) and the majority of ruminal Zn disappeared in the first 20 min of incubation (Figure 2). Similar to Exp. 1, data from the control buffer were not statistically evaluated due to very low and inconsistent Zn concentrations.

Figure 2.

Effect of dietary Zn concentration1 on ruminal Zn as a percent of initial Zn concentration (raw data, average +/− SD) over time in steers receiving a 10:90 forage:concentrate diet in experiment 2. Zn treatments: supplemental Zn (0 mg [CON] or 120 mg [Zn120] supplemental Zn/kg diet DM) from ZnSO₄.

Experiment 3

There was no effect of ZINC or the DIET × ZINC interaction on wether performance over the 60-d experiment (Table 6; P ≥ 0.16). Wethers on the HG diet had greater final BW, ADG, and feed efficiency (P ≤ 0.02), and tended to have greater DMI (P = 0.08) than wethers on the HF diet.

Table 6.

Effect dietary Zn concentration on growth performance of wethers consuming a 45:55 or 10:90 forage:concentrate diet in experiment 3

	DIET
	HF		HG
	ZINC2					Treatment P-value
	CON	ZN	CON	ZN	SEM	DIET	ZINC	DIET × ZINC
Initial BW, kg	38	37	33	32	1.8	0.02	0.74	0.74
Final BW3, kg	44	46	51	52	2.4	0.01	0.48	0.83
ADG, kg/d	0.16	0.19	0.29	0.30	0.027	0.01	0.47	0.81
Average DMI, kg/d	1.1	1.1	1.2	1.2	0.09	0.08	0.89	0.91
G:F	0.151	0.182	0.231	0.247	0.0162	0.01	0.16	0.66

¹DIET: 45% forage and 55% grain (HF) or 90% concentrate and 10% forage (HF).

²ZINC: supplemental Zn (0 mg [CON] or 120 mg [ZN] supplemental Zn/kg diet DM) from ZnSO_4.

³Initial BW was used as a covariate in analysis, final BW was collected on day 56.

There was a tendency for ZN to have greater plasma Cu (Table 7; P = 0.08) and greater plasma Zn concentrations (P = 0.01). The HG animals had greater plasma Fe and Zn concentrations (P ≤ 0.01).

Table 7.

Effect dietary Zn concentration on plasma, liver, and rumen tissue trace mineral concentrations in wethers consuming a 45:55 or 10:90 forage:concentrate diet in experiment 3

	DIET1
	HF		HG
	ZINC2					Treatment P-value
	CON	ZN	CON	ZN	SEM	DIET	ZINC	DIET × ZINC
Plasma, mg/L
Cu	0.98	1.03	0.91	1.22	0.095	0.50	0.08	0.19
Fe	1.36	1.28	2.01	1.76	0.131	0.01	0.24	0.51
Zn	0.51	0.71	0.71	0.83	0.048	0.01	0.01	0.48
Liver, mg/kg
Cu	542ab	407b	564ab	666a	64.1	0.04	0.80	0.08
Fe	203	179	136	125	20.9	0.01	0.42	0.76
Zn	140	161	119	163	22.4	0.68	0.17	0.61
Rumen epithelia, mg/kg
Cu	15	16	18	20	1.0	0.01	0.16	0.41
Fe	96	93	60	56	10.6	0.01	0.75	0.99
Zn	129c	261b	159bc	480a	45.1	0.01	0.01	0.05

¹DIET: 45% forage and 55% grain (HF) or 90% concentrate and 10% forage (HG).

²ZINC: supplemental Zn (0 mg [CON] or 120 mg [ZN] supplemental Zn/kg diet DM) from ZnSO_4.

^a-cLSmeans with different superscripts differ, P ≤ 0.05.

There was a tendency for an interaction between DIET and ZINC on liver Cu (P = 0.08) where HG + ZN wethers had the greatest liver Cu and HF + ZN had the least, while HG + CON and HF + CON were intermediate. There was no effect of either diet or zinc concentration on liver Zn concentration (P ≥ 0.17). The HG wethers had lesser liver Fe than HF wethers (P = 0.01).

The HG wethers had greater rumen epithelial Cu (P = 0.01) and lesser Fe concentration (P = 0.01) than HF wethers. There was no effect of Zn concentration on ruminal epithelial Cu or Fe concentrations (P ≥ 0.16). There was a DIET × ZINC interaction (P = 0.05) where HG + ZN had the greatest ruminal epithelial Zn, followed by HF + CON, HG + CON was intermediate, and HF + ZN had the least.

There was no effect of DIET or ZINC on ruminal papillae width, epithelial layer, non-keratinized layer, and keratinized layer thicknesses (Table 8; P > 0.13). However, there was an interaction between DIET and ZINC on papillae length (P = 0.05), where papillae length was shorter in HF than HG, and within HG, ZN animals had shorter papillae than CON (P = 0.01).

Table 8.

Effect dietary Zn concentration on rumen ventral sac morphology in wethers consuming a 45:55 or 10:90 forage:concentrate diet in experiment 3

	DIET1
	HF		HG
	ZINC2					Treatment P-value
	CON	ZN	CON	ZN	SEM	DIET	ZINC	DIET × ZINC
Rumen
Papillae length, μm	1,285c	1,276c	2,084a	1,706b	89.0	0.01	0.04	0.05
Papillae width, μm	385	379	378	356	20.8	0.48	0.51	0.70
Epithelial layer, μm	125	113	118	122	4.9	0.84	0.43	0.13
Non-keratinized layer, μm	81	71	77	74	6.0	0.87	0.27	0.60
Keratinized layer, μm	43	42	41	48	5.4	0.72	0.60	0.42

¹DIET: 45% forage and 55% grain (HF) or 90% concentrate and 10% forage (HG).

²ZINC: supplemental Zn (0 mg [CON] or 120 mg [ZN] supplemental Zn/kg diet DM) from ZnSO_4.

^a-cLSmeans with different superscripts differ, P ≤ 0.05.

There were no DIET × ZINC interactions on rumen epithelial TJ protein expression (Table 9; P ≥ 0.25). There was no effect of diet type or diet Zn concentration on claudin-1, claudin-4, or occludin protein expression (P ≥ 0.32). Claudin-7 expression was greater in HG (P = 0.01) and tended to be lesser in ZINC (P = 0.09).

Table 9.

Effect dietary Zn concentration on ventral sac ruminal protein expression of tight junction proteins in wethers consuming a 45:55 or 10:90 forage:concentrate diet in experiment 3

	DIET1
	HF		HG
	ZINC2					Treatment P-value
	CON	ZN	CON	ZN	SEM	DIET	ZINC	DIET × ZINC
Relative protein expression 3
Claudin 1	1.18	1.08	1.06	0.97	0.123	0.32	0.43	0.96
Claudin 4	1.04	1.07	1.26	1.10	0.154	0.38	0.67	0.52
Claudin 7	0.64	0.57	1.21	0.86	0.124	0.01	0.09	0.25
Occludin	1.08	0.90	1.02	0.87	0.321	0.89	0.58	0.95
β-actin	1.60	1.48	0.93	1.13	0.154	0.01	0.79	0.31

¹DIET: 45% forage and 55% grain (HF) or 90% concentrate and 10% forage (HG).

²ZINC: supplemental Zn (0 mg [CON] or 120 mg [ZN] supplemental Zn/kg diet DM) from ZnSO_4.

³Relative protein expression is a ratio of expression compared with a pooled control sample.

Minimal expression of ZIP4 and ZIP5 prevented statistical analysis of these data, so means are presented (Table 10). Expression of ZnT1 was decreased by dietary Zn concentration across DIET (Table 10; P < 0.001) within the stratum basale layer of the ruminal epithelium. A DIET × ZINC effect was detected for ZnT1 RNAscope score within the stratum spinosum (P = 0.01) where HG + CON wethers had greater ZnT1 RNAscope score than other treatments (P ≤ 0.006).

Table 10.

Effect dietary Zn concentration on rumen Zn transporter mRNA in situ hybridization score in wethers consuming a 45:55 or 10:90 forage:concentrate diet in experiment 3

	DIET1
	HF		HG
	ZINC2					Treatment P-value
	CON	ZN	CON	ZN	SEM	DIET	ZINC	DIET × ZINC
In situ hybridization score 3
ZnT14
Stratum basale	2.09	1.72	2.2	1.6	0.099	0.99	< 0.001	0.25
Stratum spinosum	0.27b	0.22b	0.56a	0.15b	0.072	0.14	0.001	0.01
Stratum granulosum5	0.18	0.28	0.47	0.3	—	—	—	—
Stratum corneum5	0.02	0.38	0.26	0.18	—	—	—	—
ZIP46	0.18	0.27	0.27	0.22	—	—	—	—
ZIP57	0.11	0.16	0.09	0.08	—	—	—	—

¹DIET: 45% forage and 55% grain (HF) or 90% concentrate and 10% forage (HG).

²ZINC: supplemental Zn (0 mg [CON] or 120 mg [ZN] supplemental Zn/kg diet DM) from ZnSO_4.

³In situ hybridization score: 0 = no staining; 1 = 0 to 1 stained spot per cell, not all cells stained; 2 = 1 to 2 stained spots per cell; 3 = 3 to 4 stained spots per cell; 4 = 5 to 6 stained spots per cell; 5 = 7 + stained spots per cell. Scoring guidelines adapted from ACDBio (Washington, DC). Although ZIP4 and.

⁴ZnT1: Zn transporter SLC30A1.

⁵Not enough expression to statistically analyze. Numerical means within the epithelial layer are presented.

⁶ZIP4: Zn transporter SLC39A4; not enough expression to statistically analyze, numerical means across rumen epithelium are presented.

⁷ZIP5: Zn transporter SLC39A5; not enough expression to statistically analyze, numerical means across rumen epithelium are presented.

^a-cLSmeans with different superscripts differ, P ≤ 0.05.

Discussion

As an adaptational response, dietary concentrate inclusion influences rumen structure, function, and mineral absorption (Kabaija and Smith, 1988; Genther and Hansen, 2013; Daniel et al., 2020; Guimaraes, 2020). Feeding high-concentrate diets is common for finishing cattle (Samuelson et al., 2016) and improves growth performance but can negatively impact rumen epithelium integrity due to lower ruminal pH, increased osmolality, and increased concentrations of microbial-associated molecular patterns such as lipopolysaccharide (Kennedy and Bunting, 1991; Emmanuel et al., 2007). Zinc plays an important role in epithelial permeability and these experiments aimed to characterize: 1) ruminal Zn disappearance rates as influenced by dietary Zn and ruminal Zn concentrations (experiments 1 and 2); and 2) the effects of dietary concentrate and Zn levels on ruminal epithelial morphology, Zn transport mechanisms, and TJ protein expression in lambs (experiment 3).

Zinc homeostasis in mammals relies on specialized transporter proteins, primarily the SLC39 (ZIP) family, which imports Zn into the cytosol, and the SLC30 (ZnT) family, which exports Zn out of the cytosol (Kambe et al., 2015). To date, these transporters have not been characterized in ruminal epithelial tissue. Expression of ZIP4 and ZIP5 in the ruminal epithelium was relatively low and localized mainly in the stratum basale (approximately 65% to 87% of total expression across all measured layers). In situ hybridization results from this study suggest that the significance of these transporters in ruminal Zn transport may be limited when compared to ZnT1. ZIP4 (SLC39A4) is a primary apical absorptive mechanism for Zn in epithelial cells (Dufner-Beattie et al., 2003). Given wethers fed HG + ZN had the greatest ruminal tissue Zn concentration, while the least was HF + CON, alteration of apical Zn transporter expression was expected. However, its absence in the ruminal epithelium may suggest apical absorption of Zn is limited or, alternatively, other Zn importers may be at play, such as ZIP14. ZIP14 is a potential candidate for future investigation given the diverse roles ZIP14 plays in hepatic tissue and the intestine (Liuzzi et al., 2005; Girijashanker et al., 2008). To date, ZIP14 has not been characterized in the ruminal epithelium. Minimal expression of ZIP5, a basolateral transporter responsible for transporting Zn into epithelial cells from circulation, may indicate limited Zn movement from circulation into the ruminal epithelium. This is consistent with the lack of increase for Zn into the ruminal buffers in experiments 1 and 2. The expression of these transporters was not influenced by dietary treatment. This finding may indicate a lesser role in ruminal Zn metabolism or perhaps a critical role that is not easily modulated by diet.

Interestingly, in contrast with prior work (Wright et al., 2008), the quantitatively greater relative expression of ZnT1 localized in the stratum basale (compared to ZIP4 and 5 in all other regions) of wethers in experiment 3 may indicate that Zn is transported from the rumen epithelium into circulation. Although protein expression could not be evaluated, mRNA expression nearest to the vasculature within the rumen epithelium may suggest that these transporters are involved in Zn movement into blood vessels. The mRNA expression of ZnT1 was decreased by dietary Zn in the stratum basale and affected by the diet type and dietary Zn concentration where HG + CON wethers had a greater mean ZnT1 RNAscope score than other treatments. Thus, it appears that greater dietary Zn concentrations, perhaps more so in high-grain diets, decrease ruminal epithelium Znt1 expression. This is the strongest indication that the ruminal epithelium may play a role in ruminal Zn metabolism and that ZnT1, a protein likely responsible for transporting Zn into circulation from the ruminal epithelium, responds to dietary or ruminal Zn concentrations by reducing transport of Zn from the ruminal epithelium into circulation. This idea is further supported by the greater ruminal epithelial Zn concentration of animals fed diets with greater Zn concentrations. The combination of increased dietary Zn concentrations, and decreased expression of the basolateral Zn transporter ZnT1 and likely reduced the movement of Zn out of the rumen epithelium through basolateral transport mechanisms, thus resulting in increased rumen epithelial Zn concentrations. The combination of the in situ hybridization and WRR data indicates that the ruminal epithelium is involved in Zn metabolism, and Zn is being absorbed into the rumen epithelium and is likely crossing through the rumen epithelium into the systemic circulation.

Early work demonstrated apparent absorption and Zn uptake from ovine ruminal tissue in vivo using ⁶⁵Zn (Arora et al., 1969). Wright et al. (2008) also observed significant Zn uptake in ovine rumen epithelium within 30 min ex vivo. However, results from Wright et al. (2008) suggest basolateral transport from rumen epithelium into circulation does not occur. Regardless, it is clear Zn disappears from rumen contents. Results from this study revealed Zn disappearance is not linear. In the WRR, Zn rapidly disappeared between 5 and 20 min, then plateaued. While the majority of Zn disappeared from the rumen buffer after 15 min, this was unaffected by dietary Zn concentration. Even though the rumen was carefully cleaned and thoroughly rinsed before incubation buffers were added, the variation in Zn concentration in CON samples suggests fragments of remaining feed stuck to papillae may have contaminated the CON buffer. However, when compared with results from the ZN buffers, it was clear that Zn was not being excreted into the rumen in a measurable quantity. Neither the rate (k) nor the plateau (B) of disappearance was influenced by dietary Zn concentration in either experiment. Overall, the results for Zn disappearance in experiment 1 were quite similar to the results from experiment 2. However, B (plateau), as a percentage of initial Zn concentration, in experiment 2 was numerically lesser than the observed value in experiment 1, i.e., more Zn disappeared when steers were receiving high-concentrate diets. This contrasts with Kennedy and Bunting (1991), who found decreased ruminal Zn absorption with higher concentrate diets in cannulated wethers. Under the conditions of the WRR, the potential interaction of Zn with rumen contents and microbes is avoided, and the results are more indicative of the relationship between the ruminal epithelium and Zn. This difference, and the much greater basal diet Zn, and therefore greater Zn intake, may contribute to the differences between studies. While results from experiments 1 and 2 may not be directly compared, numerically, these data indicate that 10% more Zn disappeared from rumen contents in experiment 2 when steers were fed a high-concentrate diet. The rate of disappearance (k) was numerically similar between experiments.

In experiment 3, lambs fed the high-concentrate (HG) diets had improved ADG, G:F, and final BW. High-concentrate diets offer increased diet energy density and ruminally fermentable carbohydrates improving growth performance and feed efficiency (Loerch, 1990; Owens, 2010). Along with greater energy density, HG diets promote longer ruminal papillae due to increased SCFA concentrations (Shen et al., 2004). As expected, papillae length was greater in both HG treatments compared to HF, with HG + CON wethers having longer papillae than HG + ZN wethers. While not measured in the present study, high dietary Zn has been reported to reduce overall DM and fiber digestion (Eryavuz and Dehority, 2009; Genther and Hansen, 2013), altering SCFA production rates and affecting maximal ruminal pH and ruminal papillae growth, as partially supported by the higher maximal ruminal pH in the Zn120 steers from days 11 to 16 in experiment 1. In experiment 2, the average and maximum ruminal pH was numerically lower with the HG, aligning with the expected effects of diet type on ruminal fermentation dynamics (Chibisa et al., 2016). Increased rumen soluble Zn can interfere with SCFA production (Durand and Kawashima, 1980; Spears et al., 2004). Other ruminal epithelial characteristics were unaffected by DIET or ZINC. Liu et al. (2013) reported that goats fed 65% concentrate diets have a greater keratinized layer (stratum corneum) thickness compared to a hay-only diet. However, relative to diets fed by Liu et al. (2013), the lesser difference in forage-to-concentrate ratio and the higher grain concentration (45:55 forage-to-concentrate) of the HF diet in the present study may have contributed to the lack of observed differences in rumen morphology and ruminal tight-cell junction protein expression.

TJs regulate the paracellular movement of ions and molecules between cells, coordinated by transmembrane proteins such as claudins (Angelow et al., 2008). Claudin assembly into TJs involves complex interactions with other transmembrane and scaffold proteins (Koval, 2006). Previous studies identified the expression of occludin and claudins 1, 4, 7, and ZO-1 in the ruminal epithelia of sheep (Graham and Simmons, 2005; Stumpff et al., 2011). The vital role of Zn in TJs has been explored primarily in the small and large intestines (Sturniolo et al., 2002; Zhang and Guo, 2009; Opgenorth et al., 2021). Given the differences in structural characteristics between the ruminal and small intestinal epithelium (stratified squamous versus single-layer columnar, respectively) assessing how dietary Zn concentration influences markers of rumen epithelial integrity in lambs fed high or low-concentrate diets is pertinent.

Results from ventral sac ruminal protein expression of TJ proteins either suggest that diet type did not interrupt rumen integrity and expression of these proteins, or may have altered the localization of the proteins within the rumen epithelial structure without changing overall protein expression. However, in this study, β-actin protein expression was greater in HF-fed sheep than in HG sheep. Although unexpected based on the use of β-actin to normalize ruminal TJ protein expression in other concentrate:forage ratio studies (Liu et al., 2013), β-actin can be a component of supporting TJ structure (Gutierrez et al., 2014) and may have been negatively affected by the HG diets. Prior work indicates grain-fed goats displayed irregular staining and decreased protein expression of TJ proteins claudin-1, claudin-4, and occludin throughout the layers of the rumen epithelium (Liu et al., 2013). In experiment 3, claudin-7 expression was lesser in high-forage-fed sheep compared to those on a high-concentrate diet. Claudin-7 plays a crucial role in forming TJs and regulating gastrointestinal permeability (Ding et al., 2012; Günzel and Yu, 2013). Claudin-7 has previously been found to be expressed in rumen epithelium primarily localized in the stratum corneum (keratinized layer; Stumpff et al., 2011). In the present study, Claudin-7 expression tended to be lower in sheep on high Zn diets. Animals fed diets with higher proportions of forage to concentrate, typically exhibit ruminal morphological changes as an adaptational response compared to ruminant animals consuming higher grain diets (Liu et al., 2013). Numerically, Claudin-7 expression for HG + ZN aligns more closely with the sheep consuming diets higher in forage, suggesting a potential improvement in ruminal integrity or protective effect of Zn on the rumen epithelium. However, it should also be noted that claudin-7 expression in ex vivo rumen tissue has been shown to decrease in response to low mucosal pH (Meissner et al., 2017).

Conclusion

In summary, these 3 experiments provide new insights into Zn metabolism and disappearance in the rumen. Zinc disappears rapidly from the rumen, and dietary Zn concentration has minimal influence on the absolute rate of Zn disappearance. Zinc disappearance was numerically greater in steers fed high-concentrate diets. Although ruminal epithelial barrier integrity was not assessed directly in this study, summative results from these experiments indicate both diet composition and Zn intake can influence characteristics related to ruminal epithelial barrier integrity, highlighting Zn’s crucial role in maintaining epithelial health. Furthermore, the non-linear disappearance rates of Zn, along with the differential expression of Zn transporters (ZIP4, ZIP5, and ZnT1) underscore the complex and regulated nature of Zn metabolism in the rumen. The increased stratum spinosum expression of ZnT1 for lambs fed high grain diets in combination with lower dietary Zn suggests that the ruminal epithelia may adapt to varying Zn concentrations to regulate Zn transport. The combination of the in situ hybridization and the WRR results indicate that the ruminal epithelia are involved in Zn metabolism, and Zn is being absorbed into the rumen epithelia. Future research further characterizing transporters like ZIP14 could help elucidate the mechanisms governing Zn homeostasis in the rumen. Overall, the findings characterize the dynamic nature of ruminal Zn metabolism and its importance for rumen health under differing nutritional conditions.

Glossary

Abbreviations

ADG

average daily gain

Akt

protein kinase B

BW

bodyweight

CO2

Carbon dioxide

DM

dry matter

DMI

dry matter intake

EDTA

ethylenediaminetetraacetic acid

ICP-OES

inductively coupled plasma optical emission spectroscopy

SCFA

short-chain fatty acids

TJ

tight junction

TM

trace mineral

TMR

total mixed ration

WRR

washed reticulo-rumen technique

ZIP

Zrt/Irt-like proteins (SLC39 family)

Zn

Zinc

ZnT

Zn transporter (SLC30 family)

Conflict of interest statement

The authors Olivia N. Genther-Schroeder, Dathan T. Smerchek, Gregory B. Penner, and Stephanie L. Hansen declare no conflicts of interest.

Author Contributions

Olivia Genther-Schroeder (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing—original draft, Writing—review & editing), Dathan Smerchek (Data curation, Writing—original draft, Writing—review & editing), Gregory Penner (Methodology, Resources, Supervision, Writing—review & editing), and Stephanie Hansen (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing—review & editing)

Data Availability

The data supporting the findings of this study are available from the corresponding author, Dr. Stephanie Hansen, upon reasonable request.