Source: NORTH DAKOTA STATE UNIV submitted to NRP
MATERNAL NUTRITION, EPIGENETIC MODIFIERS, AND PROGRAMMING OF FETAL ORGAN DEVELOPMENT IN BEEF CATTLE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1020973
Grant No.
2020-67016-31341
Cumulative Award Amt.
$540,000.00
Proposal No.
2018-07055
Multistate No.
(N/A)
Project Start Date
May 1, 2020
Project End Date
Apr 30, 2026
Grant Year
2021
Program Code
[A1231]- Animal Health and Production and Animal Products: Improved Nutritional Performance, Growth, and Lactation of Animals
Recipient Organization
NORTH DAKOTA STATE UNIV
1310 BOLLEY DR
FARGO,ND 58105-5750
Performing Department
Animal Sciences
Non Technical Summary
Feed costs are the largest economic burden for beef cattle producers. For beef cattle, more feed resources are dedicated to maintaining the breeding cow herd than to their calves that finished for the beef market. Additionally, in cattle production systems there is potential for periods of inadequate nutrition during pregnancy due to factors such as poor forage quality or limited quantity, like during periods of drought. Recent evidence suggests that a mother's nutrition during pregnancy can have life-long impacts on the growth and metabolism of her offspring through a process called developmental programming. Compromised growth and metabolism are major detriments to efficient and sustainable livestock production systems. We hypothesize that strategic changes to the cow's diet during early pregnancy will alter the growth, development, and metabolism of her developing calf. Our objective is to study the interaction between cow plane of nutrition (adequate versus restricted) and supplementation of key nutrients (methionine, choline, folate, and vitamin B12) on calf growth, metabolism, and gene expression during pregnancy. We expect to observe differences in the developing calf's liver and muscle growth, metabolic function, and gene expression due to maternal nutrition and strategic supplementation. These efforts will provide critical data that will positively impact whole herd beef cattle production efficiencies via management practices that will increase economic and environmental sustainability, and ultimately contribute to meeting world food demands with nutrient dense healthy animal products.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3023310101025%
3023310102025%
3023310104025%
3023310105025%
Knowledge Area
302 - Nutrient Utilization in Animals;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1040 - Molecular biology; 1010 - Nutrition and metabolism; 1020 - Physiology; 1050 - Developmental biology;
Goals / Objectives
Our long-term goals are to ascertain the underlying mechanisms regulating developmental programming in livestock species and to develop successful approaches to mitigate impacts of compromised fetal development. This will increase competitiveness of U.S. animal agriculture and ultimately contribute to meeting world food demands. Our short-term goals of this project are to determine the mechanisms associated with impacts of maternal nutrition and strategic supplementation of one-carbon metabolites on developmental programming events in fetal liver and muscle in female beef cattle.Specific Objectives: to assess the impacts of maternal nutrition and inclusion of OCM from breeding to d 63 of gestation on: 1) the transcriptome and identity of differentially expressed genes (and associated pathways) in fetal liver and muscle; 2) the metabolic phenotype of fetal liver and muscle, mitochondrial and glycolytic functionality, and cell fuel use; and 3) organ growth and morphology, as measured by mass and muscle fiber formation and size.
Project Methods
Experiment 1: Angus-cross beef heifers (n=144) will be estrus-synchronized and bred via artificial insemination using female sexed semen from a single sire. At breeding (d 0) heifers will be assigned to one of four dietary treatments arranged in a 2 (plane of nutrition) x 2 (supplementation) factorial design:CON - OCM (control level of feed intake, no additional supplementation)CON + OCM (control feed intake plus supplementation of one-carbon metabolites [methionine, choline, folate, and vitamin B12)RES - OCM (restricted feed intake, no additional supplementation)RES + OCM (restricted feed intake plus supplementation of one-carbon metabolites)Dietary treatments will be fed until d63 of gestation, after which all animals will receive CON intake with no supplementation for the remainder of the study. Pregnancy will be confirmed via ultrasound on d 35. Non-pregnant animals will be removed from the study. We anticipate a pregnancy rate of 45-55% and therefore n=16 per treatment.Blood samples will be collected on days 0, 35, 63, and 91 of gestation analyzed for glucose, amino acids, NEFA, B-vitamins, and homocysteine, IGF-1, insulin, and growth hormone concentrations.At d 63 half of the heifers (n=8 per treatment) will undergo a ovariohysterectomy procedure to collect fetal and reproductive tissues. The other half of the heifers (n=8 per treatment) will all be reverted to control intake without supplementation (CON - OCM) from d 63 to d 91. At d 91 these remaining pregnant heifers will also undergo ovariohysterectomy for tissue collection.Fetal allantoic and amniotic fluid and arterial umbilical cord blood samples will be collected and analyzed for glucose, fructose, amino acids, B-vitamins, and homocysteine concentrations. The fetus will be isolated and crown-rump length and fetal mass will be measured. Fetuses will be dissected under visualization with a stereoscope and organs, including brain, liver, biceps femoris muscle, and intestines will be dissected and weighed. Fetal muscle and liver samples will be analyzed by RNA seq using GridIONx5 sequencing to measure global gene expression and identify differentially-expressed genes. Further analysis will identify cellular pathways and processes altered by maternal dietary treatment. Fetal liver and muscle tissue will also be analyzed for key parameters of mitochondrial function using the Agilent Seahorse XFe Cell Mito Stress Test to directly measure the oxygen consumption rate of cells. Sequential compound injections of Oligomycin (inhibitor of ATP Synthase), FCCP (uncouples ATP synthesis from ATP Synthase), and Rotenone-Antimycin A (inhibitors of complex 1 and 3, respectively) measure basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity, and nonmitochondrial respiration.Experiment 2: Angus-cross beef heifers (n=72) will be estrus-synchronized and bred via artificial insemination using female sexed semen from a single sire. At breeding (d 0) heifers will be assigned to one of four dietary treatments arranged in a 2 (plane of nutrition) x 2 (supplementation) factorial as detailed in Experiment 1.Dietary treatments will be fed until d63 of gestation, after which all animals will receive CON intake with no supplementation for the remainder of the study. Pregnancy will be confirmed via ultrasound on d 35. Non-pregnant animals will be removed from the study. We anticipate a pregnancy rate of 45-55% and therefore n=8 per treatment.Blood samples will be collected on days 0, 35, 63, 91, 175 and 259 and analyzed as described in Experiment 1.Heifers will be euthanized on d 259 (90% of full gestation length). Fetal allantoic and amniotic fluid and arterial umbilical cord blood samples will be collected at necropsy and analyzed for glucose, fructose, amino acids, B-vitamins, and homocysteine concentrations. Fetuses will be measured for gross morphological differences. Fetal muscle and liver samples will be analyzed for differential gene expression and mitochondrial function as described in Experiment 1.Data within each Experiment will be analyzed as a 2 (plane of nutrition) × 2 (supplementation) factorial design. Differentially expressed genes will be identified using Ballgown with significance set at q ≤ 0.10. Pathway analysis will be performed using iPathwayGuide (Advaita Corporation) with significance set at P ≤ 0.05. All other data will be analyzed using the GLM procedure of SAS (SAS Institute Inc., Cary, NC) with P ≤ 0.05 considered significant. Heifer cohort, age, and day of collection will be included as co-variates and removed from the model if not significant. Mean separation will be performed for significant main effects using the LSMEANS procedure of SAS.The results from this study will advance the understanding of the impacts of maternal nutrition in early pregnancy on fetal growth and development. It will demonstrate if fetal changes observed in early gestation persist throughout pregnancy. It is also the first step towards developing management practices of heifer and cow nutrition to enhance calf performance through developmental programming.

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:The primary target audience is the scientific community and other researchers, particularly in the field of Animal Science. Additional target audiences include students and emerging professionals (undergraduate, graduate, and postdoctoral) as results from these efforts are presented in multiple venues. Producers and industry partners are also a target audience as these stakeholders have vested interests in the outcomes and new knowledge being discovered. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided professional development and training opportunities for five graduate students (2 MS and 5 PhD), This includes writing abstracts, extension research reports, and grant proposals; learning new techniques; and presenting their research at conferences. This project also provided the opportunity for visiting scientist from Brazil to learn new techniques and improve her scientific writing skills. How have the results been disseminated to communities of interest?Results have been disseminated through abstracts, presentations at conferences, and journal publications. Preparation of manuscripts of additional peer reviewed manuscripts is in progress. What do you plan to do during the next reporting period to accomplish the goals?The major goals for the next reporting period are to continue with analysis of Exp.1, 2, and 3. The field work for Exp 3 was completed in May 2024.

Impacts
What was accomplished under these goals? This year we continued to analyze and publish data from Exp. 1 and 2, and completed the animal portion of Exp. 3. We hypothesized that: 1) a low maternal plane of nutrition would impair fetal growth in early gestation and that supplementation of one-carbon metabolites (OCM) would rescue growth restricted offspring at d 63 and 161 of gestation, Exp. 1, and Exp. 2, respectively; 2) that low maternal plane of nutrition would impair antioxidant production, and that supplementation of one-carbon metabolites (OCM) in early gestation would rescue/increase total antioxidant capacity in beef heifers at d 63 and 161 of gestation, Exp. 1, and Exp. 2, respectively; and 3) that low maternal plane of nutrition will cause differential expression of fetal genes in early gestation and that supplementation of one-carbon metabolites (OCM) will mitigate differential expression in offspring at d 63. Both experiments had a 2 × 2 factorial arrangement of treatments with two levels of average daily gain (ADG), each with or without supplementation of OCM. In each experiment, seventy-two crossbred heifers were bred with female-sexed semen from a single sire. At breeding (d 0), beef heifers were individually fed using an electronic headgate system and randomly assigned to one of four treatments: Control (targeted: 0.45 kg/d ADG) without OCM (CON-OCM), CON with OCM (CON+OCM), Restricted (targeted: -0.23 kg/d ADG) without OCM (RES-OCM), or RES with OCM (RES+OCM). The basal diet given to all heifers for both experiments consisted of alfalfa-grass hay, corn silage, and ground corn. The OCM supplement consisted of ruminal-protected choline (44.4 g/d) and methionine (7.4 g/d) in a fine-ground corn carrier top-dressed daily, and weekly injections of folate (320 mg) and vitamin B12 (20 mg). The -OCM heifers received the corn carrier and saline injections. In Exp 1, heifers were slaughtered and fetal tissues were collected on d 63. In Exp. 2, heifers received treatment up to d 63 then were all placed on a common control diet with no supplement until slaughter and tissues were collected on d 161. Maternal serum samples were collected on d 0 (prior to treatment), d 35 (middle of treatment period), d 63 (end of treatment period), and d 154 (prior to slaughter). The serum was analyzed for total antioxidant capacity (CRE) measured by copper reduction equivalent and CRE change between days. Tissues were immediately placed in RNAlater and stored in -80 C until analysis. Extraction of RNA and quality checks were performed at North Dakota State University and eukaryotic mRNA-seq was performed on samples with data output of 6 Gb/20M PE150 read-pairs per sample via Novogene. In Exp. 1 heifer data, there were pancreas gain × OCM interactions where CON+OCM weights were greater when compared to each of the three remaining treatments (P < 0.01). Dam liver, live slaughter weight, and hot carcass weight had gain effects where CON heifers were heavier (P < 0.01). When looking at the fetus, fetal body weight tended (P = 0.07) to be lower and stomach complex weight was lower (P = 0.026) in RES compared with CON. When organ weights were normalized to total brain weight to assess allometric growth, OCM × gain interactions were observed in heart (P = 0.04), left longissimus dorsi (P = 0.04), and right hemisphere (P = 0.01) with CON+OCM greater than CON-OCM. The normalized left hemisphere tended to be greater in RES+OCM than RES-OCM (P = 0.079). In Exp. 2 heifer data, gain × OCM interactions were not present (P > 0.05). Live slaughter and hot carcass weight had gain effects where CON heifers were heavier than RES (P < 0.01). The 12th rib fat depth was larger in CON (P = 0.03) with a tendency (P = 0.08) for CON heifers to have a larger ribeye area. The +OCM heifers had an average yield grade of 1.69 while -OCM heifers had 2.02 (P = 0.017). In Exp. 2 fetal data, there was no differences in fetal body weight. However, trachea was heavier (P = 0.04) and spleen was lighter (P = 0.05) in fetuses from +OCM than -OCM. The right hindlimb muscle was influenced by a gain × OCM interaction (P = 0.04), where RES+OCM was heavier than CON+OCM. With allometric adjustments, right longissimus dorsi, rumen complex, and liver were heavier (P ≤ 0.05) in RES compared with CON. Day 63 is within the period of peak primary myogenesis in cattle; therefore, differences in muscle growth during this time could have lasting implications in post-natal calf growth and performance. Furthermore, nutrient partitioning in liver and muscle tissue can affect many postnatal production traits such as lactation, reproduction, and carcass quality. These data suggest that providing OCM during early gestation may rescue skeletal muscle tissue growth in nutrient restricted offspring, and that restricting nutrients to dams results in prioritization of allometric growth of critical fetal organs such as liver and lungs. In Exp. 1, when the percent change in CRE was calculated, there was a difference in change between the two gain levels (P = 0.05). CON heifers had a greater % change in CRE from d-2 to d63 than the RES heifers (7.2% vs 0.12%). In Exp. 2, heifers on CON treatments had a greater CRE compared with those receiving RES (1,971.2 vs 1,837.8 ± 17.9 µM, P < 0.01). When observing the data within specific days, there was a tendency for CRE in CON heifers to be greater (P = 0.07) at d 63 when compared with RES. At day 154 of gestation, CON heifers had a greater CRE capacity when compared with RES (2,080.2 vs. 1,898.6 ± 42.6 µM, P = 0.02). There were tendencies for +OCM heifers to have a greater total antioxidant capacity compared with controls when expressing the data as CRE change and percent change from d 0 until d 64 (P = 0.09 and 0.08, respectively). These data suggest that adequate amount of nutrients and the supply of OCM improved the overall antioxidant capacity in maternal serum. A total of 9 genes were downregulated in fetal muscle tissue from CON heifers and 1 gene was upregulated when compared with RES. Additionally, in fetal muscle, 4 genes were downregulated in fetuses from heifers not receiving OCM supplementation when compared with provided OCM. In fetal liver tissue, 5 genes were downregulated in CON compared with RES. Two genes were upregulated and three downregulated in liver tissue +OCM fetuses compared to -OCM. The genes mentioned are involved in pathways such as mTOR signaling, ROS production, eukaryotic translation initiation factor 4 (eIF4) and serine/threonine protein kinase (p70S6K) signaling, and more. Thus, OCM supplementation and maternal rate of gain did influence transcript abundance of genes associated with cellular processes such as cell death, growth, free radical production, and calcium signaling.

Publications

  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Daneshi, M., Borowicz, P. P., Entzie, Y. L., Syring, J. G., King, L. E., Safain, K. S., Anas, M., Reynolds, L. P., Ward, A. K., Dahlen, C. R., Crouse, M. S., and Caton, J. S. 2024. Influence of Maternal Nutrition and One-Carbon Metabolites Supplementation during Early Pregnancy on Bovine Fetal Small Intestine Vascularity and Cell Proliferation. Veterinary sciences, 11(4), 146. https://doi.org/10.3390/vetsci11040146
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Anas M, Diniz WJS, Menezes ACB, Reynolds LP, Caton JS, Dahlen CR, Ward AK. Maternal Mineral Nutrition Regulates Fetal Genomic Programming in Cattle: A Review. Metabolites. 2023 Apr 26;13(5):593. doi: 10.3390/metabo13050593. PMID: 37233634; PMCID: PMC10223334
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Daneshi M, Caton JS, Caixeta LS, Eftekhari Z, Ward AK. 2023. Expression, Regulation, and Function of ?-Defensins in the Bovine Mammary Glands: Current Knowledge and Future Perspectives. Animals (Basel). 2023 Oct 31;13(21):3372. doi: 10.3390/ani13213372. PMID: 37958127; PMCID: PMC10650070.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Reynolds LP, Dahlen CR, Ward AK, Crouse MS, Borowicz PP, Davila-Ruiz BJ, Kanjanaruch C, Bochantin KA, McLean KJ, McCarthy KL, Menezes ACB, Diniz WJS, Cushman RA, Caton JS. 2023. (Invited Review) Role of the placenta in developmental programming: Observations from models using large animals. Anim Reprod Sci. 2023 Oct;257:107322. doi: 10.1016/j.anireprosci.2023.107322. Epub 2023 Aug 30. PMID: 37696224; PMCID: PMC10591737.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Chutikun Kanjanaruch, Kerri A Bochantin, Bethania J D�vila Ruiz, Jessica Syring, Yssi Entzie, Layla King, Pawel P Borowicz, Matthew S Crouse, Joel S Caton, Carl R Dahlen, Alison K Ward, and Lawrence P Reynolds. 2024. Short Communications: One-carbon metabolite supplementation to nutrient restricted beef heifers affects placental vascularity during early pregnancy, Journal of Animal Science, 2024;, skae044, https://doi.org/10.1093/jas/skae044
  • Type: Theses/Dissertations Status: Published Year Published: 2023 Citation: Syring, J. G. 2023. Effects of moderate nutrient restriction in beef cattle during early gestation on one-carbon metabolite concentrations in maternal and fetal tissues. M.S. Thesis. North Dakota State University. Fargo, ND.
  • Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: King, L. E. 2024. One-Carbon metabolites, fetal growth, maternal oxidative stress, and fetal programming during early gestation. Ph.D. Dissertation. North Dakota State University. Fargo. ND.


Progress 05/01/22 to 04/30/23

Outputs
Target Audience:The primary target audience is the scientific community and other researchers, particularly in the field of Animal Science. Additional target audiences include students and emerging professionals (undergraduate, graduate, and postdoctoral) as results from these efforts are presented in multiple venues. Producers and industry partners are also a target audience as these stakeholders have vested interests in the outcomes and new knowledge being discovered. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided professional development and training opportunities for four graduate students (2 MS and 5 PhD), This includes writing abstracts, extension research reports, and grant proposals; learning new techniques; and presenting their research at conferences. This project also provided the opportunity for visiting scientist from Iran to learn new techniques and improve her scientific writing skills. How have the results been disseminated to communities of interest?Results have been disseminated through abstracts, presentations at conferences, and journal publications. Preparation of manuscripts of additional peer reviewed manuscripts is in progress. What do you plan to do during the next reporting period to accomplish the goals?The major goals for the next reporting period are to continue with analysis of Exp.1 and 2. We will also initiate and complete data collections associated with Exp.2.

Impacts
What was accomplished under these goals? This year we continued to analyze and publish data from Exp. 1 and completed the animal portion of Exp. 2. Data from Exp. 1 show that restricted maternal nutrition increased (P = 0.03) B12 concentrations in maternal serum, increased (P ≤ 0.05) 5,10-methylenetetrahydrofolate and 5,10-methenyltetrahydrofolate concentrations in maternal liver, decreased (P = 0.05) 5,10-methylenetetrahydrofolate concentrations in fetal liver, increased (P = 0.0082) concentrations of cystathionine in fetal liver, and altered (P ≤ 0.03) concentrations of other metabolites in maternal liver. Supplementing OCM increased (P ≤ 0.05) maternal B12, folate and folate intermediate concentrations, increased (P ≤ 0.03) ALF and AMF 5-methyltetrahydrofolate concentrations, and altered (P ≤ 0.02) other maternal liver intermediates. We interpret these data to show our OCM supplementation was effective at altering maternal OCM status, and restricting nutrition also affected maternal OCM status. Data from Exp. 1 and 2 show that in Exp. 1 heifer data, there were pancreas gain × OCM interactions where CON+OCM weights were greater when compared to each of the three remaining treatments (P < 0.01). Furthermore, CON and +OCM heifers had larger pancreas weights (P ≤ 0.05). Dam liver, ribeye area, live slaughter weight, and hot carcass weight had gain effects where CON heifers had a heavier weight (P < 0.01). When looking at the fetus, fetal body weight tended (P = 0.07) to be lower and stomach complex weight was lower (P = 0.026) in RES compared with CON. When organ weights were normalized to total brain weight to assess allometric growth, OCM × gain interactions were observed in heart (P = 0.04), left longissimus dorsi (P = 0.04), and right hemisphere (P = 0.01) with CON+OCM greater than CON-OCM. The normalized left hemisphere tended to be greater in RES+OCM than RES-OCM (P = 0.079). In Exp. 2 heifer data, gain × OCM interactions were not present (P > 0.05). Live slaughter and hot carcass weight had gain effects where CON heifers were heavier than RES (P < 0.01). The 12th rib fat depth was larger in CON (P = 0.03) and a tendency (P = 0.08) for CON heifers to have a larger ribeye area. The +OCM heifers had an average yield grade of 1.69 while -OCM heifers had 2.02 (P = 0.017). In Exp. 2 fetal data, there was no differences in fetal body weight. However, trachea was heavier (P = 0.04) and spleen was lighter (P = 0.05) in fetuses from +OCM than -OCM. Right hindlimb muscle was influenced by a gain × OCM interaction (P = 0.04), where RES+OCM was heavier than CON+OCM. With allometric adjustments, right longissimus dorsi, rumen complex, and liver were heavier (P ≤ 0.05) in RES compared with CON. Day 63 is within the period of peak primary myogenesis in cattle; therefore, differences in muscle growth during this time could have lasting implications in post-natal calf growth and performance. Furthermore, nutrient partitioning in liver and muscle tissue can affect many postnatal production traits such as lactation, reproduction, and carcass quality. These data suggest that providing OCM during early gestation may rescue skeletal muscle tissue growth in nutrient restricted offspring, and that restricting nutrients to dams results in prioritization of allometric growth of critical fetal organs such as liver and lungs.Supplementation with OCM did not rescue all organ growth, but alterations in muscle and metabolic organ weight observed could have lasting postnatal implications. Data from Exp. 2 indicate that supplementation of OCM improved the mitochondrial function in the liver of control beef heifers, which could suggest changes in the function of mitochondrial respiration and a more favorable metabolic control.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Dahlen CR, Reynolds LP, Caton JS. 2022. Selenium supplementation and pregnancy outcomes. Front Nutr. 2022 Oct 31;9:1011850. doi: 10.3389/fnut.2022.1011850. PMID: 36386927; PMCID: PMC9659920. (Invited Review)
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Diniz WJS, Reynolds LP, Ward AK, Borowicz PP, Sedivec KK, McCarthy KL, Kassetas CJ, Baumgaertner F, Kirsch JD, Dorsam ST, Neville TL, Forcherio JC, Scott RR, Caton JS, Dahlen CR. 2022. Untangling the placentome gene network of beef heifers in early gestation. Genomics. 2022 Mar;114(2):110274. doi: 10.1016/j.ygeno.2022.110274. Epub 2022 Jan 25. PMID: 35090995
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Reynolds, LP, WJS Diniz, MS Crouse, JS Caton, CR Dahlen, PP Borowicz, and AK Ward. 2022. Maternal nutrition and developmental programming of offspring. Reproduction, Fertility, and Development 35(1-2). 19-26. doi:10.1071/RD22234. (Invited Review).
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Syring JG, Crouse MS, Neville TL, Ward AK, Dahlen CR, Reynolds LP, Borowicz PP, McLean KJ, Neville BW, Caton JS. Concentrations of vitamin B12 and folate in maternal serum and fetal fluids, metabolite interrelationships, and hepatic transcript abundance of key folate and methionine cycle genes: the impacts of maternal nutrition during the first 50 d of gestation. J Anim Sci. 2023 Jan 3;101:skad139. doi: 10.1093/jas/skad139. PMID: 37129588; PMCID: PMC10199783.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Diniz WJS, Ward AK, McCarthy KL, Kassetas CJ, Baumgaertner F, Reynolds LP, Borowicz PP, Sedivec KK, Kirsch JD, Dorsam ST, Neville TL, Forcherio JC, Scott R, Caton JS, Dahlen CR. Periconceptual Maternal Nutrition Affects Fetal Liver Programming of Energy- and Lipid-Related Genes. Animals (Basel). 2023 Feb 9;13(4):600. doi: 10.3390/ani13040600. PMID: 36830387; PMCID: PMC9951695.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Hammer CJ, Caton JS, Dahlen CR, Ward AK, Borowicz PP, Reynolds LP. DOHaD: A MENAGERIE OF ADAPTATIONS AND PERSPECTIVES: Large animal models of developmental programming: sustenance, stress, and sex matter. Reproduction. 2023 Apr 21;165(6):F1-F13. doi: 10.1530/REP-22-0453. PMID: 36951791.


Progress 05/01/21 to 04/30/22

Outputs
Target Audience:The primary target audience is the scientific community and other researchers, particularly in the field of Animal Science.The primary target audience is the scientific community and other researchers, particularly in the field of Animal Science. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided professional development and training opportunities for four graduate students (3 MS and 1 PhD). This includes writing abstracts, extension research reports, and grant proposals; learning new techniques; and presenting their research at conferences. This project also provided the opportunity for visiting PhD student from Thailand to learn new techniques and improve her scientific writing skills. How have the results been disseminated to communities of interest?Results have been disseminated through abstracts and presentations at conferences. Preparation of manuscripts for peer-reviewed journals has begun and is in progress. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In beef cattle production systems, inadequate maternal nutrition during pregnancy is a common stressor that occurs, particularly during periods of low forage quality or availability. Undernutrition during pregnancy can have negative impacts on fetal development that persist through out the life of the calf. This developmental programming impacts calf growth, efficiency, and body composition. Our long-term goal is to develop strategic supplementation strategies that producers can employ during periods of poor forage availability to ensure the optimal growth and efficiency of their calf crop. This study investigated the effects of One-Carbon Metabolites (OCM) supplementation on fetal growth and development in feed restricted beef cattle during pregnancy. The OCM studied are methionine, choline, folate, and vitamin B12. We hypothesize that maternal feed restriction will impair fetal growth in early pregnancy and that supplementation of OCM will rescue the growth restriction. We have completed the first of three experiments designed to study this hypothesis across three stages of fetal development (early, mid, and late pregnancy). Experiment 1 was designed in a 2 x 2 factorial arrangement of dietary treatments, with two levels of feed intake (adequate [CON] or restricted [RES]) and two levels of strategic OCM supplementation (with supplement [+OCM] or without supplement [-OCM]). Angus-cross heifers were randomly assigned to treatment at breeding (via artificial insemination) for a total of n = 8 heifers per treatment. The pregnant heifers were maintained on their treatments until day 63 of pregnancy (within the first trimester), at which time they were harvested and maternal and fetal tissues were collected. Maternal blood samples were collected at the start and end of the study for analysis of nutrient and hormone concentrations and hematology. Red blood cell count was greater (P = 0.0019) in RES than CON (9.29 vs 8.30 ± 0.186 × 106 cells/mm3). Hematocrit (P = 0.08) and packed cell volume tended (P = 0.09) to be greater in RES than CON. The percentage of monocytes was lower (P = 0.04) in +OCM than -OCM (1.5 vs 3.0 ± 0.50 %). The percentage of segmented neutrophils was greater (P = 0.01) in CON than RES (24.6 vs 16.8 ± 1.93 %). These results indicate that supplementation with OCM did not increase red blood cell production in early gestion in heifers receiving an adequate or restricted plane of nutrition. Serum glucose was greater in CON than RES (3.44 vs 3.21 ± 0.07 mM; P = 0.03). The gain by day interaction was significant for blood urea nitrogen and non-esterified fatty acid concentration (P < 0.05), with nitrogen concentrations increasing and fatty acid decreasing over time for CON but not for RES. Amniotic fluid fructose concentration was greater in CON-OCM than CON+OCM and RES-OCM, with RES+OCM intermediate (P = 0.005). Allantoic glucose concentration tended to be greater (P = 0.09) for CON-OCM than CON+OCM. We conclude that supplementation of OCM in early gestation increases concentrations of energy metabolites glucose and fructose in fetal fluids of RES heifers. Fetal organ weights were measured at collection. Fetal body weight tended (P = 0.07) to be lower and stomach complex weight was lower (P = 0.026) in RES than CON. When organ weights were normalized to total brain weight to assess allometric growth, feed intake x supplementation interactions were observed in heart (P = 0.04), loin muscle (P = 0.04), and right brain hemisphere (P = 0.01) with CON+OCM greater than CON-OCM. The normalized left hemisphere was greater in RES+OCM than RES-OCM (P = 0.079). No effect of treatment was found on pancreas, kidneys, lungs, liver, mammary gland, reproductive organs, hindbrain, and hindlimb weights. Fetal liver and muscle samples were analyzed for mitochondrial efficiency. No effect of treatment was found on mitochondrial function. Fetal liver and muscle samples were also collected for analysis of global gene expression. The RNA has been extracted and sequencing and analysis is in progress.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Amat, S., C. R. Dahlen, K. C. Swanson, A. K. Ward, L. P. Reynolds, and J. S. Caton. 2022. Bovine animal model for studying the maternal microbiome, in utero microbial colonization and their role in offspring development and fetal programming. Front. Microbiol. 13:854453. doi:10.3389/fmicb.2022.854453
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Entzie, Y., L. King, S. Syring, M. R. Hirchert, J. Caton, C. R. Dahlen, A. K. Ward, and M. S. Crouse. 2022. PSIII-7 The effect of one-carbon metabolite supplementation in combination with adequate or restricted feed intake during early gestation on maternal hematology. J. Anim. Sci. 100 (Supp. 2):130-131. doi: 10.1093/jas/skac064.223
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: King, L., J. Syring., Y. Entzie., M. R. Hirchert, M. S. Crouse, J. Caton, C. R. Dahlen, and A. K. Ward. 2022. 180 Supplementation of one-carbon metabolites to beef heifers during early gestation increased fetal organ weight at day 63 of gestation. J. Anim. Sci. 100(Supp. 2):87-88. doi:10.1093/jas/skac064.142
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Syring, J., J. Caton, M. S. Crouse, and A. K. Ward. 2022. PSIV-1 Differential gene expression of methionine-folate cycle enzymes in heifers supplemented with one-carbon metabolites. J. Anim. Sci. 100(Supp. 2):190. doi: 10.1093/jas/skac064.320
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Entzie. Y. L., L. E. King, J. G. Syring, M. Hirchert, J. S. Caton, M. S. Crouse, C. R. Dahlen, and A. K. Ward. 2022. The effect of one-carbon metabolite supplementation in combination with plane of nutrition during early gestation on maternal serum and fetal fluids. 2022 Perinatal Biology Symposium, Aspen, CO. Aug 28-31, 2022.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Kanjanaruch, C., K. A. Bochantin, P. P. Borowicz, L. P. Reynolds, M. S. Crouse, J. S. Caton, C. R. Dahlen, and A. K. Ward. 2022. Supplementing one-carbon metabolites to nutrient-restricted cows during early pregnancy affects placental vascularity. 2022 Perinatal Biology Symposium, Aspen, CO. Aug 28-31, 2022.


Progress 05/01/20 to 04/30/21

Outputs
Target Audience:The target audience for this period was scientific colleagues, who were consulted for advice in developing assays. Changes/Problems: In the proposal we had stated that we were going to use a standing-flank ovariohysterectomy surgery to collect fetal calf tissues at days 63 and 91 of gestation. This procedure permits the rapid collection and preservation of tissues and a significant cost savings as heifers are returned to the production herd after the end of the study. At the time the proposal was submitted, our surgical protocol was approved by the North Dakota State University IACUC and had been successfully used on over 100 heifers. After the proposal was submitted we began another study using the ovariohysterectomy procedure. Near the end of the study three heifers were lost due to post-surgical complications (internal bleeding and sepsis from a gut laceration). These complications were listed as potential complications in the IACUC protocol and the number of animals lost was within our projected death loss (8%). Even though the adverse events were within the parameters of the approved IACUC protocol, out of an abundance of caution the IACUC has suspended the ovariohysterectomy procedure. As a result of IACUC suspension of the ovariohysterectomy procedure we will not be able to use this technique to collect fetal tissues as we had originally proposed. As an alternative we will harvest the heifers at their respective gestational timepoints and collect fetal tissues via necropsy. This change results in a significant increase in the cost of the study as the heifers must now be purchased rather than returned to the production herd after the completion of the study. We have secured additional funding from our institution as well as an award supplement from USDA-NIFA which will cover the additional costs. We will therefore be able to successfully proceed with the objectives of the study. We had originally planned to begin Experiment 1 in spring 2020. Due to COVID-19 restrictions we were unable to initiate the experiment and it has been delayed to spring 2021. We plan initiating the second half (day 93 collection) of Experiment 1 as well as Experiment 2 (day 259 collection) in spring/summer 2022. This will keep us on track to complete the project on schedule. What opportunities for training and professional development has the project provided?We currently have two PhD students and one MS student working on the project. Additional staff associated with the project include two laboratory technicians and a postdoctoral fellow. We have had the opportunity to work with our collaborators at USDA-MARC on a pilot dose titration study. Our students and Co-PIs visited USDA-MARC and learned how to perform tissue sampling, mitochondria extraction, and mitochondrial function using a Seahorse XF Analyzer. This training significantly aided in the development of our mitochondria and Seahorse XF Anaylzer protocols. How have the results been disseminated to communities of interest?We have communicated with colleagues via email and video conference to discuss planning of laboratory assays. We have not yet generated any results to disseminate. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will conduct and complete the day 63 fetal timepoint of Experiment 1. In this experiment heifers will be fed a control or restricted diet with or without OCM supplementation from breeding until day 63 of gestation, at which time fetal tissue will be collected. Laboratory analyses to be completed during the next reporting period include serum metabolite and hormone concentrations, fetal transcriptome, and fetal mitochondrial function, and fetal muscle fiber typing.

Impacts
What was accomplished under these goals? The goals of our study are to understand how maternal nutrition during early pregnancy alters fetal development in beef cattle and if strategic nutrient supplementation can improve fetal outcomes. The long-term goal is to develop management strategies for pregnant beef cattle to improve calf performance, thereby increasing the efficiency of beef production. We have begun preparations to initiate our first experiment to study strategic supplementation of one carbon metabolites (OCM; choline, methionine, folate, and vitamin B12) in early pregnancy. In this experiment heifers will receive one of four dietary treatments from breeding until day 63 of pregnancy: control or restricted level of feed intake, each with or without OCM supplementation. The fetal calves will be collected at day 63 and analyses will be performed to study their metabolism and development. In conjunction with our collaborators at USDA-MARC, we have completed a pilot dose-titration study of one carbon metabolite (OCM; choline, methionine, folate, and vitamin B12) supplementation in heifers. The objective was to confirm that our proposed levels of OCM supplementation resulted in increased serum concentration in heifers. Preliminary results indicate that our proposed levels of supplementation are sufficient to increase the serum concentrations of these nutrients.

Publications