Examination of Wnt signaling as a therapeutic target for pancreatic ductal adenocarcinoma (PDAC) using a pancreatic tumor organoid library (PTOL).

Pancreatic ductal adenocarcinoma (PDAC) presents at advanced stages and is refractory to most treatment modalities. Wnt signaling activation plays a critical role in proliferation and chemotherapeutic resistance. Minimal media conditions, growth factor dependency, and Wnt dependency were determined via Wnt inhibition for seven patient derived organoids (PDOs) derived from pancreatic tumor organoid libraries (PTOL). Organoids demonstrating response in vitro were assessed in vivo using patient-derived xenografts. Wnt (in)dependent gene signatures were identified for each organoid. Panc269 demonstrated a trend of reduced organoid growth when treated with ETC-159 in combination with paclitaxel or gemcitabine as compared with chemotherapy or ETC-159 alone. Panc320 demonstrated a more pronounced anti-proliferative effect in the combination of ETC-159 and paclitaxel but not with gemcitabine. Panc269 and Panc320 were implanted into nude mice and treated with ETC-159, paclitaxel, and gemcitabine as single agents and in combination. The combination of ETC-159 and paclitaxel demonstrated an anti-tumor effect greater than ETC-159 alone. Extent of combinatory treatment effect were observed to a lesser extent in the Panc320 xenograft. Wnt (in)dependent gene signatures of Panc269 and 320 were consistent with the phenotypes displayed. Gene expression of several key Wnt genes assessed via RT-PCR demonstrated notable fold change following treatment in vivo. Each pancreatic organoid demonstrated varied niche factor dependencies, providing an avenue for targeted therapy, supported through growth analysis following combinatory treatment of Wnt inhibitor and standard chemotherapy in vitro. The clinical utilization of this combinatory treatment modality in pancreatic cancer PDOs has thus far been supported in our patient-derived xenograft models treated with Wnt inhibitor plus paclitaxel or gemcitabine. Gene expression analysis suggests there are key Wnt genes that contribute to the Wnt (in)dependent phenotypes of pancreatic tumors, providing plausible mechanistic explanation for Wnt (in)dependency and susceptibility or resistance to treatment on the genotypic level.

Cabozantinib sensitizes microsatellite stable colorectal cancer to immune checkpoint blockade by immune modulation in human immune system mouse models.

Immune checkpoint inhibitors have been found to be effective in metastatic MSI-high colorectal cancers (CRC), however, have no efficacy in microsatellite stable (MSS) cancers, which comprise the majority of mCRC cases. Cabozantinib is a small molecule multi-tyrosine kinase inhibitor that is FDA approved in advanced renal cell, medullary thyroid, and hepatocellular carcinoma. Using Human Immune System (HIS) mice, we tested the ability of cabozantinib to prime MSS-CRC tumors to enhance the potency of immune checkpoint inhibitor nivolumab. In four independent experiments, we implanted distinct MSS-CRC patient-derived xenografts (PDXs) into the flanks of humanized BALB/c-Rag2nullIl2rγnullSirpαNOD (BRGS) mice that had been engrafted with human hematopoietic stem cells at birth. For each PDX, HIS-mice cohorts were treated with vehicle, nivolumab, cabozantinib, or the combination. In three out of the four models, the combination had a lower tumor growth rate compared to vehicle or nivolumab-treated groups. Furthermore, interrogation of the HIS in immune organs and tumors by flow cytometry revealed increased Granzyme B+, TNFα+ and IFNγ+ CD4+ T cells among the human tumor infiltrating leukocytes (TIL) that correlated with reduced tumor growth in the combination-treated HIS-mice. Notably, slower growth correlated with increased expression of the CD4+ T cell ligand, HLA-DR, on the tumor cells themselves. Finally, the cabozantinib/nivolumab combination was tested in comparison to cobimetinib/atezolizumab. Although both combinations showed tumor growth inhibition, cabozantinib/nivolumab had enhanced cytotoxic IFNγ and TNFα+ T cells. This pre-clinical in vivo data warrants testing the combination in clinical trials for patients with MSS-CRC.

ATM kinase inhibitor AZD0156 in combination with irinotecan and 5-fluorouracil in preclinical models of colorectal cancer.

BACKGROUND: AZD0156 is an oral inhibitor of ATM, a serine threonine kinase that plays a key role in DNA damage response (DDR) associated with double-strand breaks. Topoisomerase-I inhibitor irinotecan is used clinically to treat colorectal cancer (CRC), often in combination with 5-fluorouracil (5FU). AZD0156 in combination with irinotecan and 5FU was evaluated in preclinical models of CRC to determine whether low doses of AZD0156 enhance the cytotoxicity of irinotecan in chemotherapy regimens used in the clinic. METHODS: Anti-proliferative effects of single-agent AZD0156, the active metabolite of irinotecan (SN38), and combination therapy were evaluated in 12 CRC cell lines. Additional assessment with clonogenic assay, cell cycle analysis, and immunoblotting were performed in 4 selected cell lines. Four colorectal cancer patient derived xenograft (PDX) models were treated with AZD0156, irinotecan, or 5FU alone and in combination for assessment of tumor growth inhibition (TGI). Immunofluorescence was performed on tumor tissues. The DDR mutation profile was compared across in vitro and in vivo models. RESULTS: Enhanced effects on cellular proliferation and regrowth were observed with the combination of AZD0156 and SN38 in select models. In cell cycle analysis of these models, increased G2/M arrest was observed with combination treatment over either single agent. Immunoblotting results suggest an increase in DDR associated with irinotecan therapy, with a reduced effect noted when combined with AZD0156, which is more pronounced in some models. Increased TGI was observed with the combination of AZD0156 and irinotecan as compared to single-agent therapy in some PDX models. The DDR mutation profile was variable across models. CONCLUSIONS: AZD0156 and irinotecan provide a rational and active combination in preclinical colorectal cancer models. Variability across in vivo and in vitro results may be related to the variable DDR mutation profiles of the models evaluated. Further understanding of the implications of individual DDR mutation profiles may help better identify patients more likely to benefit from treatment with the combination of AZD0156 and irinotecan in the clinical setting.

Preclinical Development of the Class-I-Selective Histone Deacetylase Inhibitor OKI-179 for the Treatment of Solid Tumors.

Histone deacetylases (HDACs) play critical roles in epigenomic regulation, and histone acetylation is dysregulated in many human cancers. Although HDAC inhibitors are active in T-cell lymphomas, poor isoform selectivity, narrow therapeutic indices, and a deficiency of reliable biomarkers may contribute to the lack of efficacy in solid tumors. In this article, we report the discovery and preclinical development of the novel, orally bioavailable, class-I-selective HDAC inhibitor, OKI-179. OKI-179 and its cell active predecessor OKI-005 are thioester prodrugs of the active metabolite OKI-006, a unique congener of the natural product HDAC inhibitor largazole. OKI-006, OKI-005, and subsequently OKI-179, were developed through a lead candidate optimization program designed to enhance physiochemical properties without eroding potency and selectivity relative to largazole. OKI-005 displays antiproliferative activity in vitro with induction of apoptosis and increased histone acetylation, consistent with target engagement. OKI-179 showed antitumor activity in preclinical cancer models with a favorable pharmacokinetic profile and on-target pharmacodynamic effects. Based on its potency, desirable class I HDAC inhibition profile, oral bioavailability, and efficacy against a broad range of solid tumors, OKI-179 is currently being evaluated in a first-in-human phase I clinical trial with plans for continued clinical development in solid tumor and hematologic malignancies.

Investigating the effects of a novel rumen-protected folic acid supplement on feedlot performance and carcass characteristics of beef steers.

Angus-crossbred steers (n = 180; 292 ± 18 kg) from a single ranch were used to investigate the effects of a novel rumen-protected folic acid (RPFA) supplement on feedlot performance and carcass characteristics. On d 0, steers were blocked by body weight to pens (5 steers/pen), and pens within a block were randomly assigned to dietary treatments (n = 6 pens/treatment): target intake of 0 (CON), 30 (RPFA-30), 60 (RPFA-60), 90 (RPFA-90), 120 (RPFA-120), or 150 (RPFA-150) mg RPFA·steer-1·d-1. Steers were weighed before feeding on d -1, 0, 55, 56, 86, 87, 181, and 182. Pen average daily gain (ADG), dry matter intake (DMI), and gain:feed (G:F) were calculated for growing (d 0 to 56), dietary transition (d 56 to 87), finishing (d 87 to 182), and overall (d 0 to 182). Liver and blood samples were collected from two steers/pen before trial initiation and at the end of growing and finishing. Steers were slaughtered on d 183, and carcass data were collected after a 48-h chill. Data were analyzed as a randomized complete block design using ProcMixed of SAS 9.4 (fixed effects of treatment and block; experimental unit of pen). Liver abscess scores were analyzed using the Genmod Procedure of SAS 9.4. Contrast statements assessed the polynomial effects of RPFA. Supplemental RPFA linearly increased plasma folate at the end of growing and finishing (P < 0.01), and linearly decreased plasma glucose at the end of growing (P = 0.01). There was a cubic effect of RPFA on liver folate at the end of growing (P = 0.01), driven by lesser concentrations for RPFA-30, RPFA-60, and RPFA-150. Growing period ADG and G:F were greatest for CON and RPFA-120 (cubic P ≤ 0.03). Transition period DMI was linearly increased due to RPFA (P = 0.05). There was a tendency for a cubic effect of RPFA on the percentage of livers with no abscesses (P = 0.06), driven by a greater percentage of non-abscessed livers in RPFA-30 and RPFA-60. Despite supplementing 1 mg Co/kg DM, and regardless of treatment, plasma vitamin B12 concentrations were low (<200 pg/mL), which may have influenced the response to RPFA as vitamin B12 is essential for recycling of folate.

The effects of feeding ferric citrate on ruminal bacteria, methanogenic archaea and methane production in growing beef steers.

Methane produced by cattle is one of the contributors of anthropogenic greenhouse gas. Methods to lessen methane emissions from cattle have been met with varying success; thus establishing consistent methods for decreasing methane production are imperative. Ferric iron may possibly act to decrease methane by acting as an alternative electron acceptor. The objective of this study was to assess the effect of ferric citrate on the rumen bacterial and archaeal communities and its impact on methane production. In this study, eight steers were used in a repeated Latin square design with 0, 250, 500 or 750 mg Fe/kg DM of ferric iron (as ferric citrate) in four different periods. Each period consisted of a 16 day adaptation period and 5 day sampling period. During each sampling period, methane production was measured, and rumen content was collected for bacterial and archaeal community analyses. Normally distributed data were analysed using a mixed model ANOVA using the GLIMMIX procedure of SAS, and non-normally distributed data were analysed in the same manner following ranking. Ferric citrate did not have any effect on bacterial community composition, methanogenic archaea nor methane production (P>0.05). Ferric citrate may not be a viable option to observe a ruminal response for decreases in enteric methane production.

WEE1 Inhibition in Combination With Targeted Agents and Standard Chemotherapy in Preclinical Models of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with high incidences of p53 mutations. AZD1775 (adavosertib, previously MK-1775) is a small molecule WEE1 inhibitor that abrogates the G2M checkpoint and can potentially synergize with DNA damaging therapies commonly used in PDAC treatment. The purpose of this study was to identify combination partners for AZD1775, including standard chemotherapy or targeted agents, in PDAC preclinical models. Low powered preliminary screens demonstrated that two of the four PDX models responded better to the combinations of AZD1775 with irinotecan or capecitabine than to either single agent. Following the screens, two full powered PDAC PDX models of differing p53 status were tested with the combinations of AZD1775 and irinotecan or capecitabine. The combinations of AZD1775 and SN38 or 5-FU were also tested on PDAC cell lines. Cellular proliferation was measured using an IncuCyte Live Cell Imager and apoptosis was measured using a Caspase-Glo 3/7 assay. Flow cytometry was conducted to measure alterations in cell cycle distribution. Western blot analysis was used to determine the effects of the drug combinations on downstream effectors. In PDX models with mutated p53 status, there was significant tumor growth inhibition from the combination of AZD1775 with irinotecan or capecitabine (P ≤ 0.03), while PDX models with wild type p53 did not show anti-tumor synergy from the same combinations (P ≥ 0.08). The combination of AZD1775 with SN38 or 5-FU significantly decreased proliferation in all PDAC cell lines, and enhanced apoptosis in multiple cell lines. Cell cycle distribution was disrupted from the combination of AZD1775 with SN38 or 5-FU which was recorded as G2M arrest and decreased G1 phase. AZD1775 inhibited phospho-CDC2 and increased the expression of γH2AX that was either maintained or enhanced after combination with SN38 or 5-FU. The combination of AZD1775 with irinotecan/SN38 or capecitabine/5-FU showed anti-tumor effects in vivo and in vitro in PDAC models. These results support further investigation for these combination strategies to enhance outcomes for PDAC patients.

Testing Cancer Immunotherapy in a Human Immune System Mouse Model: Correlating Treatment Responses to Human Chimerism, Therapeutic Variables and Immune Cell Phenotypes.

Over the past decade, immunotherapies have revolutionized the treatment of cancer. Although the success of immunotherapy is remarkable, it is still limited to a subset of patients. More than 1500 clinical trials are currently ongoing with a goal of improving the efficacy of immunotherapy through co-administration of other agents. Preclinical, small-animal models are strongly desired to increase the pace of scientific discovery, while reducing the cost of combination drug testing in humans. Human immune system (HIS) mice are highly immune-deficient mouse recipients rtpeconstituted with human hematopoietic stem cells. These HIS-mice are capable of growing human tumor cell lines and patient-derived tumor xenografts. This model allows rapid testing of multiple, immune-related therapeutics for tumors originating from unique clinical samples. Using a cord blood-derived HIS-BALB/c-Rag2nullIl2rγnullSIRPαNOD (BRGS) mouse model, we summarize our experiments testing immune checkpoint blockade combinations in these mice bearing a variety of human tumors, including breast, colorectal, pancreatic, lung, adrenocortical, melanoma and hematological malignancies. We present in-depth characterization of the kinetics and subsets of the HIS in lymph and non-lymph organs and relate these to protocol development and immune-related treatment responses. Furthermore, we compare the phenotype of the HIS in lymph tissues and tumors. We show that the immunotype and amount of tumor infiltrating leukocytes are widely-variable and that this phenotype is tumor-dependent in the HIS-BRGS model. We further present flow cytometric analyses of immune cell subsets, activation state, cytokine production and inhibitory receptor expression in peripheral lymph organs and tumors. We show that responding tumors bear human infiltrating T cells with a more inflammatory signature compared to non-responding tumors, similar to reports of "responding" patients in human immunotherapy clinical trials. Collectively these data support the use of HIS mice as a preclinical model to test combination immunotherapies for human cancers, if careful attention is taken to both protocol details and data analysis.

RX-5902, a novel ß-catenin modulator, potentiates the efficacy of immune checkpoint inhibitors in preclinical models of triple-negative breast Cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited systemic treatment options. RX-5902 is a novel anti-cancer agent that inhibits phosphorylated-p68 and thus attenuates nuclear ß-catenin signaling. The purpose of this study was to evaluate the ability of ß-catenin signaling blockade to enhance the efficacy of anti-CTLA-4 and anti-PD-1 immune checkpoint blockade in immunocompetent, preclinical models of TNBC. METHODS: Treatment with RX-5902, anti-PD-1, anti-CTLA-4 or the combination was investigated in BALB/c mice injected with the 4 T1 TNBC cell line. Humanized BALB/c-Rag2nullIl2rγnullSIRPαNOD (hu-CB-BRGS) mice transplanted with a human immune system were implanted with MDA-MB-231 cells. Mice were randomized into treatment groups according to human hematopoietic chimerism and treated with RX-5902, anti-PD-1 or the combination. At sacrifice, bone marrow, lymph nodes, spleen and tumors were harvested for flow cytometry analysis of human immune cells. RESULTS: The addition of RX-5902 to CTLA-4 or PD-1 inhibitors resulted in decreased tumor growth in the 4 T1 and human immune system and MDA-MB-231 xenograft models. Immunologic analyses demonstrated a significant increase in the number of activated T cells in tumor infiltrating lymphocytes (TILs) with RX-5902 treatment compared to vehicle (p < 0.05). In the RX-5902/nivolumab combination group, there was a significant increase in the percentage of CD4+ T cells in TILs and increased systemic granzyme B production (p < 0.01). CONCLUSIONS: Conclusions: RX-5902 enhanced the efficacy of nivolumab in a humanized, preclinical model of TNBC. Several changes in immunologic profiles were noted in mice treated with RX-5902 and the combination, including an increase in activated TILs and a decrease in human myeloid populations, that are often associated with immunosuppression in a tumor microenvironment. RX-5902 also was shown to potentiate the effects of checkpoint inhibitors of CTLA4 and the PD-1 inhibitor in the 4 T-1 murine TNBC model. These findings indicate that RX-5902 may have important immunomodulatory, as well as anti-tumor activity, in TNBC when combined with a checkpoint inhibitor.

Wee1 Inhibition Enhances the Anti-Tumor Effects of Capecitabine in Preclinical Models of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype defined by lack of hormone receptor expression and non-amplified HER2. Adavosertib (AZD1775) is a potent, small-molecule, ATP-competitive inhibitor of the Wee1 kinase that potentiates the activity of many DNA-damaging chemotherapeutics and is currently in clinical development for multiple indications. The purpose of this study was to investigate the combination of AZD1775 and capecitabine/5FU in preclinical TNBC models. TNBC cell lines were treated with AZD1775 and 5FU and cellular proliferation was assessed in real-time using IncuCyte® Live Cell Analysis. Apoptosis was assessed via the Caspase-Glo 3/7 assay system. Western blotting was used to assess changes in expression of downstream effectors. TNBC patient-derived xenograft (PDX) models were treated with AZD1775, capecitabine, or the combination and assessed for tumor growth inhibition. From the initial PDX screen, two of the four TNBC PDX models demonstrated a better response in the combination treatment than either of the single agents. As confirmation, two PDX models were expanded for statistical comparison. Both PDX models demonstrated a significant growth inhibition in the combination versus either of the single agents. (TNBC012, p < 0.05 combo vs. adavosertib or capecitabine, TNBC013, p < 0.01 combo vs. adavosertib or capecitabine.) An enhanced anti-proliferative effect was observed in the adavosertib/5FU combination treatment as measured by live cell analysis. An increase in apoptosis was observed in two of the four cell lines in the combination when compared to single-agent treatment. Treatment with adavosertib as a single agent resulted in a decrease in p-CDC2 in a dose-dependent manner that was also observed in the combination treatment. An increase in γH2AX in two of the four cell lines tested was also observed. No significant changes were observed in Bcl-xL following treatment in any of the cell lines. The combination of adavosertib and capecitabine/5FU demonstrated enhanced combination effects both in vitro and in vivo in preclinical models of TNBC. These results support the clinical investigation of this combination in patients with TNBC, including those with brain metastasis given the CNS penetration of both agents.

First-in-Class Phosphorylated-p68 Inhibitor RX-5902 Inhibits ß-Catenin Signaling and Demonstrates Antitumor Activity in Triple-Negative Breast Cancer.

RX-5902 is a first-in-class anticancer agent targeting phosphorylated-p68 and attenuating nuclear shuttling of ß-catenin. The purpose of this study was to evaluate the efficacy of RX-5902 in preclinical models of triple-negative breast cancer (TNBC) and to explore effects on ß-catenin expression. A panel of 18 TNBC cell lines was exposed to RX-5902, and changes in proliferation, apoptosis, cellular ploidy, and effector protein expression were assessed. Gene expression profiling was used in sensitive and resistant cell lines with pathway analysis to explore pathways associated with sensitivity to RX-5902. The activity of RX-5902 was confirmed in vivo in cell line and patient-derived tumor xenograft (PDX) models. RX-5902 demonstrated potent antiproliferative activity in vitro against TNBC cell lines with an average IC50 of 56 nmol/L in sensitive cell lines. RX-5902 treatment resulted in the induction of apoptosis, G2-M cell-cycle arrest, and aneuploidy in a subset of cell lines. RX-5902 was active in vivo against TNBC PDX models, and treatment resulted in a decrease in nuclear ß-catenin. RX-5902 exhibited dose-proportional pharmacokinetics and plasma and tumor tissue in nude mice. Pathway analysis demonstrated an increase in the epithelial-to-mesenchymal transformation (EMT), TGFß, and Wnt/ß-catenin pathways associated with sensitivity to RX-5902. RX-5902 is active against in vitro and in vivo preclinical models of TNBC. Target engagement was confirmed with decreases in nuclear ß-catenin and MCL-1 observed, confirming the proposed mechanism of action. This study supports the continued investigation of RX-5902 in TNBC and combinations with immunotherapy.

Influence of dietary zinc concentration and supplemental zinc source on nutrient digestibility, zinc absorption, and retention in sheep.

The objective of this study was to assess whether supplemental Zn source or concentration would affect ruminant Zn retention and nutrient digestibility. Thirty-six weaned crossbred Polypay wethers, were sorted by BW to 3 periods and stagger started on a common diet (22 mg Zn/kg DM) for a 52-d depletion period. Day 52 BW was used to assign Zn treatments (3 lambs/treatment/period): no supplemental Zn (CON), or supplemental Zn (40 mg Zn/d) from Zn sulfate (ING; Zinc Nacional, Monterrey, SA, Mexico), Zn methionine (ORG; Zinpro 120; Zinpro, Eden Prairie, MN), or Zn hydroxychloride (HYD; IntelliBond Z; Micronutrients USA LLC, Indianapolis, IN). On day 53 (day 1 of Zn treatments), lambs were moved to metabolism crates for 10 d of adaptation and 5 d of total fecal and urine collection. Blood for plasma Zn analysis was collected on day 52 and day 68. Data were analyzed as a randomized complete design with fixed effects of treatment, period and the interaction, which was significant (P ≥ 0.19) for day 68 plasma Zn but was removed for all other variables. Contrast statements were used to separate treatment means: CON vs. ZINC (ING, ORG, HYD), ING vs. HYD, and ORG vs. HYD. Day 52 plasma Zn concentrations were similar when CON was compared with ZINC (P = 0.84), and when ING and ORG were compared with HYD (P ≥ 0.19). Dry matter and neutral detergent fiber digestibility were lesser in ORG compared with HYD (P = 0.05) and organic matter and acid detergent fiber digestibility tended (P ≤ 0.08) to be lesser in ORG compared with HYD. Intake and fecal excretion of Zn was lesser, while apparent absorption of Zn was greater, in CON compared with ZINC (P ≤ 0.001). Zinc retained as a percent of Zn intake was greater in CON compared with ZINC (P = 0.001). Zinc retained (mg/d) was similar in CON compared with ZINC (P = 0.58) and when ING or ORG were compared with HYD (P ≥ 0.83). There was a treatment × period interaction for day 68 plasma Zn where treatments did not differ for periods 1 and 3 but ORG lambs had increased plasma Zn in period 2 compared with other treatments (P = 0.02). Lambs receiving no supplemental Zn had increased apparent absorption, suggesting Zn absorption may be upregulated in these lambs. Similarities in Zn retention across treatments suggests Zn requirements of these lambs were met regardless of supplementation concentration or source. Nutrient digestibility was improved in HYD lambs compared with ORG, and further work is needed to clarify the influence of supplemental Zn source on nutrient digestion.

The influence of supplemental zinc and ractopamine hydrochloride on trace mineral and nitrogen retention of beef steers.

The study objective was to determine whether N retention was improved with supplemental Zn above NRC concentrations with or without ractopamine hydrochloride inclusion. Angus crossbred steers (n = 32, 485 ± 26 kg BW) with Genemax gain scores of 4 or 5 were utilized in a 2 × 2 factorial arrangement (8 steers/treatment). Steers were blocked by BW to a finishing diet with 1 of 2 mineral supplementation strategies (ZNTRT), no supplemental Zn (analyzed 32 mg Zn/kg DM; CON) or supranutritional Zn (CON + 60 ppm ZnSO4 + 60 ppm Zn-amino acid complex; analyzed 145 mg Zn/kg DM; SUPZN), fed 56 days in pens equipped with GrowSafe bunks and assigned to ß-agonist (BA) supplementation strategies of 0 (NON) or 300 mg steer-1 d-1 ractopamine hydrochloride (RAC) fed the last 30 d before harvest. Initial 56-d ADG was not affected by ZNTRT (P = 0.66), but DMI was greater in CON vs. SUPZN (P < 0.01). On day 56 (day 1 of BA supplementation), steers (4 groups; 8 steers/group; 2 steers/treatment) were moved to metabolism crates and adapted for 10 d, followed by 5 d of total fecal and urine collection. Total retention of Zn, Mn, Fe, Cu, and N were calculated. Data were analyzed as a 2 × 2 factorial arrangement, with group as a fixed effect and the 3-way interaction of ZNTRT × BA × group as random. No interactions between ZNTRT and BA were noted for any data (P ≥ 0.19). Collection DMI did not differ among treatments (P ≥ 0.23); however, Zn intake was lesser in CON vs. SUPZN (P < 0.01). Fecal and urinary Zn excretion and Zn and Mn retention were lesser in CON vs. SUPZN (P ≤ 0.03); however, Zn retention was not different between NON and RAC (P = 0.43). Retention of Cu and Fe was unaffected by strategies (P ≥ 0.49). Urine output and urine N excretion were greater in NON vs. RAC (P ≤ 0.05). Nitrogen retention (as percent of N intake) was lesser (P = 0.05) in CON (40.0%) vs. SUPZN (44.3%) and lesser (P = 0.02) in NON (39.5%) vs. RAC (44.8%). Zinc and N retention were found to be positively correlated (r = 0.46, P < 0.01). Average daily gain and G:F across the 86-d trial were lesser in NON vs. RAC (P < 0.03). Overall, SUPZN appears to improve N retention, suggesting that increasing dietary Zn may be important for cattle growth beyond that induced by ractopamine hydrochloride.

Comparison of trace mineral repletion strategies in feedlot steers to overcome diets containing high concentrations of sulfur and molybdenum.

To compare trace mineral (TM) repletion in feedlot steers after depletion by S and Mo, 72 Red Angus steers blocked by BW (253 ± 14 kg) were assigned (6 steers per pen, fed via GrowSafe bunks) to corn silage depletion diets (depletion, DEP) supplemented with NRC (1996) recommended concentrations of Cu, Mn, Se, and Zn (CON) or supplemented with 0.3% S (CaSO4), 2 mg of Mo/kg dry matter (DM), and no added Cu, Mn, Zn, or Se (antagonist, ANT). Three 62 d TM repletion strategies (repletion, REP) were applied within DEP diets on day 89: 1) Multimin90 injection (contains Cu, Mn, Se, Zn) and 100% of recommended Cu, Mn, Zn, and Se from inorganic sources (ITM), 2) saline injection and 150% of recommended TM from inorganic sources (ING), or 3) saline injection and 150% of recommended TM provided as 25% organic and 75% inorganic sources (BLEND). Subcutaneous injections were given at 1 mL/68 kg BW. Inorganic sources were Cu, Mn, and Zn SO4, and sodium selenite, and organic sources were Availa Cu, Mn and Zn, and SelPlex Se. Repletion period liver and blood were collected on day -10, 14, 28, and 42 and data were analyzed as a 2 × 3 factorial (n = 12 steers per treatment) using Proc Glimmix of SAS with plasma and liver analytes analyzed as repeated measures. Liver Cu, Se, and Mn were decreased (P < 0.01) by ANT during DEP. There were no DEP × REP × day interactions in liver TM (P ≥ 0.18). A DEP × day effect was noted for liver Cu (P < 0.01) and Mn (P = 0.07), where ANT Cu increased linearly from day 0 to day 42, CON Cu was slightly increased on day 14 and day 28, and ANT Mn was lesser than CON Mn on all days except day 42. There were REP × day effects on liver Cu (P < 0.01) and Se (P < 0.01) where status was improved by ITM by day 14, increased in BLEND by day 28, and not different by day 42. Liver Se concentrations were lesser (P < 0.01) in ANT vs. CON throughout repletion. Liver Zn was greater (P < 0.01) on day 0 than day 14, 28, and 42, and concentrations were greater on day 42 than day 28. Glutathione peroxidase activity tended to be lesser (P = 0.07) on day 14 relative to other days. Manganese superoxide dismutase activity was lesser (P < 0.01) on day 14 and 28 compared to day 0 and 42, and tended to be lesser (P = 0.06) in ANT than CON during repletion. Final body weight (BW) and average daily gain (ADG) were not affected by treatment (P ≥ 0.60), and ANT decreased dry matter intake (DMI) (P = 0.04) and improved G:F (P < 0.01) during repletion. All repletion strategies were effective at increasing TM status of steers, and ITM had the most rapid recovery of Cu and Se status, followed by BLEND, and ING.