Performing Department
(N/A)
Non Technical Summary
Pregnant livestock are often grazed through the fall and winter when forage is less abundant and lower quality. As maternal metabolic demands increase and pastures become dormant, dams are often nutrient restricted due to an inability to consume enough nutrition for themselves, the developing fetus, and placenta. Producers commonly manage these situations with strategic supplementation at the end of gestation when demands are the highest and peak fetal growth is occurring. While this may be beneficial to recover fetal growth by size, we hypothesize that periods of restriction earlier in gestation may cause tissue specific differences not evident by birthweight. Thus, the objective of this project is to evaluate the long-term impacts of this nutritional strategy on ewe lamb development and reproductive capacity. After restricting ewes from 30-125 days of gestation, our first objective is to determine how maternal nutrition impacts ewe lambs related to puberty attainment, follicle development, and uterine morphology. Our second objective will delve into the molecular indicators of oocyte and uterine competency that contribute to normal estrous cyclicity and pregnancy. We expect the results of this study to indicate whether common nutritional practices have unapparent impacts on tissues that determine reproductive success. If our hypothesis is correct, this will provide key fundamental information that warrants further investigation and may influence how livestock producers manage their pregnant animals to have more reproductive success in replacement females.
Animal Health Component
30%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
0%
Goals / Objectives
Our long-term goal is to identify how developmental programming manifests in reproductivetissues and develop targeted strategies to overcome these deficits to support prolific breeding stock.The goal of this project is to evaluate if common nutrient challenges experienced by grazing dams impacts ewe lamb development and reproductive efficiency.Our hypothesis is that while developmental programming may not be evident by birthweight differences, thrifty tissue-specific adaptations underlie differences in animal reproductive capacity.As a preliminary study, we aim to substantiate our hypothesis through the following objectives:Objective 1: Determine the impact of maternal nutrient restriction during gestation on ewe lamb puberty attainment, response to estrous synchronization, and reproductive dynamicsObjective 2: Investigate transcript expression in the oocyte and endometrial tissue of pubertal lambs born from nutrient-restricted dams
Project Methods
Upon pregnancy confirmation by blood at ~25 days of gestation (dGA), timed pregnant ewes will be randomized by bodyweight, allocated to a treatment, and placed in individual housing to control the quality and quantity of feed intake. Control ewes will receive a diet that meets 100% of National Research Council (NRC) requirements for gestating ewes throughout pregnancy. From 30-125 dGA, nutrient-restricted ewes will receive a diet mimicking poor winter forage quality with <6% crude protein and <60% total digestible nutrients. At 125 dGA ewes will be realimented to 100% NRC requirements to align with common late-gestation supplementation strategies. Nutrient restricted ewes will be pair-fed with control ewes. Throughout gestation, individual feed intake, bodyweight, and body condition scores will be collected to evaluate maternal performance. Ewes will lamb out naturally to produce control (C) and nutrient-restricted (NR) lambs. Daily weights and weekly morphometrics will be collected from birth until weaning (70 d) to evaluate growth and symmetry. Puberty attainment. Beginning at 6 mo of age, ewe lambs will be bled bi-weekly and housed with a vasectomized ram fitted with a marking harness. Marking will be evaluated daily during routine health checks and compared to serum progesterone concentrations. Serum P4 will be determined by commercially available ELISA. A female will be considered pubertal when serum P4 concentrations align with marking and exceed 1ng/mL in two consecutive estrous cycles. After puberty confirmation, ewe lambs will be synchronized by a 10-day CIDR protocol and then harvested on day 15 for tissue collection. Uterine luminal fluid analysis: The uterine horns will be flushed with phosphate-buffered saline (PBS) to determine uterine luminal fluid (ULF) glucose concentrations via immobilized enzyme system. Uterine gland density. After flushing, the uterus will be dissected, opened, and caruncle counts will be performed. A sample of intercaruncular endometrial tissue from the mid-section of the horn ipsilateral to the CL will be collected, cyroembeded, and IHC performed to determine uterine gland density. RNA Sequencing. An additional intercaruncular endometrium sample will be collected from each animal for RNA sequencing to evaluate gene expression in pathways related to cyclicity and pregnancy. Antral follicle counts. A cross section of the ovary contralateral to the CL will be cryoembeded and immunohistochemistry (IHC) performed to determine histological follicle counts (primordial, primary, secondary, and antral stages). A TUNEL assay will be concurrently performed to determine a ratio of apoptotic:total cells and only healthy follicles will be included in the follicle counts. Oocyte Transcript Abundance. Small antral follicles will be aspirated, and cumulus oocyte complexes (COC) will be collected. COC will be combined with hyaluronidase and vortexed to separate oocytes from cumulus cells. Oocytes will be collected using a stereomicroscope and RNA will be isolated for quantitative RT-PCR evaluating prostaglandin-endoperoxide synthase 2 (PTGS2), steroidogenic acute regulatory protein (STAR), and B-cell CLL/lymphoma 2 (BCL2), with reference targets glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein L19 (RPL19).