Walter Gay Bottje

Background: The goal of this study was to evaluate gene expression of water homeostasis-related genes in the intestines of male and female broilers individually phenotyped for low and high water conversion ratio (LWCR and HWCR, respectively). Methods: A 2x2 factorial experiment was conducted with male and female broilers from fourth generation LWCR and HWCR lines. From 4 to 6 wk, broilers were housed in cages to phenotype birds for WCR (g water intake/g body weight gain [BWG]) and feed conversion ratio (FCR, g feed intake/BWG). At 6 wk, samples obtained from the upper duodenum, ileum, and cecum (n=5/group) were frozen in liquid nitrogen. Gene expression was determined by RT-PCR for: 1) the arginine vasotocin ( AVT ) system ( AVT , mesotocin [ MT ] and receptors [ VR2, VR1a, VR1b , MTR ]), 2) the renin-angiotensin system (angiotensinogen [ AGT ], angiotensin receptors [ AT1, AT2 ], renin [ REN ], AGT converting enzyme [ ACE ], and Na+/K+ ATPase [ ATP1B1 ], 3) aquaporins ( AQP1–5 and 11), and 4) genes encoding tight junction proteins (occludin, [ OCLN ], and claudins [ CLDN2, CLDN15 ]). Results: Gene (mRNA) expression of AVT, VR1a, ACE , and AQP 1,3 and 11 was lower and CLDN2 higher in the LWCR compared to the HWCR line in the upper duodenum. In the ileum, mRNA expression of VR1a and AQP11 was lower, and REN , ATP1B1 and CLDN2 higher in LWCR compared to HWCR broilers. In the cecum, the LWCR line exhibited higher expression of VR2 , ACE , and CLDN15 and lower expression of AGT and AQP 5 compared to HWCR broilers. Female broilers exhibited lower expression of AQP4 in the upper duodenum and lower expression of AGT, REN, ACE, AT1 , and AT2 in the ileum compared to males. In the cecum, females had elevated levels of MTR , AGT , ACE , AT1 , AT2 , AQP1,2,3,4 , and 11, and OCLN and CLDN 2 in the cecum compared to males. Conclusion: The results of this study provide insight into water homeostasis gene expression in intestines of male and female broilers divergently selected for LWCR and HWCR. We hope this information will contribute to greater understanding of water homeostasis in broilers and help sustain the broiler industry as water scarcity issues continue worldwide.

Background/Objectives: Seasonal crude protein (CP) and phosphorus (P) deficiency in northern Australian pastures reduces feed intake and growth of grazing ruminants, but the hepatic mitochondrial mechanisms underlying this response remain unclear. We characterized the hepatic mitochondrial transcriptome of sheep exposed to CP-P deficiency or matched-intake feed restriction. Methods: Merino wethers were assigned for 63 d to one of three treatments (n = 8/group): High CP-P, Low CP-P, or Restricted, in which High CP-P feed was offered at the same energy intake as the Low CP-P group. Liver RNA was sequenced, and transcripts encoding mitochondrial proteins were identified using MitoCarta 3.0. Differentially expressed genes (DEGs) were defined as adjusted p < 0.05 and |log2FC| ≥ 0.585. Results: Of 804 mitochondrial genes detected, 83 were differentially expressed in at least one pairwise comparison. The greatest transcriptional response occurred in contrasts against High CP-P (Low CP-P vs. High CP-P: 38 DEGs in 8 enriched pathways; Restricted vs. High CP-P: 37 DEGs in 10 enriched pathways). In both low-intake treatments, ALDH1L2, ALDH1L1, SHMT2, and DMGDH were upregulated, suggesting altered folate-mediated one-carbon metabolism. Restricted sheep also showed higher expression of several SLC25A transporters (SLC25A4, SLC25A28, SLC25A29, SLC25A33, and SLC25A34), indicative of enhanced mitochondrial nucleotide and metabolite exchange under CP-P adequate energy restriction. In contrast, Low CP-P sheep showed higher expression of SLC25A15 and SLC25A25 relative to either High CP-P or Restricted sheep, a nutrient-deficiency specific transporter response. CKMT2 expression was also higher in Restricted sheep than in both other groups. Conclusions: These findings suggest that reduced metabolizable energy intake was associated with the bulk of the hepatic mitochondrial transcriptional response, particularly in folate-mediated one-carbon metabolism, whereas CP-P deficiency was associated with a smaller but distinct transporter signature. The liver mitochondrial transcriptome may provide mechanistic insight into nutritional adaptation under CP and P deficiency in grazing sheep.

Background: The major goal of this study was to compare mRNA expression associated with water homeostasis in intestines of high water efficient (HWE) broilers to expression in unselected Modern Random Bred (MRB) broilers under thermoneutral (TN) and heat stress (HS) environments. Methods: A 2 × 2 factorial study was conducted with two lines (MRB and HWE) and environments (TN and HS). Males were raised in environmental chambers and maintained in TN conditions from 0 to 4 weeks. From 4 to 7 weeks, broilers were maintained under TN conditions (25C) or exposed to cyclic HS conditions (35C, 8 h/d). At 7 weeks, upper and lower duodenum, and cecum samples were obtained. mRNA was determined by RT-PCR for; 1) heat shock proteins (HSP70, HSP90), 2) the arginine vasotocin (AVT), mesotocin (MT) and receptors (VR2, VR1a, VR1b, MTR), 3) the renin angiotensin system (RAS): angiotensinogen (AGT), angiotensin receptors, (AT1, AT2), renin (REN), AGT converting enzyme (ACE), and Na ⁺ / ⁺ ATPase (ATP1B1), 4) aquaporins (AQP1-5 and 11), and 5) tight junction proteins occludin (OCLN), and claudins (CLDN2, CLDN15). Results: A main effect of HS was observed for HSP70 and HSP90 expression in all three intestinal-segments. In lower duodenum, HS-induced upregulation of AVT was accompanied by elevations in VR2, VR1a, VRb, AT1, AQPs (1, 3, 11) OCLN, and CLND2, CLDN15. Main effects of broiler line were observed with HWE exhibiting increased mRNA expression of VR2, VR1a, VR1b, AQP1, AQP3 and AQP5 and CLDN2 in the upper duodenum. There was also higher VR2, MT and AQP5 and lower expression of CLDN15 in the lower duodenum but there were no differences in gene expression between MRB and HWE lines in the cecum. Heat stress upregulated AVT, VR1a, MTR, AVT/MT ratio, AQP1, and AQP3 but downregulated ACE in the upper duodenum. Heat stress also upregulated AVT, VR2, VR1a, VR1b, MTR, AT1, AT2 AQP3, ACP5, OCLN, CLDN2, CLDN15 in the lower duodenum and cecum. Conclusion: The results provide insight into mechanisms responsible for water efficiency in HWE broilers and provide new information in gene expression in the gastrointestinal tract of broilers exposed to chronic cyclic heat stress conditions.

(Bottje and Harrison, 1985ab, 1986ab, 1987). However, the nearly 2-year delay was lucky because I ended up doing a post-doc that ultimately led to an NIH grant a couple years later. I interviewed at the University of New Hampshire but (luckily) I wasn't hired for that position. One thing that I lacked was post-doc experience.When I put my PhD committee together, the 'normal' number of committee members was 5. I added a sixth member, Dr. Ken Holmes, in the Dept. of Veterinary Biosciences (U of I). Dr. Holmes had developed a new method of measuring blood flow in tissue called a thermal pulse decay (TPD) system. I was lucky enough to have been hired into his lab as a post-doc to work on further development and validation of the TPD system (see Arkin et al., 1986). The TPD method could measure time course changes in blood flow (tissue perfusion) at 3 min intervals in 6 areas in a single organ or multiple organs over several hours. Placement of probes in the renal cortex and medulla, enabled us to take repeated measurements in these regions of the kidney -something that had never been done before.Getting started.While working in Dr. Holmes' lab, Dr. Hassan, (in an adjacent lab), came in one day and mentioned that while working with a chemical that rapidly depletes hepatic glutathione (GSH)foot_0 levels, he noticed that the rat's ears turned red. He said, 'I think there are changes in blood flow happening -you should see if it affects liver blood flow'. We conducted a set of studies on rats that revealed an inverse relationship between hepatic blood flow and tissue GSH. This study eventually led to an NIH grant.I took a chance and gave my interview seminar for the Dept. Animal Science (University of Arkansas, UA) on the GSH and liver blood flow study in rats (even though the position was for an Environmental Physiologist in poultry). I was hired and started at the UA, Division of Agriculture in July of 1985. That fall, I attended the American Physiology Society (APS) fall meeting (Niagra Falls, CN) and presented a poster on the apparent inverse relationship between GSH and liver blood flow. Two people stopped by my poster that had a huge impact on me. The first one was Dr. Aubrey Taylor who was President of APS who thought the research was interesting and novel and encouraged me to pursue this rigorously. I submitted the GSH-liver blood flow manuscript one more time and got it accepted in Biochemical Pharmacology (Bottje et al., 1986). The second person I met was Dr. Bob Wideman (at Pennsylvannia State University) who was interested in the TPD method and how it might be used for studying blood flow mechanisms in the avian kidney.We ended up doing a series of studies a few years later using his one lobe avian kidney model. That was a very good piece of luck for me.In early 1986, I gave a seminar for the Chemistry Department at UA the liver blood flow and GSH interrelationship. After the seminar, Dr. Collis Geren (Dept. Chair and later the Dean of the Graduate School) offered to help me package an NIH grant which was extremely fortunate because I had not written a grant proposal before -another piece of luck. Dr. Geren was world renown for his fundamental work in spider venom toxicity. Dr. Geren was also one of those people who looks out for the general good of the community and never self-serving. In 1987, the grant was ranked in the top 5% and fully funded by NIEHS. Starting off as an Assistant Professor with a 5 year large federal grant was extremely lucky. I was able to hire a couple of post doc and graduate students without relying on departmental funds.Antioxidants, Oxidative Stress, Blood flow and Prostaglandins:To our knowledge, the inverse relationship between GSH and hepatic blood in vivo (Bottje et al., 1986) was the first time this was reported. Subsequently, we later reported that indomethacin, an inhibitor of prostaglandin synthase, attenuated both the increase in celiac blood flow in broilers (Beers et al., 1990) and hepatic blood flow and elevations in 6-keto PGF1a (prostacyclin) in rabbits (Bottje et al., 1991) and swine (Bottje et al., 1992). There were also inverse relationships between GSH levels and prostaglandin synthesis in renal medullary homogenates (Nejad et al., 1990). The increased blood flow in tissues following toxic insult could contribute to tissue damage as well as help in tissue repair and recovery.A fundamental study by Anderson et al. (1981) described interorgan circulation of GSH that entailed synthesis in the liver followed by export into the general circulation and taken up by extrahepatic tissues. In that study, blood samples were taken at a single time point; ~ 20 from a systemic artery and hepatic portal vein (representing afferent sources of blood entering the liver) with only 4 samples obtained from the hepatic vein which is difficult to reach due to its location next to the diaphragm within the thoracic cavity. Birds, however, do not have a diaphragm, thus it was possible to obtain repeated blood samples from the hepatic vein. This was facilitated with a hooked needle that Bob Wideman used in his avian kidney studies. In the study by Wang et al. (1998), interorgan circulation of GSH was clearly confirmed and was documented in the avian liver for the first time. This technique also enabled assessment effects of a stress hormone (norepinephrine) that stimulated GSH release from the liver (Song et al., 2000) and hepatic extraction of circulating amino acids and impact of methionine infusion across the hepatic vasculature (Song et al., 2001).Oxidative stress, Mitochondria, and Pulmonary Hypertension Syndrome (PHS).At the fall physiology meeting in 1986, Bob Wideman mentioned a new problem in the poultry industry that he had seen at altitude and was now showing up at sea level; ascites (PHS). I visited his lab after the meeting and he showed me evidence of lung damage in day old chicks. The GSHoxidative stress studies associated with the NIEHS studies...

Selection for water consumption could impact broiler breeders’ immune capabilities. To assess these impacts of selection based on the water conversion ratio (WCR), three trials were conducted using broiler breeders from the modern random bred (MRB), low (L)WCR, and high (H)WCR lines. Ten- to 11-week-old male broilers received intradermal (i.d.) growing feather (GF)-pulp injections of LPS (Trial 1) or PGN (Trial 2), to assess local (GF-pulp) and systemic (blood) inflammatory responses over 24 h and 72 h p.i., respectively. Measurements included leukocyte profiles in GF-pulps and blood, GF cytokine mRNA expression and reactive oxygen species (ROS) generation, and plasma concentrations of α1-acid glycoprotein (AGP-1). In Trial 3, 14-week-old pullets were immunized by i.d. GF-pulp injection of SEV (10⁸ CFU/mL). Leukocyte profiles in the GF-pulp and blood were measured over 72 h and plasma levels of SEV-specific IgM, IgY(G), and IgA antibodies over 4 weeks p.i. Independent of the line, phagocytes infiltrated GF-pulps by 6 h post-LPS injection (p ≤ 0.05), while lymphocytes were the major leukocyte recruited in response to PGN (p ≤ 0.05). However, with both LPS and PGN, HWCR broilers were less effective in recruiting lymphocytes than MRB and LWCR broilers, which had similar lymphocyte infiltration levels. There were no line differences in GF-pulp cytokine mRNA expression and ROS generation, nor in blood leukocyte and AGP-1 concentrations, following LPS injections. Independent of the line, SEV immunization stimulated similar phagocyte recruitment profiles; however, the LWCR and MRB lines had a higher infiltration of lymphocytes (esp. B cells) than the HWCR line (p ≤ 0.05). Independent of the line, SEV immunization triggered a robust, high-quality, primary SE-specific antibody response (p ≤ 0.05). Collectively, selection for improved water efficiency in the LWCR broiler breeder lines did not negatively impact immune response capabilities to LPS, PGN, and a killed SEV.

Fenugreek seeds (FSs) are a natural source of bioactive compounds that may modulate the immune system and gut microbiota in broilers. This study examined the effects of dietary fenugreek seed powder on immune-related gene expression and cecal microbiota composition in broilers. A total of 144 broiler chickens were randomly allocated to three dietary groups, CON (0 g/kg FS, FS5 (5 g/kg FS) and FS10 (10 g/kg FS), each with 6 replicates of 8 birds. Ileum tissues and cecal contents were collected on day 42 for the mRNA expression of inflammation and antimicrobial defense-related genes and cecal microbiome diversity, respectively. The results indicated that fenugreek seeds downregulated mRNA-level inflammation and antimicrobial defense-related genes: IL6, IL8L2, CASP6, PTGS2, IRF7, AvBD9, AvBD10, and AvBD11. Moreover, fenugreek seeds altered the cecal microbial community by increasing the population of Firmicutes and decreasing the population of Actinobacteriota, Gemmatimonadota and Verrucomicrobiota at the phylum level and increasing Alistipes, Bacteriodes and Prevotellaceae at the genera level. These findings suggest that fenugreek seeds have a positive impact on the immunological profile and microbiome of broiler chickens, possibly through the interplay of the immune system and the gut microbiome.

Two experiments were conducted to evaluate the effects of fenugreek seeds (FS) as a potential alternative to antibiotic growth promoters in broiler chickens. In the first experiment, one-day-old Ross (n = 160) straight-run broilers were fed FS at 0 g, 2.5 g, 5 g, and 10 g/kg of diet during the starter (from 1 to 21 days) and finisher phase (from 22 to 35 days) with four replicates of ten birds each. In the second experiment, one-day-old Ross (n = 144) male broilers were fed 0 g, 5 g, and 10 g FS per kilogram of diet during the starter (from 1 to 21 days) and finisher phase (from 22 to 42 days) with six replicates of eight birds each. In addition to growth performance, hematological parameters and intestinal histomorphology were measured in the second experiment. FS linearly reduced the body weight gain (BWG) (p < 0.001), feed intake (FI) (p < 0.05), and increased feed conversion ratio (FCR) (p < 0.05) during the starter phase in both experiments. However, no significant effects on BWG, FI, and FCR were observed during the finisher phase. Moreover, the overall BWG and FI were linearly reduced (p < 0.05) with the increasing levels of FS, but BWG and FI were similar in the 5 g/kg FS group and control group. The inclusion of FS had a linear increase in white blood cell (WBC), heterophil, and lymphocyte count (p < 0.005) and the decrease in hematocrit % (p = 0.004) and total bilirubin (p = 0.001). The villus height and villus height: crypt depth ratio of jejunum and ileum were significantly lower in 5 g FS and 10 g FS treatments (p < 0.001) compared to the control. The result indicates that the dietary inclusion of FS reduces the early growth performance, increases the WBC counts, and negatively affects the intestinal morphology of broiler chickens.