M.S. - Animal Sciences

Permanent URI for this collectionhttps://hdl.handle.net/10125/589

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Current management practices of local small ruminant producers and survey of the trace mineral status of locally harvested sheep in Hawaiʻi
(University of Hawai'i at Manoa, 2025) Nishimura, Shaye; Reichhardt, Caleb C.; Animal Sciences
The small ruminant industry in Hawaiʻi has experienced notable growth due to increasing consumer demand. However, limited research exists regarding the management practices and nutritional health of sheep and goat herds across the islands. This study aimed to characterize the current practices of small ruminant producers in Hawaiʻi and evaluate trace mineral concentration in locally raised sheep. A survey was administered to collect data on producer demographics, herd management, feeding practices, health protocols, and marketing strategies. Results showed that 78% of respondents were female, 79% held at least a college degree, and 70% owned their land instead of leasing it. Of all respondents, 64% only raised sheep, 15% only raised goats, and 21% raised both sheep and goats together. Dorper, Katahdin, and Barbados were the common sheep breeds. Feeding strategies, grazing systems, and production goals varied. Health protocols such as deworming were widely used, but 54% of respondents did not implement reproductive practices. To assess trace mineral status, 83 liver and 79 plasma samples were collected from 11 farms across Hawaiʻi and Oʻahu. A high percentage of liver samples were deficient in copper (47%), iron (46%), and cobalt (31%), while 11% showed toxic manganese concentration. Significant correlations were observed between plasma zinc and magnesium (r = 0.814, p <0.0001) and between liver molybdenum and selenium (r = 0.72, p < 0.0001). Trace mineral concentrations varied significantly between farms, suggesting that farm-specific management or environmental factors influence mineral status. These findings provide valuable baseline data for improving trace mineral nutrition and herd health, further highlighting the need for locally tailored management strategies to support the sustainability and productivity of Hawaiʻi’s small ruminant industry.
Supplementation of orally drenched ascorbic acid and injectable α-tocopherol reduces markers of naturally acquired gastrointestinal parasites in grazing hair sheep
(University of Hawai'i at Manoa, 2025) Rojas-Reyes, Jesus A.; Reichhardt, Caleb C.; Animal Sciences
The objective of this study was to evaluate the effects of exogenous vitamin E and C supplementation in grazing hair sheep on markers of naturally acquired parasites, hypothesizing that supplementing vitamins may be effective as anthelmintics in controlling parasites. Twenty-six lambs and thirty-one ewes were stratified by FAMACHA® scores and placed in one of five treatment groups: (1) control (CON; Lambs: n = 6, Ewes: n = 4), (2) dewormer (DW; Levamed; 0.08 mg/kg; Lambs: n = 5, Ewes: n = 7), (3) vitamin C (VC; Vitamin C;0.56 ml/kg of BW; Lambs: n = 5, Ewes: n = 6), (4) vitamin E (VE; Vitamin E 300; 22 IU/kg of BW; Lambs: n = 6, Ewes: n = 7), and (5) combination of vitamins E and C (E+C; same dosages as vitamins C and E; Lambs: n = 4, Ewes: n = 7). Physiological, parasitology and blood metabolite parameters samples were collected on day 0, 14, 28, 56, and 77. The following parameters and treatments are compared to the CON, unless otherwise mentioned. FAMACHA® scores improved in lambs treated with DW (P = 0.0001), E+C (P = 0.04), VC (P = 0.02), and VE (P = 0.01). Eggs per gram (EPG) decreased in lambs treated with DW (P = 0.001), VC (P = 0.001) and VE (P = 0.008). Alanine transaminase (ALT) decreased (P = 0.04) in lambs treated with DW and VE treated lambs increased (P = 0.02) in ALT compared to the dewormer. Packed cell volume (PCV) in the ewes improved with E+C (P = 0.02) and VE (P = 0.03) compared to DW. The data suggests that vitamin E and C supplementation can be used as a tool to assist in managing parasites in grazing hair sheep.
Evaluating the impact of increasing vitamin C and E concentration on primary ovine satellite cells
(University of Hawai'i at Manoa, 2025) Blad , Taya J N; Reichhardt, Caleb C.; Animal Sciences
Skeletal muscle becomes meat through a series of biochemical processes post-harvest. Since sheep producers are typically paid based on carcass weight, a goal is to optimize skeletal muscle growth, as well as the yield and quality of the final meat product. Satellite cells are stem cell-like cells responsible for mammalian postnatal skeletal muscle growth capacity through myonuclear accretion. Satellite cells proliferate and then differentiate into myoblasts before fusing with existing myotubes. Inflammation, as well as free radicals, stimulate skeletal muscle growth. Antioxidants such as vitamins C and E combat unstable free radicals to maintain a healthy concentration to promote skeletal muscle growth. Despite potential involvement in sheep skeletal muscle growth, ideal concentrations of vitamin E and C for optimal skeletal muscle growth are unknown. The objective of this project is to determine if increasing concentrations of α-tocopherol or ascorbic acid affects the proliferation rates of primary ovine satellite cells (OSC) or protein synthesis rates of fused primary OSC. Primary OSC were isolated from the hind leg of 9-mo old commercial hair sheep wethers (n=8). For proliferation, cultures were grown to approximately 70% confluency, followed by treatment with 5% fetal bovine serum (FBS) or 5% FBS and concentrations of α-tocopherol (4.6 µM, 9.2 µM, or 13.8 µM) or ascorbic acid (8 µM, 16 µM, or 24 µM). Protein synthesis cultures were induced to differentiate at approximately 85% confluency and treated 48 h later with serum free media (SFM) or SFM and concentrations of α-tocopherol (4.6 µM, 9.2 µM, or 13.8 µM) or ascorbic acid (8 µM, 16 µM, or 24 µM). Proliferation rates were measured at 24 h post-treatment, and protein synthesis rates were measured at 6 h post treatment. Fused primary OSC protein synthesis rates linearly increased (P = 0.007) with increasing α-tocopherol concentrations. Increasing ascorbic acid treatment led to a linear increase (P = 0.05) of proliferation rates, while fused primary OSC treatment saw a linear (P = 0.08) and quadratic (P = 0.06) tendency for an increase in protein synthesis rates. These findings suggest that increasing vitamin C supplementation could increase myonuclear accretion potential, and increasing vitamin E and C supplementation could increase skeletal muscle growth.
EVALUATION OF EGGSHELLS USING A SCANNING ELECTRON MICROSCOPE AND ENERGY DISPERSIVE SPECTROMETRY
(University of Hawai'i at Manoa, 2024) Mahato, Prem Lal; Mishra, Birendra; Animal Sciences
The eggshell is the outermost covering of an egg that provides physical and chemical protection. It is a major source of calcium and minerals for the growing embryo during incubation. The egg industry suffers from a considerable economic loss due to poor eggshell quality. Therefore, developing an accurate and precise method of determining eggshell quality is crucial in improving eggshells in subsequent generations of breeding stock. Hence, this study aimed to develop a method to accurately and precisely determine 1) eggshell thickness using a scanning electron microscope (SEM) and 2) eggshell mineral components using an SEM-Energy Dispersive Spectrometry (EDS) system. Four types of table eggs (N = 48; 12 eggs/group): Cage-Free Organic from the US Mainland (CFO-M) and Hawaii (CFO-H), Caged Non-Organic from the US Mainland (CNO-M), and Hawaii (CNO-H) were sourced from the grocery store. Approximately 0.5 mm2 pieces of eggshells from the equator region of the egg were taken and processed for visualization under the SEM. Three distinct layers of eggshell were identified under SEM: the outermost cuticle, the middle palisade, and the innermost mammillary region. The results showed that CFO-H eggs have a greater eggshell thickness (380.43 ± 2.69 µm) and effective thickness (306.28 ± 4.15 µm). Similarly, the mammillary knob count was denser in CNO-H eggs (186 ± 23.02 knobs/0.5 mm2). Calcium (97.36 ± 0.17%) was the highest among minerals in lower palisades (LP). The magnesium concentration was lowest in the LP region, whereas the phosphorus concentration was highest in the upper palisades. Our study established a scientific method to assess the eggshell quality and biochemical characteristics of eggs through SEM and EDS. This method can be used as a marker for selecting superior parent stock to improve eggshell quality in subsequent generations of breeding stock.
THE STRESS RESPONSE OF THE BRASSY CHUB, A LOCAL REEF FISH AND CANDIDATE FOR SUSTAINABLE AQUACULTURE
(University of Hawai'i at Manoa, 2024) Merlo, Reilly Stanton; Seale, Andre P.; Animal Sciences
The brassy chub, Kyphosus vaigiensis, is a reef herbivore that has been shown to growrapidly in captivity, though no information is available on how they respond to maintenance- related stress, a major component of captive fish welfare. Generally, in fish, the stress response has been shown to disrupt hydromineral balance and activate endocrine systems including the hypothalamus-pituitary-interrenal (HPI) axis, which are characteristic responses of the alarm phase. During the resistance phase of the stress response, typical changes involve energy metabolism affecting glucose availability and antioxidant enzyme activity. In this study, I characterized the effects of acute handling stress on the physiological responses of brassy chub over a 24-hour (h) time course, by measuring endpoints associated with both the alarm and resistance phases of the stress response. These included plasma and mucus cortisol, plasma glucose and osmolality, and hepatic activity of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPX), and hepatic lipid peroxidation. The results indicated that plasma and mucus cortisol levels were transiently increased in stressed fish by 1 h, suggesting activation of the HPI axis. Moreover, plasma glucose increased by 1, 12, and 24 h while plasma osmolality increased at 1 and 6 h and decreased at 12 and 24 h. SOD activity decreased in the stressed treatment at 6 and 12 h and returned to control levels by 24 h. GPX activity and lipid peroxidation were unaffected by the handling stressor throughout the 24 h period. In addition to validating the use of mucus as a minimally invasive technique to detect HPI axis activation, this study provides fundamental insight on the effects of a maintenance- related stressor on osmoregulatory function, energy mobilization, and the antioxidant defense system. Understanding these responses provides critical insights needed to inform management practices for the welfare and production of the brassy chub.
MITIGATION OF HEAT STRESS IN BROILER CHICKENS USING DIETARY SUPPLEMENTATION OF MICROALGAE
(University of Hawai'i at Manoa, 2023) Chaudhary, Ajay; Mishra, Birendra BM; Animal Sciences
Heat stress is a serious concern in the poultry industry, affecting their health and productivity. Prolonged exposure to high environmental temperatures can disrupt the basic cellular mechanisms, including the redox system, thereby impeding the production potential of broilers. This phenomenon can consequently lead to oxidative stress, predominantly resulting from the excessive formation of free radicals and reduced antioxidant enzymes. To effectively address the issue of heat stress, it is essential to include antioxidant-rich compounds in the poultry diet to ensure the proper functioning of the redox system. Microalgae (Spirulina platensis) are rich in antioxidants and have several health benefits in humans and animals. However, the effects of microalgae on the health and production in heat-stressed broilers, as well as the underlying mechanism, remains poorly understood. The primary objective of this study was to determine the effect of microalgae supplementation on the health and production of heat-stressed broilers. Cobb500 day-old chicks (N=144) were raised in litter floor pens (6 pens/treatment and 8 birds/pen). The chicks were divided into 3 treatment groups: a) no heat stress (NHS), b) heat stress (HS), and c) heat stress+3% microalgae (HS+MAG). The broilers in the HS+MAG group were fed a diet supplemented with 3% microalgae, whereas NHS and HS groups were fed a standard broiler diet. Broilers in the NHS were raised under standard temperature (20-24⁰C), while HS and HS+MAG broilers were subjected to cyclic heat stress from day 22-35 (32-33⁰C for 8 hours). All data are presented as mean ± SEM after performing one-way ANOVA Tukey’s post hoc test using GraphPad Prism. Microbial bioinformatics was performed in CLC Workbench and Spearman’s rank correlation was performed to establish correlations between microbiota and other parameters. The significance of the experiment was set at P<0.05. Heat stress significantly decreased the final body weight, whereas the supplementation of microalgae increased the final body weight of broilers (P<0.05). The expressions of ileal antioxidant (GPX3), immune-related (IL4), and tight-junction (CLDN2) genes were significantly increased in microalgae-supplemented broilers compared to heat-stressed broilers (P<0.05). The ileal villus height to crypt depth ratio was improved in microalgae-supplemented broilers (P<0.05). In addition, microbial alpha, and beta diversities were higher in the HS+MAG group compared to the HS group (P<0.05). The group that received microalgae supplementation exhibited an increase in volatile fatty acid-producing bacteria at the genus level, including Ruminococcus, Ocillospira, Lactobacillus, Oscillobacter, Flavonifractor, and Colidextribacter. In conclusion, dietary supplementation of microalgae improved the growth performances of heat-stressed broilers by improving their physiogenomics and gut microbiota composition. Thus, the dietary inclusion of microalgae can potentially mitigate heat stress in broilers.
Effects Of Microalgae, With Or Without Xylanase Supplementation, On Growth Performance And Gut Health Parameters Of Broiler Chickens
(University of Hawaii at Manoa, 2023) Mishra, Pravin; Jha, Rajesh; Animal Sciences
Modern broilers are selected for a fast growth rate in a shorter period. The rapid increase in body weight leads to the possibility of gut disorders like poor gastrointestinal tract development, poor nutrient utilization, and gut dysbiosis if not managed properly. Nutritional programming using several feedstuffs and co-products has been studied to manage gut health without compromising growth performance. Microalgae are becoming potential sustainable feed ingredients, while terrestrial feedstuffs are becoming scarce and costly. They are rich in nutritional and functional values but have lower digestibility. Therefore, the study evaluated the effects of microalgae with or without xylanase supplementation on growth performance and gut health parameters of broiler chickens. A total of 162-day-old Cobb 500 chicks were raised for 35 days. Birds were fed with three dietary treatments: a) corn-soybean meal-based diet (CON), b) CON + 3% microalgae (MAG), and c) MAG + xylanase (MAG+XYN) in two phases (starter: d0-21 and finisher: d22-35). Weekly body weight (BW) and feed intake were recorded to calculate average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR). On d35, carcass and organ weight were recorded. Tissue samples were collected for histomorphology (duodenum, jejunum, and ileum) and total RNA isolation (ileum) on d3 and d35. In addition, blood was collected for serum immunoglobulin (on d35) and cecal digest for short-chain fatty acids, microbiota characterization, and functional metabolic pathways determination (on d3 and d35). Ileal gene expression related to the gut barrier, immunity, antioxidant, and nutrient transporter was determined using qPCR. Bioinformatics analyses were performed in CLC Genomic Workbench and STAMP. All other data were analyzed in GraphPad Prism or RStudio using one-way ANOVA followed by Tukey’s multiple comparison test. The significance level was set at p<0.05, and the results are expressed as the mean ± SEM. The BW, ADG, and ADFI were significantly higher (p<0.05) in MAG and MAG+XYN compared to the CON group. No significant difference (p>0.05) was found for FCR. Relative carcass and organ weight also showed no significant differences among treatments (p>0.05). The expressions of ZO1, CD56, and SLC7A7 were significantly higher (p<0.05) in the MAG group. Statistically insignificant, however, villi height (VH), crypt depth (CD), VH:CD, and villi surface area (VSA) tended to increase in MAG and MAG+XYN groups both on d3 and d35. Relative microbial abundance at the genus level showed that MAG and MAG+XYN groups had a diverse microbial community on d3 and d35. However, no bacterial genus has a significant difference in their relative abundance on d3, but sixteen genera showed significant differences in their relative abundance among the dietary treatments on d35. Most of these bacteria are short-chain fatty acid-producing bacteria. Moreover, MAG and MAG+XYN-fed broilers had better responses than CON groups for metabolic pathways (D-mannose degradation, Pectin degradation I and II, β-1-4-mannan degradation, tetrahydrofolate biosynthesis, glutathione biosynthesis, glutathione-peroxide redox reactions, lactate fermentation to propanoate, acetate, and hydrogen, etc.) both on d3 and d35. In conclusion, the use of microalgae with or without xylanase in a corn-soybean meal-based broilers diet promotes growth performance, immunity, and gut health of broiler chickens. Improvement in SCFA production, microbial diversity, and metabolic pathways is a sign of improved gut health. Most of the statistically changed pathways are related to fiber utilization and oxidative stress reduction. In addition, using microalgae in feeding programs may help to produce healthy broilers.
The Stress Response Of Mozambique Tilapia (Oreochromis Mossambicus) Transferred To Dynamically Changing Salinities
(University of Hawaii at Manoa, 2022) Chang, Ryan James Akira; Seale, Andre P.; Animal Sciences
An organism’s survival depends on effectively responding to stressors. One of the many stressors aquatic organisms face is osmotic stress due to differences between their internal and environmental solute concentrations. In estuarine environments, euryhaline fish can maintain a stable internal osmolality despite daily changes in environmental salinity. The capacity of euryhaline fish to maintain homeostasis in a wide range of salinities is largely facilitated by the endocrine system. The hypothalamus-pituitary-interrenal (HPI) axis plays a role in the hypo- and hyperosmotic stress responses by facilitating ion and water transport at the gill while also modifying glucose metabolism to provide energy. I characterized responses of the HPI axis and glycogen metabolism in the gill and liver of the euryhaline Mozambique tilapia (Oreochromis mossambicus) under salinity stress. Specifically, in one study I analyzed fish transferred from freshwater (FW) to seawater (SW) and from SW to FW and, in the other, from steady state salinities to salinities that changed between FW and SW every 6 h to simulate a tidal environment. In both studies, I found that the effects of salinity in the HPI-axis were mainly characterized by changes in branchial corticosteroid receptor expression. Moreover, a robust rise in plasma glucose was observed when fish were transferred to FW, and in the FW phase of the tidal cycle, suggesting that glucose may play an osmotic role in compensating for the fall in plasma osmolality triggered by FW environments. Given the natural changes in salinity that occur in a range of wild and controlled environments, identifying changes in the underlying components of the stress response to perturbations in salinity can help mitigate the maladaptive effects of stress, thereby informing environmental management and aquaculture practices.
Transcriptomic Analysis Of Bovine Endometrium With Short Uterine Tract Leading To Infertility
(University of Hawaii at Manoa, 2022) ADNAN, MAHFUZ RAHMAN; Mishra, Birendra; Animal Sciences
Background: Beef is one of the most highly consumed meat worldwide. With the increasing human population, the demand for beef will be increased by 43% in 2050. However, the production of beef cattle has declined in recent years. Infertility/subfertility is one of the main reasons causing a decline in beef production. Infertility is the diminished or incapability of females to produce viable offspring, causing a substantial economic loss. Since 1980, infertility has been increasing among the cattle population worldwide. Infertility is triggered by several factors, including developmental defects of the reproductive tract. Recently, Hawaii beef producers reported that heifers selected for breeding had good body condition scores and were bred several times with fertile bulls but failed to conceive. After slaughtering at a local abbatoir, these heifers had small uterine tract with normal ovaries. The uterus provides the biological environment for the embryo’s growth and development. Bovine endometrium consists of caruncular (CAR) and Intercaruncular (ICAR) regions. The caruncular region serves as the implantation and placentation site, whereas the intercaruncular region contains numerous glands for preparing the biological environment for the embryo's growth and development. Studies have suggested that secretions from the ICAR are necessary for the conceptus growth, development, and survival. So far, there is no report on infertility induced by smaller reproductive tract in bovines. Therefore, we hypothesized that the heifers with short uterine tract might have abnormal uterine transcriptomic profiles and developmental defects leading to infertility. The objectives of this study are to 1) analyze the morphological difference between the short and normal uterine heifers, and 2) determine the genes and biological pathways in the intercaruncular endometrium of the heifers with short uterine tract compared to normal heifers.Methods: We examined the reproductive tracts of over 171 cattle from local slaughterhouses; among them, 51 heifers had a smaller uterine tract. Uterine tissues were collected from a subset of infertile heifers (n=6, designated as the short uterine tract). Uterine tissues were also collected from cyclic heifers having normal uterine tract (n=6, cyclic heifers, designated as the normal uterine tract). Samples were selected, matching the heifers' corpus luteum stage (II). For the histological study, uterine tissues were collected and fixed in 10% Neutral buffered formalin. The fixed tissues were sectioned and stained with Hematoxyline and Eosin. For the transcriptomic study, intercaruncular tissues were collected in RNAlater solution. Total RNAs were isolated and were subjected to high-throughput RNA-sequencing (n=5/group). The differentially expressed genes (DEGs) were determined between the short uterine tract compared to normal. To delineate the DEGs and associated biological pathways, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Ingenuity Pathway Analysis were done. The most highly upregulated and downregulated genes expression were validated using real-time quantitative PCR (qPCR). Results and conclusion: Total uterine layer length, endometrial layer length, and gland numbers were significantly lower in the short uterine tract than normal. A total of 521 genes were differentially expressed in the short uterine tract compared to normal. The top 20 downregulated genes in the short bovine endometrium were NLRP12, ENSBTAG00000052892, ENSBTAG00000055111, ENSBTAG00000036102, LOC112444733, LOC100847874, OTOG, CLCNKA, NPPC, CYP26A1, LOC788915, VNN2, PRKCG, LOC782061, MYOT, PLA2G3, ANKS1B, GPR183, ST8SIA6, and TMIE. Metabolic pathways, carbon metabolism, and Wnt signaling pathways were the top three enriched pathways by the downregulated DEGs. The qPCR result confirmed the expression of the DEGs in short uteri compared to normal. In conclusion, heifers with short uterine tract had significantly decreased endometrial layers, uterine glands, and altered transcriptomic profiles. The decrease in uterine glands probably resulted in lower uterine secretions necessary to support embryo growth and development. As a result, heifers with short uteri were infertile even when they were bred by fertile bulls.
Thermal-induced changes in cell volume and hormone release in prolactin cells of Mozambique tilapia
(University of Hawaii at Manoa, 2021) Woo, Daniel Wan; Seale, Andre P.; Animal Sciences
The multifunctional pituitary hormone prolactin (PRL), is critical for survival when fish acclimate to freshwater environments by stimulating ion retention, ion absorption, and water secretion. Due to the important role PRL plays in osmoreception, examining the effect of temperature on this fundamental process can offer insight into how natural, climate-change- driven, or controlled temperatures may alter a process that underlies salinity acclimation in fish. Consistent with their role in fresh water (FW)-acclimation, PRL cells of the euryhaline and eurythermal Mozambique tilapia (Oreochromis mossambicus), respond to a drop in osmolality with a short-term release of PRL. Hyposmotic-induced PRL release is triggered by an increase in cell volume that enables extracellular Ca2+ to enter the cell through the transient receptor potential vanilloid 4 (TRPV4), a stretch gated cation channel shown to be thermosensitive in some vertebrates. While the cell-volume-dependent secretory response of tilapia PRL cells to a hyposmotic stimulus is well established, the effects of temperature on PRL cells have not been characterized. PRL cells were dispersed from the rostral pars distalis (RPD) of Mozambique tilapia and incubated at 26 and 32 °C in isosmotic and hyposmotic media. A delayed rise in PRL release from dispersed PRL cells was observed in response to an increase in temperature while hyposmotic-induced PRL release occurred rapidly. When dispersed PRL cells were subjected to a temperature increase from 26 to 32 oC, ~50% of the total cells gradually increased in volume by ~8%; all cells were responsive to a hyposmotic stimulus (280 mOsm/kg), increasing in volume by ~20%. These findings indicate that thermal-induced PRL release may be mediated, at least in part, through a cell-volume-dependent mechanism, similar to how osmoreception is achieved. Moreover, a possible mechanism through which many of the functions of PRL are affected by temperature is proposed.
The Effects Of Maternal Immunization Against Myostatin On Post-hatch Chicken Growth
(University of Hawaii at Manoa, 2021) Mishra, Rajeev Kumar; Kim, Yong Soo; Animal Sciences
Myostatin (MSTN) is a negative regulator of skeletal muscle growth. A previous study showed that in-ovo administration of monoclonal anti-MSTN antibody increased post-hatch body weight and muscle mass, demonstrating the potential of in-ovo MSTN suppression to improve the growth performance of chicks. Therefore, we hypothesized that active immunization of hens against MSTN will produce anti-MSTN antibodies, and this antibody will be passively transferred into fertilized eggs, leading to improvement of skeletal muscle growth of the first-generation chicks. Recombinant myostatin (chMSTN) was produced from transformed E. coli and was processed for hen immunization. Two experiments examined the effects of maternal immunization against chMSTN and Myo2, a synthetic MSTN fragment, on the growth performance and muscle mass of chicks from immunized hens. In Experiment 1, twelve 18 weeks old Hy line brown hens were randomly allocated into four pens with a breeder’s feeding program plus freely available water, each pen with 3 hens and 1 White Leghorn rooster of the same age. At 24 weeks of age, the 4 pens were randomly divided into two groups with two pens in each group: Control and chMSTN. Hens in the Control and chMSTN groups were immunized by subcutaneous injection of 0, and 0.5 mg of chMSTN emulsified with adjuvant on days 0, 7, and 21, respectively. The peak antibody titer was seen around day 35 from the first immunization, and the peak egg yolk antibody titer was day 41. About 60 eggs were collected from each group between day 38 to 48 after the first immunization and were incubated under standard conditions. Chicks were reared under a deep litter system for 7 weeks with weekly bodyweight monitoring. At sacrifice, viscera were removed, breast muscle and bone-in legs were separated and weighed. There was no difference in the body weight and carcass parameters between the two groups. In Experiment 2, the two pens of the chMSTN group used in Experiment 1 were divided into chMSTN and Myo2 groups while the two pens of the Control group remain as the Control group. At 43 weeks of age, Control and chMSTN groups were immunized in the same way as the experiment 1 on days 0 and 7. The Myo2 group was immunized with 2 mg of Myo2 peptide (synthetic MSTN fragment) conjugated to KLH /100 μl of PBS emulsified with the same volume of adjuvant. The peak serum antibody titer was seen around day 21 from the first immunization, and the peak egg yolk antibody titer was day 28. Thirty-three eggs were collected from each group between days 24 to 34 after the first immunization and were incubated and chicks were reared for 11 weeks and sacrificed in the same manner as was performed in Experiment 1. The body weight of the chMSTN was not different from that of the Control, but the body weight of the Myo2-KLH was significantly lower than that of the Control. The dressing % and breast muscle mass of the chMSTN and Myo2 were significantly lower than those of the control, and the breast muscle mass of Myo2 was significantly lower than that of the chMSTN, indicating that maternal immunization against chMSTN or Myo2 suppressed muscle growth of offspring with immunization against Myo2 being more suppressive than immunization against chMSTN. In Western blot analysis, IgY from the chMSTN and Myo2 groups showed binding affinity to the chMSTN, Myo2, and commercial MSTN. However, the binding affinity of egg yolk IgY to commercial MSTN was much lower than to chMSTN, Myo2-KLH, or Myo2-BSA. The weak binding affinity to bioactive MSTN of the IgY partly explains the reason why the maternal immunization against chMSTN and Myo2 did not increase the muscle mass of offspring, but the reason for reduced muscle mass of the offspring from immunized hens is not clear. In summary, the maternal immunization against chMSTN and Myo2 did not increase but decrease the body weight and muscle mass of offspring from the immunized hens. The results are not as expected since the inhibition of MSTN via maternal immunization could not enhance the body weight and muscle mass of offspring. However, the results show that the body weight and muscle mass of offspring could be controlled by maternal immunization against molecules having physiological functions.
RNA-sequencing Based Analysis Of Bovine Endometrium During The Maternal Recognition Of Pregnancy
(University of Hawaii at Manoa, 2020) Adhikari, Bindu; Mishra, Birendra; Animal Sciences
Background: Reproductive efficiency is crucial to the production of food animals and overall profitability of the farm. The majority of pregnancy losses occur in the first month, especially around Day-19 of gestation, mainly due to the inability of the uterus to support conceptus growth or abnormal development of conceptus. Since the incidence of pregnancy failure does not occur in a single day of Day-19, then days 15-17 is a critical period for the maternal recognition and establishment of pregnancy. We hypothesize that RNA-Sequencing based analysis of bovine endometrial tissues during the critical period of maternal recognition of pregnancy will reveal important genes and biological pathways required for the conceptus growth and development. Objectives: Hence, the objectives of the current study are: 1) To identify the important differentially expressed genes (DEGs) and biological pathways in the bovine caruncular endometrium among the groups (Pregnant vs. Cyclic) and (Pregnant vs. Non-Pregnant), and 2) To validate the most highly up-regulated DEGs using quantitative polymerase chain reaction (qPCR). Methods: Grass-fed Angus heifers (2-3 years old) were used for sampling. The estrous cycles of heifers (n=21) were synchronized using the intramuscular injection of a Prostaglandin F2 alpha (on Day-0 and -11). Fifteen heifers were bred by natural mating at estrus. Endometrial samples were collected at Day 15-17 of gestation (pregnant), of estrous cycle (cyclic), and absence of conceptus (nonpregnant) heifers. Total RNAs were isolated and were subjected to high throughput RNA-sequencing (n=4/group). The genes with at least two-fold change, and Benjamini and Hochberg q-value <= 0.05 were considered as differentially expressed. The mRNA expression of selected candidate genes in the bovine endometrium was also validated using qPCR. Results: A total of 107 genes (pregnant vs. cyclic), and 98 genes (pregnant vs. Nonpregnant) were differentially expressed (FDR <0.05) in the pregnant endometrium. The most highly up-regulated genes in the pregnant endometrium were MRS2, CST6, FOS, VLDLR, ISG15, IFI6, MX2, C15H11ORF34, EIF3M, PENK, PRSS22, MS4A8, CLDN4, TINAGL1, and R3HDM1. Gene ontology analysis revealed that the biological process related to Type-1 interferon signaling (MX1, MX2, IFI6, IRF1, and ISG15), immune response (IL23A, and RSAD2), extracellular matrix organization (COL1A1, COL1A2, COL3A1, and TIMP2) and ion transporters (SLC34A2, SLC2A1, SLC16A11, SLC16A4 and ATP1B1) were significantly enriched in the pregnant endometrium. The qPCR results confirmed the significantly higher (P <0.05) mRNA expression of MRS2, CST6, FOS, VLDLR, ISG15, IFI6, MX2, C15H11ORF34, PRSS22, TINAGL1, and MS4A8 in the presence of conceptus in the bovine endometrium. Conclusions: Both the RNA-Seq and qPCR confirmed the differential expression of several pre-discovered and novel genes, and their biological pathways during the maternal recognition of pregnancy (Day 15-17 of gestation) compared to cyclic and non-pregnant endometrium. Interferon signaling, immune response, nutrient transporter, synthesis, and secretion of proteins are crucial pathways during the maternal recognition of pregnancy. Overall, this study identified the differentially expressed genes and their pathways in the pregnant caruncular endometrium compared to cyclic and non-pregnant. In this study, using RNA-sequencing, we found some novel genes (MRS2, C15H11ORF34, and PRSS22). The study demonstrated that the presence of conceptus on day 15-17 of gestation could actively affect the endometrial gene expression during the maternal recognition of pregnancy. In summary, this study provides a comprehensive dataset of transcripts associated with maternal recognition of pregnancy.
Mitigation Of Heat Stress In Poultry Using Dried Plum Or Alpha-lipoic Acid Supplement
(University of Hawaii at Manoa, 2020) Wasti, Sanjeev; Mishra, Birendra; Animal Sciences
Introduction: Heat stress is a significant problem in the poultry industry, causing a severe economic loss due to its detrimental effect on the health and performances of chickens. Dried plum (DP) is a good source of minerals, vitamins, antioxidants, and phenolic compounds, and plays a role in calcium homeostasis and cardiovascular dysfunctions. Alpha-lipoic acid (ALA), on the other hand, is water and fat-soluble antioxidant, which can be readily absorbed from the intestine resulting in maximum bioavailability. Moreover, ALA acts as a coenzyme in glucose metabolism and helps generate other antioxidants. Considering these health benefits and properties, we hypothesized that the dietary supplementation of DP or ALA would help to mitigate heat stress in poultry. Objectives: The purposes of this study were to: 1) to determine the effects of DP supplementation on growth performance, gut health and immune parameters of heat-stressed broiler chickens, and 2) to determine the effects of ALA supplementation on growth performance, gut microbiota, gut health and immune parameters of heat-stressed broiler chickens. Methods: Study 1: Day-old Cob-500 unsexed chicks (n=72) were randomly placed into three treatment groups (n=24/group): No heat stress (NHS), 2) Heat stress with basal diet (HS), and 3) Heat stress with dried plum (HS+DP). Birds were raised under the standard broiler rearing guidelines for the first 21 days. Afterward, birds in the HS and HS+DP groups were exposed to heat stress conditions (33°C for 8 hours during daylight) for 3 weeks, while those in those in the NHS group were reared under normal conditions. Inclusion of 2.5% DP was made on the diet of the HS+DP group from 14 d onwards. Weekly body weight and feed intake were measured to calculate the average daily growth rate (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR). On day 42, birds were euthanized, a portion of ileum was excised for the gene expression and histomorphometry analysis. Cecum digesta was collected for the volatile fatty acids (VFAs) and microbial population analysis using 16S rRNA sequencing. Study 2: Day-old Cob-500 unsexed chicks (n=72) were randomly placed into three treatment groups (n=24/group): No heat stress (NHS), 2) Heat stress with basal diet (HS), and 3) Heat stress with alpha-lipoic acid (HS+ALA) and were reared under the standard broiler rearing guidelines for the first 21 days. Afterward, birds in the HS and HS+ALA groups were exposed to heat stress conditions (33°C for 8 hours during daylight) for 3 weeks, while those in the NHS group were reared under normal conditions. Supplementation of ALA (500 mg/kg) was made on the diet of the HS+ALA group from 14 d onwards. All other experimental procedures and analyses were carried out as mentioned in the DP study. Results: Study 1: Supplementation of DP in the heat-stressed broilers significantly improved the final body weight, ADG, ADFI, efficiency; the expression of heat shock protein-related genes (HSF1, HSF3, HSP70, HSP90), antioxidant-related genes (SOD1, SOD2, GPX1, GPX3, PRDX1, TXN), tight junction-related genes (CLDN1, OCLN), immune-related genes (IL4, MUC2) and major VFAs. The microbial analysis revealed significant enrichment of beneficial bacteria in DP supplemented broilers. Study 2: Supplementation of ALA in the heat-stressed broilers significantly improve the final body weight, ADG, expression of HSP90, PRDX1, GPX3, SOD2, OCLN, MUC2, and major VFAs. Finally, the microbial analysis revealed the significant abundance of beneficial bacteria Lactobacillus and Peptostreptococcaceae in the ALA supplemented broilers. Conclusion: This study identified dietary supplementation of DP to be the novel strategy to mitigate heat stress in poultry. Dietary supplementation of the DP improved both the growth performance and overall gastrointestinal physiology in heat-stressed broilers. Similarly, ALA improved body weight, gut microbiota, and other gut health parameters. Thus, the DP, and ALA supplementation can be considered as a potential remedy for heat stress in poultry.
Transcriptomic Regulation Of Egg Formation In The Oviduct Of Hens
(University of Hawaii at Manoa, 2018-12) Sah, Nirvay; Mishra, Birendra; Animal Sciences
Introduction: The oviduct of a hen provides a conducive environment for egg formation. Each part of the oviduct has a unique function: infundibulum (receives egg), magnum (deposits albumen), isthmus (synthesizes eggshell membranes), uterus (forms mineralized eggshell), and vagina (helps in oviposition). Any functional anomaly of the oviduct will lead to small-sized, misshapened, soft-shelled or cracked eggs, and taken together; these defects account for lost revenue in approximately 10% of the total number of eggs produced. At present, the cellular processes and biological pathways involved in the albumen synthesis, and eggshell formation are not clearly understood. Objective: The purposes of this study were to: (1) identify the novel differentially expressed genes (DEGs) and important biological pathways in the oviduct (laying vs. non-laying), and (2) validate the identified novel genes in the laying (3 h and 15-20 h post-ovulation; p.o.), non-laying (no growing ovarian follicles), and molting (reproductive rest) hens. Methods: Hy-line white hens, including laying (n=12) of 35 weeks, non-laying (n=6) of 35-60 weeks, and molting (n=6) hens of 60 weeks were used for this study. Magnum and uterine tissues were collected from laying hens at 3 h p.o. (egg present in the magnum), and 15-20 h p.o. (egg present in the uterus), molting, and non-laying hens for total RNA isolation. Total RNA (n=3/group) from the magnum of laying (3 h p.o.) versus non-laying, and total RNA (n=3/group) from uteri of laying (15-20 h p.o.) versus non-laying hens were subjected to next-generation sequencing (NGS). Raw reads of the sequences were filtered and trimmed (to remove low-quality reads), then aligned with the chicken genome (Galgal 5.0) and analyzed for differential gene expression. The mRNA expression of selected candidate genes in the magnum and uterus of laying, non-laying, and molting hens were also validated using real-time quantitative PCR (qPCR). Results: A total of 152 genes in the magnum, and 229 genes in the uterus of laying hens were up-regulated (FDR_BH <0.05) with the presence of egg in the oviduct. In the magnum, the glycine, serine and threonine metabolism was the most-enriched pathway; whereas, in the uterus, calcium signaling was amongst the most prevalent pathways. Results of qPCR confirmed the higher (P <0.05) mRNA expression of AVD and AvBD11 (antimicrobials); CAPN2, TMPRSS9, MMP1, and MMP9 (ECM remodeling and angiogenesis); REN and RLN3 (albumen secretion and oviposition); and CGN (vascular permeability) in the magnum of layers compared to both molters and non-layers. In the uterus, mRNA expression of MEPE (regulator of mineralization), OTOP2 (modulator of cellular calcium influx), CALCB (intracellular release of Ca-ions), STC2 (increases alkaline phosphatase activity), and ATP2C2 (cellular import of Ca-ions) were significantly higher (P <0.05) in laying hens, when compared to either molting or non-laying hens. Conclusions: This study identified several novel genes and biological pathways involved in the albumen synthesis and eggshell formation. These molecules can potentially be used as markers to improve the egg-qualities through nutritional modulation, and genetic improvement.
Survey of Disease, Management and Biosecurity Practices of Hawai‘i Swine Farmers
(University of Hawaii at Manoa, 2018-12) Castle, Brittany Amber; Zaleski, Halina M.; Animal Sciences
Although swine diseases and parasites cause significant losses to producers in Hawai‘i, limited information is available on changing disease patterns and related farm practices. The objectives of this study were to identify practices used on Hawai‘i swine farms and to determine if there is a relationship between those practices and the absence or presence of a disease. A management and biosecurity practices survey was administered to farmers (n=27). Survey questions were analyzed by region, sow population, and disease presence. Most common practices included cooking food waste (94% of farmers feeding food waste), feral pig exclusion (74%), and administering an anthelmintic (63%). Challenges faced by farmers include biosecurity concerns of on-farm sales, limited access to veterinary specialists, and excluding vermin from the production area. In addition, serological samples (n=414) from swine farms (n=57 out of 200 farms) were tested and found positive for antibodies against Porcine Circovirus Type 2b (PCV ELISA; 98% positive), Senecavirus (SVA IFA; 58%), Porcine Epidemic Diarrhea (PED IFA; 33%) and Porcine Reproductive and Respiratory Syndrome (PRRS ELISA; 16%). Fecal flotation detected coccidia oocysts (63%) on every island; Oesophagostomum dentatum (26%), Ascaris suum (18%), Strongyloides (11%), Metastrongylus spp. (8%), and Trichuris suis (8%) ova were on a subset of islands. Analysis indicates that disease prevalence is regionally distributed. Kaua‘i, which is protected by a quarantine order, has remained negative for PED, and Moloka‘i, which sees less interisland traffic, is negative for PRRS, PED, and SVA. Geographical patterns in disease distribution assist biosecurity and management practice recommendations, the design of vaccination protocols, and the judicious use of antibiotics.
The Effect of Neonatal Administration of Recombinant Myostatin Propeptide on Skeletal Muscle Growth in Mice
(University of Hawaii at Manoa, 2018-08) Xu, Xiaoxing; Animal Sciences
Myostatin (MSTN) negatively regulates skeletal muscle growth by suppressing myoblast proliferation and muscle fiber hypertrophy. Effective suppression of MSTN leads to dramatic improvement of animal muscle growth. MSTN propeptide (MSTNpro) is a potent inhibitor of MSTN activity. Studies suggest that in some species like mice and rabbits, muscle fiber number is increasing during the early neonatal period. We postulated that enhancing muscle fiber hyperplasia of these animal species during early neonatal period might increase postnatal skeletal muscle growth of these animals. Therefore, the objective of this study was to examine the effect of neonatal administration of MSTNpro on skeletal muscle growth in mice. Recombinant truncated flatfish MSTNpro fused to mouse IgG Fc domain (fMSTNpro45-100mFc) was produced in Escherichia coli (E. coli) using pMAL-c5x expression vector and purified by the amylose and protein A affinity chromatography. About 7.52 mg of purified recombinant MBP-fMSTNpro45-100mFc was obtained from 1 L culture. The MSTN-inhibitory capacity of the purified recombinant MSTNpro was similar to that of a commercial MSTNpro produced from eukaryotic cells in a pGL3-(CAGA)12-Luciferase repeater gene assay. In an oral administration study, eight female mice (4-month-old) were mated to two males (4-month-old), and the female mice were randomly divided into two groups: control and treatment. New-born pups in the treatment and control groups were fed with MBP-fMSTNpro45-100mFc (10 μg/g pup) and PBS, respectively, twice one day apart. The pups were weaned at 4 weeks, and their body weight were measured weekly for 7 weeks after weaning. At 11 weeks after weaning, animals were sacrificed, and gastrocnemius complex (gastrocnemius, plantaris and soleus) muscle and organ (heart, liver, spleen, kidney) samples were collected and weighed. Following the oral administration experiment, the same male and female mice were used for an intraperitoneal administration study with the same experimental design. Newborn pups in the treatment and control groups were intraperitoneally injected MBP-fMSTNpro45-100mFc (10 μg/g pup) and PBS, respectively, on the first and second day after birth. At 10 weeks after weaning, mice were sacrificed for the muscle and organ weight. Either neonatal oral administration or intraperitoneal injection of MBP-fMSTNpro45-100mFc did not significantly affect body weight growth and gastrocnemius muscle and organ weights of mice. This result implies that the administration of MBP-fMSTNpro45-100mFc under early neonatal period did not enhance muscle hyperplasia in mice. However, this study did not examine either the transfer of recombinant MSTNpro into circulation or the muscle fiber number after the administration. Furthermore, dose-response was not examined. Further studies are needed to validate the potential of neonatal suppression of MSTN as a strategy to improve skeletal muscle growth of animals.
Effects of Multi-Enzymes on Growth Performance, and Effects of Multi-Enzymes and Probiotics on Nutrient Utilization in Broilers Fed Different Level of Fibers
(University of Hawaii at Manoa, 2018-08) Singh, Amit K.; Animal Sciences
The overall objective was to examine the effect of multi-enzymes on growth performance and that of multi-enzymes and DFM on apparent ileal (AID) and apparent total tract digestibility (ATTD) of nutrients in broilers. Two independent studies were conducted for 21 d with Cobb 500 broilers fed low, and high fiber diets. In growth study, a 2×2 factorial design (2 level fiber and 2 supplements: none or xylanase, amylase, and protease; XAP) with 8 replicate floor pens (8 birds/pen; in two batches) was used. The digestibility study was conducted in a 2×4 factorial design with 8 replicate cages (6 birds/cage; in two batches). Each fiber level diet was supplemented 1) none (control), 2) XAP, 3) DFM or 4) XAP+DFM. In study 1, in 21 d period, the high fiber increased FCR (P < 0.05) by 0.04 units compared with low fiber. The XAP supplementation increased ADG by 12% and reduced FCR by 0.09 units compared with the control (P < 0.01). In study 2, the high fiber decreased (P < 0.05) AID and ATTD of dry matter (DM), crude protein (CP), gross energy (GE) and starch, and ATTD of total NSP and nitrogen corrected apparent metabolizable energy (AMEn). The high fiber decreased (P <0.01) AID of all amino acids except AID of lysine, glutamine, phenylalanine and glycine. The combination of XAP and DFM increased (P <0.01) total tract NR and AMEn as well as the AID and ATTD of DM, CP, GE, starch and total NSP while the individual supplements had intermediate effects. The combination increased (P <0.05) the AID of isoleucine, phenylalanine, threonine, cysteine, and tyrosine. The results suggest that the multi-enzymes can improve the performance of birds raised on fibrous diets while its combination with DFM can produce enhanced improvement in digestibility of nutrients than their single use.
Effects of Animal Behavior and Core-Body Temperature on Production Efficiency of Grass-Finished Beef Cattle.
(University of Hawaii at Manoa, 2018-08) Oshiro, Melelani A.; Animal Sciences
Forage nutrient quality and consumption have major impacts on ruminant production. Energy requirements of the grazing animal are influenced by several factors such as increased foraging activity, frame size, and physiological status, but is superseded by the requirement to maintain a homeothermic balance. Therefore, we hypothesized that changes in grazing behavior activities would affect core-body temperature (CBT) and animal performance measures. A two-year study utilizing two cohorts of 24 grass-finished cattle at the University of Hawai‘i, Mealani Agricultural Experiment Station. Animal behavior, CBT, weather variables, and forage quality were assessed during three, daily observation periods (AM, NOON, PM), for fall 2013, and summer and fall 2014 seasons. Over all seasons, active grazing (63.0%), standing (15.6%) and laying while chewing (10.4%) were the predominant behaviors observed. Grazing activity across daily periods was highest during the AM period, a time when mean CBT (38.3±0.01°C) was lowest. The CBT varied for all animals across seasons and periods and averaged 38.6 (±0.03) °C in 2013 and 38.4 (±0.04) °C in 2014. We did not find any significant relationship between CBT and grazing behavior. Forage quality varied seasonally, however crude protein (CP), and total digestible nutrients (TDN) were higher in summer 2014 compared to the fall seasons. Diurnal differences were observed in water-soluble carbohydrate (WSC) and non-fiber carbohydrates (NFC), which were higher in the PM across all seasons. Average Daily Gain (ADG) was not significantly different (P=0.78) between the years 2013 (0.87±0.04 kg/d) and 2014 (0.84±0.03 kg/d). Animals were slaughtered at approximately 21 (± 0.15) months of age and had an average live body weight of 527.1 (±8.98) kg in both years. In 2013, 75% of the animals graded Choice or higher compared with 90% in 2014.
Effect of In ovo Injection of Probiotic, Prebiotic, and Synbiotic on Growth Performance and Gut Health Parameters of Broiler Chicken
(University of Hawaii at Manoa, 2018-08) Li, Linge; Animal Sciences
Due to the claimed public health concerns, use of antibiotics as growth promoters (AGP) in the chicken feed is banned or regulated in several jurisdictions. Therefore, probiotics, prebiotics, and synbiotics are being evaluated as effective alternatives to AGP to improve growth performance and health of poultry. This study aimed to investigate the effects of Bacillus coagulans, Raffinose family oligosaccharides (RFO) and their combination on growth performance and gut health of broilers when injected in ovo. A total of 285 fertilized eggs were divided into 5 groups: i) No-injection group with intact shell, ii) 0.5 ml 0.85% normal saline, iii) Probiotic (B. coagulans) (2×106 CFU/egg) in 0.5 ml 0.85% normal saline, iv) Prebiotic (4.5 mg RFO) in 0.5 ml 0.85% normal saline, and v) Synbiotic (2×106 CFU/egg B. coagulans + 4.5mg RFO) in 0.5 ml 0.85% normal saline. The injection solution was deposited into the amniotic sac on d 17 of incubation. Hatchability of eggs were recorded. Altogether, 48 day-old chicks from each treatment were randomly allocated to 6 replicate floor pens (n=8/pen). All birds were raised on a standard commercial diet and management for 42 days. Body weight and feed intake of birds were measured weekly. Ileum samples were collected on d 0 and d 7 post hatch for total RNA isolation. Expression of immune/cytokines related genes in the ileum were determined using qPCR. The in ovo injection did not affect (P > 0.05) hatchability of eggs across the treatments. There was no significant effect of treatments on body weight, average daily gain and feed intake of broilers in different experimental groups. However, birds from normal saline treatment had significantly better (P < 0.05) feed efficiency and RFO group had the poorest feed efficiency in the first week of post-hatch period. No significant difference (P > 0.05) was found on relative organ weight of birds on d 21 and d 42. At d 7 of age, ileum villus height, crypt depth, and villus height: crypt depth ratio of RFO group were significantly better than other treatments (P < 0.05). On hatch day, expression of IL4 (inducer of T-cells differentiation) was significantly higher (P < 0.001) in the ileum of probiotic group. On d 7, immune-related genes (CD56, ChB6, TLR4, MCN2) and cytokines related gene (IL10) were significantly higher (P < 0.05) in the ileum of saline-treated group, whereas glucose transporter (SGLT1) had lower expression (P < 0.05) in synbiotic group. In conclusion, in ovo injection of probiotic enhances gut immunity of chicken which would be beneficial for gut health. It is interesting to find that in ovo injection of saline also enhanced gut immunity.
Unraveling the Mystery of the Natural Farming System (Korean): Isolation of Bacteria and Determining the Effects on Growth
(University of Hawaii at Manoa, 2018-08) Keliikuli, Ana K.; Animal Sciences
KNF is a self-sufficient farming system that involves the culturing of indigenous microorganisms (IMO) – fungi, bacteria, and protozoa. It enhances soil microorganism activity and improves soil fertility. This farming approach maximizes the use of on-farm resources, recycles farm waste, and minimizes external inputs while fostering soil health. However, scientific evidence of the benefits of KNF has been limited; little is known as to how this system works, what type of indigenous microorganisms are present in the soil treated under KNF conditions, or whether the collection site plays an integral role in soil fertility. In addition, there is no information on the rate and frequency at which IMO should be re-applied. There were three studies conducted with 4 overall goals: 1) identify the bacteria present in KNF, specifically phosphorus-solubilizing and nitrogen-fixing bacteria, 2) determine whether or not the collection site plays an integral part in plant growth, 3) determine how often to re-apply IMO 4 to the soil, and 4) inoculate seeds with bacteria isolated from KNF in the hopes of providing a better understanding as to the role it may play in plant growth. The first two studies showed that Bacillus megaterium and Bacillus aryabhattai were present in all soil samples. Bacillus subtilis and Bacillus licheniformis were dominant only in the KNF system. In the 2nd study, it was determined that the collection site of IMO plays an integral role and that applying a 2nd IMO application 14 days after the initial treatment increases plant yield. In the 3rd study (seed inoculation), the results showed that B. subtilis promoted plant growth in terms of germination rate, lateral root formation, root length, and stem elongation. Inoculating seeds with P. aeruginosa on the other hand proved to have little to no effect on plant growth. Knowing where to collect/cultivate IMO and how often to apply it to the soil will be of great use to farmers who currently practice KNF. This study also provided statistical data that shows KNF to be more effective than conventional farming methods when sufficient bacteria are applied to the soil in a regular schedule. Natural farming is the key to a sustainable future.

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