Progress 05/01/20 to 04/17/25
Outputs
Target Audience:The target audience for this project is livestock producers, small ruminant producers, animal health industries, and animal/veterinary scientists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Numerous presentations on results from this research project were given at Joint Annual Meetings of the American Society of Animal Science and Canadian Animal Science Society during this research project. Results were also shared with livestock producers at various field days. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Multiple experiments were conducted to explore potential mitigation strategies for fescue toxicosis induced vasoconstriction during gestation and reduced fetal muscle growth. Experiment 1 and 2: The objective of the first experiment was to screen for compounds that stimulate relaxation using a sheep lateral saphenous artery and vein bioassay. Eighteen different compounds were evaluated for relaxation potential. The dose response to serotonin showed the greatest potential in the vein. The objectives of the second experiment were to assess serotonin (5-HT) as a vasorelaxant in sheep, identify the involved receptors in an ovine lateral saphenous vein and artery bioassay, and further determine if 5-HT can stimulate relaxation after these blood vessels are constricted with ergovaline. This is the first reported observation of stimulated relaxation of a blood vessel constricted by ergovaline. The agonist for 5-HT4 produced the greatest relaxation in the ovine lateral saphenous vein, does not appear to be antagonized by ergovaline, and should be targeted in subsequent research focused on mitigation of ergot alkaloid-induced vasoconstriction (Klotz et al., 2025). Experiment 3: The objective of this initial experiment was to determine if melatonin with or without citrulline or arginine drench would reduce ergot alkaloid induced vasoconstriction in ewe lambs. Results show that melatonin (100 ug/kg BW/d) with or without citrulline or arginine drench (81 mg/kg BW/dose; 3 doses per d) did not alter ergot alkaloid induced vasoconstriction as measured by doppler ultrasound. Melatonin and citrulline concentrations were elevated in circulation after treatment; however, arginine concentrations were not consistently elevated and neither treatment altered vasoconstriction. In a second experiment, the objective was to evaluate the supplementation dose of a serotonin precursor, 5-hydroxytryptophan (5-HTP) at 0, 1.0, and 2.5 mg/kg BW/d levels have on circulating 5-HTP and serotonin concentrations and the impact of this supplementation on behavior aspects. There was a significant increase in plasma 5-HTP and a tendency for an increase in plasma serotonin for the 5-HTP doses compared to control. Experiment 4: The objective of this experiment was to assess if 5-HTP supplementation to pregnant ewes could mitigate the effects of fescue toxicosis on fetal development. Thirty pregnant ewes carrying twins were randomly assigned to one of three treatments: 1) negative control (CON, n = 10), 2) 5-HTP (2.5 mg/kg BW) and toxic tall fescue seed containing 1.77 mg ewe-1 d-1 ergovaline/ergovalinine (5HTP, n = 10), or 3) positive control, fed toxic tall fescue seed containing 1.77 mg ewe-1 d-1 ergovaline/ergovalinine (E+, n = 10) from d 86 to d 111 of gestation. Fetal body weight was reduced by 20% for E+ compared to control; however, fetal body weight for 5HTP did not differ from control. Results suggest that 5HTP supplementation is an effective mitigation strategy to reduce the impact of fescue toxicosis on fetal development. We are also the first to confirm that ergovaline crosses the placental barrier and accumulates in the fetal blood samples when ewes are fed ergot alkaloids. Expt 5 and 6: Two experiments were conducted using mimic/agomiR or antagomiR technologies to alter miRNA expression and examine changes in myoblast proliferation in vitro and muscle hypertrophy in vivo. In vitro experiments found that antagomiR-22-3p and mimic-127 increased myoblast proliferation compared to other miRNA treatments or controls. These miRNA treatments, antagomiR-22-3p (ANT22) and agomiR-127 (AGO127), were then used for intramuscular injections in longissimus muscle. The use of agomiR and antagomiR technologies allows us to alter miRNA expression in vitro and in vivo to enhance myoblast proliferation and alter muscle fiber hypertrophy in IUGR lambs during early postnatal growth (Greene et al., 2023). Expt 7: The objective of this study was to test in vivo vascular injection of antagomir-22-3p in lateral saphenous vein on miRNA and HDAC family mRNA expression in intrauterine growth restricted (IUGR) lambs. Results demonstrate that antagomir-22-3p treatment to IUGR lambs down-regulates miR-22-3p in semitendinosus and semimembranosus muscle causing a shift to more oxidative fiber type associated with down-regulation of HDAC family genes (Duckett et al., 2025). Conclusions: Fescue toxicosis in pregnant ewes during mid-to-late gestation reduces fetal body weight by 20% compared to control. Our results demonstrate that 5-HTP supplementation mitigates these vasoconstrictive effects of fescue toxicosis to support normal fetal growth in ewes fed ergot alkaloids. Intrauterine growth restriction impacts muscle mass in lambs and our research shows that microRNA therapies that alter epigenetic regulation of gene expression can shift muscle fiber metabolism to potentially enhance protein synthesis.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Klotz, J. L., C. M. Checura, M. A. Greene, H. Ji, S. K. Duckett. 2025. Serotonin can stimulate vasorelaxation in ovine lateral saphenous veins pre-contracted with ergovaline. J. Anim. Sci. doi: /10.1093/jas/skaf108.
- Type:
Peer Reviewed Journal Articles
Status:
Under Review
Year Published:
2025
Citation:
Duckett, S. K., M. A. Greene, A. N. S. Udoka, R. R. Powell, T. F. Bruce and J. L. Klotz. 2025. Antagomir-22-3p treatment on skeletal muscle growth in intrauterine growth restricted lambs. Front. Mol. Biosci. (Under review).
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Progress 05/01/23 to 04/30/24
Outputs
Target Audience:Our target audience for this research is livestock producers, extension agents, and researchers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Results were presented at the American Society of Animal Science meetingin July 2023. An abstract is submitted for presentation at the 2024 American Society of Animal Science meeting. Presentations have also been given at local livestock producer events to update them on the research in progress. What do you plan to do during the next reporting period to accomplish the goals?We will complete all research projects and publish results in scientific journals.
Impacts
What was accomplished under these goals?
Experiment 1 has been completed and manuscript is in progress. Experiment 2 has been completed and manuscript is in progress. Experiment 3 was completed, and manuscript is in progress. Results show that melatonin (100 ug/kg BW/d) with or without citrulline or arginine drench (81 mg/kg BW/dose; 3 doses per d) did not alter ergot alkaloid induced vasoconstriction as measured by doppler ultrasound. Melatonin and citrulline concentrations were elevated in circulation after treatment; however, arginine concentrations were not altered. Experiment 4 is in progress. Suffolk ewes (n = 75), naïve to toxic tall fescue, were obtained from Clemson University Sheep Farm. Ewes are blocked by body condition score and weight into 10 groups (7-8 ewes/group). Each week, one group was synchronized and mated to a Texel ram (7 d). Ewes were confirmed pregnant using transabdominal ultrasound and lamb number estimated at gestational day 60. Ewes confirmed with twins were randomly allotted to treatments. Thirty pregnant ewes carrying twins were randomly assigned to one of three treatments: 1) negative control (CON,n = 10), 2)5HTP (2.5 mg/kg BW)and toxic tall fescue seed containing 1.77 mg ewe-1d-1ergovaline/ergovalinine (VASO,n = 10), or 3) positive control, fed toxic tall fescue seed containing 1.77 mg ewe-1d-1ergovaline/ergovalinine (E+,n = 10). Ewes were individually fed total mixed ration (TMR) from gd 70 to gd 85 of pregnancy and then TMR plus treatment (CON, E+ or VASO) from d 86 to d 111. Blood samples were collected at d 78, 85, 92, 99, and 111 of gestation.Urine was collected from each ewe on d 85, 92 and 111 for lysergic acid excretion.Carotid and umbilical blood flow were measured by doppler ultrasound at d 80, 90, 100, and 110 of gestation. On d 111, a terminal necropsy was performed on the ewes to collect gravid uterus and fetal weights, placentome number, type and weight, fetal organ and muscle weights, and umbilical artery and vein for myograph experiments. Tissue samples were collected from cotyledon, muscle, liver and kidney tissues from fetus and ewe for gene expression and metabolomics. Animal studies are near completion and sample analyses are underway. Experiment 5 and 6 are completed and published. Experiment 7 has been completed and results will be presented at ASAS meetings in summer 2024. This experiment examined the systemic injection of antagomir-22-3p to down-regulate miR-22-3p and alter muscle hypertrophy. Results demonstrate that antagomir-22-3p down-regulates miR-22-3p in heart and semimembranosus muscles. Expression of HDAC and SIRT genes was also down-regulated and muscle fiber type converted to more oxidative metabolism.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Greene MA, Worley GA, Udoka ANS, Powell RR, Bruce T, Klotz JL, Bridges WC Jr, Duckett SK. Use of AgomiR and AntagomiR technologies to alter satellite cell proliferation in vitro, miRNA expression, and muscle fiber hypertrophy in intrauterine growth-restricted lambs. Front Mol Biosci. 2023 Nov 3;10:1286890. doi: 10.3389/fmolb.2023.1286890. eCollection 2023. PMID: 38028550
- Type:
Other
Status:
Submitted
Year Published:
2024
Citation:
Duckett, S. K., G. Worley, M. A. Greene, A. N. S. Udoka-Johnson, and J. L. Klotz. 2024. AntagomiR-22-3p injection alters miRNA expression and muscle fiber type in intrauterine growth restricted lambs. Submitted for presentation at ASAS meetings in July 2024.
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Progress 05/01/22 to 04/30/23
Outputs
Target Audience:Results from this research were presented to livestock producers, allied industry and scientific community at a local, regional, and national level. Changes/Problems:Due to restrictions imposed during Covid-19, we had to adjust our timetable for experiments. Experiment 4 will start this fall and samples will be collected in the spring for analyses. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Results were presented at American Society of Animal Science Meetings, International Grassland Conference, and producer field days. What do you plan to do during the next reporting period to accomplish the goals?We will complete all on-going experiments this summer and start experiment 4 in the fall. We will present and publish results from this research project.
Impacts
What was accomplished under these goals?
For experiment 1, additional compounds were evaluated to build on what was reported last year. We have added cannabidiol and alpha-humulene to the list of compounds evaluated in the saphenous vein for vasorelaxation. This brings the total number of compounds evaluated up to 18. Building on the data set that was reported last year for saphenous artery, we have added formononetin, biochanin A, melatonin, cannabidiol, and alpha-humulene. This brings the total number of compounds evaluated in the saphenous artery model for vasorelaxation up to 11. One finding in this experiment was that serotonin caused significant vasorelaxation in the precontracted saphenous vein, but not saphenous artery. A group of experiments (n = 24 lambs) is nearing completion that will profile the serotonin receptor subtypes present on the ovine lateral saphenous artery and vein that are involved in vascular smooth contraction and relaxation. For experiment 2, serotonin has been evaluated in lateral saphenous veins preconstricted with ergovaline. For experiment 3, Suffolk ewe lambs (n = 18) were randomly divided into two groups (n = 9) to facilitate sample collection. In each group, ewe lambs are randomly assigned to one of three groups: 1) melatonin (100 ug/kg BW/d; MEL) fed at 1400 h daily, 2) MEL + citrulline drench (81 mg/kg BW/dose; 3 doses per day; MEL-CIT) and 3) MEL + arginine drench (81 mg/kg BW/dose; 3 doses per day; MEL-ARG). Ewe lambs were individually fed TMR at 1400 for 7 d prior to the start of the experiment. Starting on d -5, cross-sections and blood flow of left common carotid artery were measured daily at 1430 h. Then ewe lambs were fed TMR plus E+ fescue seed (1.77 mg/ewe/d) and doppler ultrasound measurements of cross-sections of left common carotid artery were measured daily at 1430 h for 3 d (E+). Vasorelaxation treatments (MEL, MEL-CIT, MEL-ARG) were supplemented with TMR and E+ fescue seed on d 7 to 12. Jugular blood samples were collected at 1600 to measure plasma amino acid levels and melatonin concentrations. Plasma samples were analyzed for melatonin, arginine, citrulline, ornithine, glutamine, and glutamate in USDA-ARS laboratory. The analyses of the doppler measurements collected in this study is on-going. Experiment 4 will begin in the fall of 2023. Experiment 5 is complete. For experiment 6, pregnant ewes (n = 18) with twins were nutrient restricted (60%) from gestational d 85 to parturition. On d 2 of age, lambs were randomly selected and assigned to one of two miRNA treatments, 1) agomiR-127 (n = 9) to assess gain of function or 2) antagomiR-22-3p (n = 9) to assess loss of function. AgomiR-127 or antagomir-22-3p were reconstituted in phosphate buffered saline (PBS) and injected into the center of the LM on the left side every 3-d starting at the 10th rib and moving posterior by 1.27 cm at each injection for a total of 7 injections. A sham control treatment of PBS was administered following the same protocol in the right LM to serve as the in-animal control. Lambs were harvested 4 d after the last injection and samples were collected from the injection region of each LM for miRNA and mRNA expression, histology, and proteomic analyses. The agomir-127 and antagomir-22-3p treatments up-regulated and down-regulated their targeted miRNA, respectively. Antagomir-22-3p treatment increased DNA content, ratio of protein to DNA, and altered the proteome of the injected region. Expression of predicted mRNA targets of miR-22-3p, myosin heavy chain isoforms, and other myogenic genes were also altered in the LM. The direct injection of agomiR and antagomir technologies in the LM up- and down-regulated expression, respectively, for the miRNA of interest, which altered mRNA expression of predicted targets, protein abundance and muscle fiber type. For experiment 7, Suffolk ewes carrying twins (n = 18) were used for the study. Ewes were randomly divided into two groups (n = 9/group) and assigned to treatment: 1) control (100% NRC; CON), or 2) nutrient restricted (60% of NRC; NR) from d 86 to parturition. Lambs (n = 9) from NR ewes were selected for testing of the antagomiR-22-3p (ANT22). Lambs (n = 9) from the CON ewes were selected for sham treatments (SHAM) administered similarly to ANT22. A blood flow resistance band was applied to the right leg for 5 min. and ANT22 or SHAM treatment was injected into the right lateral saphenous vein for three consecutive days. Lambs remained with their dam for 24 d and then were slaughtered for sample collection. At slaughter, leg muscles were removed from the right (ANT22 or SHAM) or left (control) side of each lamb for subsequent miRNA, mRNA, and muscle fiber type. This experiment is on-going.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Greene, M. A., R. R. Powell, T. Bruce, W. C. Bridges Jr., and S. K. Duckett. 2022. miRNA transcriptome and myofiber characteristics of lamb skeletal muscle during hypertrophic growth. Front. Genet. 13:988756. https://doi.org/10.3389/fgene.2022.988756
- Type:
Other
Status:
Submitted
Year Published:
2023
Citation:
Klotz, J. L., S. K. Duckett, M. A. Greene, and C. M. Checura. 2023. Evaluation of serotonin as a vasorelaxant in ovine saphenous artery and vein. J. Anim. Sci. Abstr (submitted).
- Type:
Other
Status:
Submitted
Year Published:
2023
Citation:
Greene, M. A., R. R. Powell, T. Bruce, J. L. Klotz, C. M. Checura and S. K. Duckett. 2023. Use of AgomiR and AntagomiR technologies to alter miRNA expression and muscle hypertrophy in intrauterine growth restricted lambs. J. Anim. Sci. Abstr (submitted).
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Greene, M. A., A. N. S. Udoka, R. Powell, R. Noorai, T. Bruce, and S. K. Duckett. 2022. Impact of fetal exposure to mycotoxins on longissimus muscle fiber hypertrophy and miRNA transcriptome. BMC Genomics 23:59. https://doi.org/10.1186/s12864-022-08794-0
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2023
Citation:
Klotz, J. L., S. K. Duckett, and D. L. Harmon. 2023. Physiological impacts of ergot alkaloid consumption in Ruminant Livestock. Proc. International Grassland Conference (In press).
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Progress 05/01/21 to 04/30/22
Outputs
Target Audience:The target audience for this research is scientists, extension agents, applied animal industries, and livestock producers. Changes/Problems:Based on our in vitro research (Expt 5), the localized miRNA therapy treatments were adjusted to utilize AgomiR and AntgomiR technolgies, which were found to be superior to miRNA mimics and inhibitors, for localized miRNA therapy in vivo (expt 6). Also the timeline was adjusted based on the in vitro results (Expt 5) for injections to every 3 d for seven injections and then harvest at 4 d post last injection. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Results from this research were presented at the National American Society of Animal Science Meetings and the International Grasslands Congress in Kenya, Africa. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we plan to finish experiments 1, 2, and 3 to evaluate compounds that mitigate ergot aklaoid induced vasoconstriction. We also plan to complete laboratory analyses for experiment 6 to examine how miRNA therapies alter muscle growth and select one miRNA for systemic treatment in experiment 7. The goal of this research is to develop mitigation strategies that have direct application to livestock producers for enhancing fetal development and postnatal muscle growth.
Impacts
What was accomplished under these goals?
For experiment 1, lateral saphenous veins (n = 42) were used in a preconstricted bioassay to assess vasorelaxation potential of 16 different compounds (genistin, genistein, daidzin, daidzein, formononetin, equol, biochanin A, bradykinin, acetylcholine, BW 723C86 [serotonin receptor 2B agonist], serotonin, n-butylphthalide, capsaicin, eugenol, curcurmin, and melatonin) for possible use in subsequent experiments as originally proposed. As a contingency for evaluation of compounds that induce vasorelaxation for the in vivo doppler ultrasound, an artery was included in the profiling experiments. Lateral saphenous arteries (n = 21) were used in a preconstricted bioassay to assess vasorelaxation potential of 10 compounds (genistin, genistein, daidzin, daidzein, equol, serotonin, and capsaicin) for possible use in subsequent experiments. This research is ongoing at this time. In addition, lateral saphenous artery and vein were collected from a group of lambs that had received resveratrol (n = 6) vs. control (n = 6) prior to slaughter. These blood vessels were evaluated for vasoactivity to phenylephrine and vasorelaxation to serotonin, equol, and capsaicin. These data are currently being analyzed for effects of test compound in relation to the animal treatment and prepared for publication. For Experiment 2, equol (n = 3 lambs) is the only compound evaluated in lateral saphenous veins preconstricted with ergovaline thus far. For experiment 5, satellite cells were isolated from lambs at d 2 of age according to Dodson et al. (1986) and Motohashi et al. (2014). In vitro experiments were conducted to examine how miRNA mimics or inhibitors altered satellite cell proliferation. Based on small RNA sequencing data, we identified miR-127 and miR-299a for up-regulation using mimics, and miR-22-3p, miR-133a-3p, miR-27a-3p, and miR-29a for down-regulation using inhibitors. Our initial testing demonstrated that AntagomiRs (Creative Biogene) worked more efficiently than inhibitors to knockdown expression of miRNAs in vitro. Satellite cell proliferation was greater for mimic-127 and antagomir-22-3p at d 4 compared to with controls or other miRNAs evaluated. Samples were subjected to mRNA sequencing to identify mRNA targets. Fibroin, IGFBP5 and others involved in extracellular matrix were up-regulated when miR-127 was overexpressed. Carnosine dipeptidase 2 (CNDP2), a non-specific dipeptidase, was up-regulated when miR-22-3p was down-regulated. We identified miR-127 and mir-22-3p as miRNA that increase satellite cell proliferation and will be used for testing in vivo (expt. 6). Abstract was submitted for presentation at ASAS meeting in July 2022. For experiment 6, Suffolk ewes carrying twins (n = 18) were used for the study. Ewes were fed at 60% of NRC requirements from d 86 to parturition. On d 2 of age, lambs (n = 18) were randomly selected for testing of the miRNA of interest, AgomiR-127 (100 nmol) or AntagomiR-22-3p (500 nmol). AgomiR-127 (n = 9) or AntagomiR-22-3p (n = 9) was directly injected (0.5 mL) into the left longissimus muscle on d 2, 5, 8, 11, 14, 17 and 20. In the right longissimus muscle, sterile PBS (0.5 mL) was injected on d 2, 5, 8, 11, 14, 17 and 20 (SHAM). Injections started at the 13th rib and then moved to the posterior by 1 cm for each injection time point. Lambs were slaughtered 4 d post the last injection (d 24 of age). These time points were selected based on the satellite cell culture in vitro experiments that showed the miRNA were up-regulated (AgomiR) or down-regulated (AntagomiR) for 3 d post transfection and satellite cell proliferation was increased on d 4 post transfection. At slaughter, longissimus muscle was removed from the left miRNA injected region, right PBS-injected region (Sham), left rib (LCON), and right rib (RCON) for subsequent miRNA, mRNA, muscle fiber type and proteomic analyses. This experiment is on-going.
Publications
- Type:
Other
Status:
Submitted
Year Published:
2022
Citation:
Greene, MA and Duckett, SK. 2022. Examination of miRNA mimics and inhibitors on satellite cell proliferation in vitro. J. Anim. Sci. (abstr., submitted).
- Type:
Other
Status:
Submitted
Year Published:
2022
Citation:
Klotz, JL, Duckett, SK, Britt, JL, Greene, MA, and Andrae, JG. 2022. Ergot alkaloid consumption alters serotonin receptor-induced vasoactivity in ovine umbilical vasculature. J. Anim. Sci. (abstr., submitted)
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Progress 05/01/20 to 04/30/21
Outputs
Target Audience:The target audience for this research is scientists, extension agents, applied animal industries, and livestock producers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Due to travel restrictions with Covid-19, we were unable to pursue training and professional development opportunities. How have the results been disseminated to communities of interest?Abstracts were submitted for presentation to American Society of Animal Science Meetings in July, 2020. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we will conduct Experiments 1, 2 and 3 to examine mitigation strategies for reducing vasoconstriction associated with ergot alkaloid consumption. Experiment 6 will also be conducted to examine miRNA mediated therapies to enhance muscle growth in intrauterine growth restricted lambs.
Impacts
What was accomplished under these goals?
The USDA ARS Forage-Animal Production Research Unit shifted to 100% max telework March, 2020 due to the Covid-19 pandemic. This has continued through March, 2021 and is currently ongoing. Consequently, all in-person laboratory activities have ceased unless deemed essential to maintain plants or animals in our care during this time. Research associated with Experiment 1 and 2 have been delayed as a result of the USDA-ARS telework requirements. For experiment 5, Suffolk ewes (n = 26) were blocked by body condition and weight, and randomly divided into two breeding groups (n = 13/group). One group was bred to a Texel (TEX; Texel Muscled with 2 copies of myostatin SNP; GeneSeek) ram and the other to a Suffolk (SUF) ram. Ewes were confirmed pregnant by ultrasound and went to term. Doppler ultrasonography of fetal blood flow was examined on pregnant ewes (n = 5/sire breed) at three stages of gestation, (60, 90, and 120 ± 3 d). Umbilical artery and fetalaorta blood-flow velocities and aorta vessel diameter were measured. Blood flow velocities were used to calculate the resistance index (RI), which was used for statistical analysis. Sire breed did not alter umbilical artery or aorta RI. For aorta diameter, the effect of sire breed tended to be significant (P = 0.054); SUF fetuses had a larger average diameter than TEX. Stage of gestation altered (P < 0.05) umbilical artery and aorta RI, and aorta diameter. These results demonstrate our ability to measure blood flow in the umbilical artery and fetal aorta, and measure aorta vessel diameter during gestation. These methods will be used in experiment 4. Male lambs (n = 4/sire/time) were harvested on d 2, 14, and 203 of age for muscle fiber type, mass and satellite cell isolation. Muscle fiber type was measured, and image analyses are on-going. Satellite cells were isolated from LM of the 2-d old lambs. Satellite cell experiments were conducted to examine proliferation and differentiation of the d 2 isolated cells. Satellite cells were propagated in proliferation to 60% confluence and plated into 24-well plates at a density of 7 x 104 per cm2. Proliferation was measured daily for 6 d using DNA assay. Differentiation was assessed at the end of the proliferation experiment by culturing for 4 d in differentiation media. Nuclei were stained with myogenin to count differentiated nuclei. Satellite cell populations per g of tissue increased between d 2 and 14 and decreased from d 14 to 203. Satellite cell proliferation capacity was altered by sire breed at d 2 of age potentially enhancing the capacity for early muscle growth. Currently, in vitro experiments are being conducted using the isolated satellite cells to ascertain the gain or loss of function for miRNA differentially regulated, as identified in miRNA sequencing projects, on satellite cell proliferation/differentiation, miRNA expression and mRNA expression using qPCR for known miRNA targets. miRNA sequencing results were obtained for longissimus muscle of lambs during skeletal muscle hypertrophy from mid-gestation to market weight. The period from prenatal (d133 of gestation) to d 40 of age had the most differentially expressed miRNA. Examination of the differentially expressed miRNA showed that 23 miRNA that were most significantly (P < 1 x 10-10) down-regulated (miR-22-3p, -let-7g, -30c, -30d, -29a, -143, -16b, -27a and -133) or up-regulated (miR-299-5p, -487b-3p, -127, -432, -299-3p, -381-3p, -410-3p, -154a-3p, -3959-5p, -376c-5p, -431, -495-3p, -493-5p, and -655-3p). Based on these results and potential targets related to myogenesis, we chose miR-22-3p, -29a, and -27a for initial experiments to examine the role of miRNA on skeletal muscle fiber hypertrophy. Isolated satellite cells are seeded into 24-well plates in duplicate and grown in proliferation medium. Preliminary experiments were conducted to examine transfection efficiency using lipofectamine transfection reagents (Lipofetamine-2000, 3000, or RNAiMAX; Invitrogen) and BLOCK-iT transfection kit (Thermo Fisher) in satellite cells according to the manufacturer. Our results show that transfection efficiency was greatest using RNAiMAX on d 1, 2, and 3 post-transfection. AntagomiR and negative controls (Creative Biogene) for each miRNA of interest (miR-22-3p, -29a, and -27a) were obtained for these experiments. LipofectamineÔ RNAiMAX transfection reagent was used according to manufacturer's directions. Satellite cell proliferation was measured daily for 6 d using a DNA assay. Satellite cells were harvested on d 1, 3, and 6 for RNA extraction and qPCR for miRNAs, MSTN and PAX7. MSTN and PAX7 protein levels are measured by western blot. Satellite cell proliferation, differentiation, miRNA and mRNA of target genes are analyzed using proc mixed with miRNA treatment (AntagomiR versus negative control for each miRNA examined) in the model. Due to delays with Covid-19, these experiments are still on-going at this time and will be completed this summer. Overall, we demonstrated that doppler ultrasonography can be used to detect differences in fetal blood flow during gestation. Satellite cell isolation established that the d 2 to 14 time period is critical for skeletal muscle growth with a doubling of body weight, longissimus muscle weight, and ribeye area in this 2-week time period. In vitro experiments are in progress to examine the role of miRNA on satellite cell proliferation and differentiation.
Publications
- Type:
Other
Status:
Submitted
Year Published:
2021
Citation:
Greene, M. A. and S. K. Duckett. 2021. Comparison of Suffolk and Texel sired lamb growth and satellite cell proliferation in the early postnatal period. J. Anim. Sci. (Abstr.; submitted).
- Type:
Other
Status:
Submitted
Year Published:
2021
Citation:
Greene, M. Greene, M. A., J. L. Britt, S. M. Justice, and S. K. Duckett. 2021. miRNA transcriptome of lamb skeletal muscle during hypertrophic growth from mid-gestation to market weight. J. Anim. Sci. (Abstr.; submitted).
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