M.S. - Animal Sciences
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Item EVALUATION OF EGGSHELLS USING A SCANNING ELECTRON MICROSCOPE AND ENERGY DISPERSIVE SPECTROMETRY(2024) Mahato, Prem Lal; Mishra, Birendra; Animal SciencesItem THE STRESS RESPONSE OF THE BRASSY CHUB, A LOCAL REEF FISH AND CANDIDATE FOR SUSTAINABLE AQUACULTURE(2024) Merlo, Reilly Stanton; Seale, Andre P.; Animal SciencesItem MITIGATION OF HEAT STRESS IN BROILER CHICKENS USING DIETARY SUPPLEMENTATION OF MICROALGAE(2023) Chaudhary, Ajay; Mishra, Birendra BM; Animal SciencesItem 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 SciencesModern 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.Item 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 SciencesAn 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.Item Transcriptomic Analysis Of Bovine Endometrium With Short Uterine Tract Leading To Infertility(University of Hawaii at Manoa, 2022) ADNAN, MAHFUZ RAHMAN; Mishra, Birendra; Animal SciencesBackground: 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.Item 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 SciencesThe 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.Item 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 SciencesMyostatin (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.Item RNA-sequencing Based Analysis Of Bovine Endometrium During The Maternal Recognition Of Pregnancy(University of Hawaii at Manoa, 2020) Adhikari, Bindu; Mishra, Birendra; Animal SciencesBackground: 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.Item 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 SciencesIntroduction: 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.