Recipient Organization
WEST TEXAS A & M UNIVERSITY
2501 4TH AVE
CANYON,TX 79016
Performing Department
(N/A)
Non Technical Summary
Melatonin is a hormone that regulates circadian rhythms, but also has been implicated to have a role as a reproductive hormone. Previous work identified roles for melatonin in oocyte quality, embryo development, pregnancy loss, and overall fertility. Oocyte quality and embryo development are the focus of this work given that poor quality oocytes generate embryos with reduced growth and viability. These observations were used to develop this central hypothesis - melatonin functions as a critical component of oocyte development in cattle, specifically in low antral follicle count (AFC) cattle and is essential for oocyte production in vivo and oocyte maturation in vitro. Two aims will be used to test this hypothesis; by supplementing high and low AFC cows with melatonin before performing ovum pick-up to evaluate oocyte quality and by supplementing oocytes from high and low AFC animals with melatonin in vitro and performing in vitro maturation to evaluate embryo development. This proposed research is novel and is anticipated to provide valuable information into oocyte development and maturation in cattle. The overall goal of this research is to identify methods to improve oocyte quality and embryo development in cattle. Extension programming will be developed to build upon this work and provide Texas dairy producers with publications to disseminate research findings, provide knowledge, and hands-on skills to improve reproductive management.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
The long-term, overarching goals of this project are to identify methods to improve oocyte quality and embryo develoment in dairy cattle. This is accomplished via 2 aims: 1) supplementing cows with melatonin prior to collection of ovum to evaluate oocyte quality and supplementing ovum after collection with melatonin and maturing in vitro to evaluate embryo development and 2) translate fundamental knowledge of oocyte and embryo development by bridging the knowledge gap anddeveloping reproductive technologies that will improve oocyte quality and embryo development.
Project Methods
Aim 1: determine if melatonin (MEL) supplemetnation to cows improves oocyte qualityCows will be sourced from a local, commercial dairy, which milks 11,000 cows with 60% registered Holstein cows. Transrectal ultrasonography will be performed on 200 Holstein cows (2+ lactation) in the herd to determine AFC. The top and bottom 15% for AFC will be selected for the high and low AFC groups. 4 experimental groups will be used:1) low AFC cows + MEL implant; 2) low AFC cows control (no implant); 3) high AFC cows + MEL implant and 4) high AFC cows control (no implant). MEL treated groups will have five 24 mg MEL implants (Melawin, USA) applied subcutaneously behind the ear. MEL will be implanted 14 d prior to the first OPU collection and every 30 days after to maintain circulating MEL concentrations. Thereafter, for each group (n = 10), OPU will be performed twice weekly at 3-4 day intervals for 10 sessions to collect cumulus-oocyte complexes (COCs) with follicles > 5 mm being collected. At OPU, oocytes will be needle aspirated via an ultrasound guided probe and follicle number and size will be recorded following standard OPU protocol. After OPU, number of COCs recovered and COC quality will be determined. Immature COCs collected over several OPU sessions from each cow will be evaluated for reactive oxygen species (ROS) as an indicator of oxidative stress. High quality COCs collected from each cow over several OPU sessions will be snap frozen in liquid nitrogen for RNA-sequencing at the VERO facilities. Data will be analyzed using SAS 9.4 to detect any differences between MEL supplementated and non-MEL supplemented cow groups on oocyte quality.Aim 2: determine if MEL supplementation improves in vitro oocyte maturation, fertilization and embryo developmentTo verify that the MEL concentration used by others (1 nM) is effective at increasing IVP embryo yields, abattoir ovaries from Caviness Beef Packers (Hereford, Texas) will be sourced and cumulus-oocyte complexes (COCs) will be collected and matured in standard culture medium (medium 199 with 10% FBS, 0.5% gentamicin, 1% sodium pyruvate, 1% glutamax, 0.1% folltropin, 0.1% estradiol, 0.1% EGF) with varying concentrations of MEL (0 nM or 1 nM). 10 oocytes from each group will be evaluated with qRT-PCR post in vitro maturation to identify the concentration that results in altered gene expression and upregulation of critical oocyte survival genes (e.g., GDF9, DNMT1, PTX3)and downregulation of apoptosis genes (e.g., BCL-2, Caspase-3). After determining the optimal MEL concentration, OPU will be performed on high and low AFC cows. High (n = 10) and low (n = 10) AFC cows will be identified as outlined in Aim 1. OPU will be performed as previously described until 1500 oocytes are collected each from the high and low AFC groups. After OPU, oocytes from each group will be matured, fertilized and cultured in vitro. Four in vitro maturation groups will be utilized to generate IVP embryos: 1) High AFC cow → IVM + BSA carrier; 2) High AFC cow → IVM + MEL; 3) Low AFC cow → IVM + BSA carrier and 4) Low AFC cow → IVM + MEL. COCs collected via OPU will be scored for quality and allowed to mature for 24 hours at 38.5 C under 5% CO2 in either standard culture media (as previously mentioned) or culture supplemented with MEL (standard culture media supplemented and MEL concentration determined in pilot study). Time of maturation evidenced by presence of the first polar body will be evaluated at 14, 17 and 21 hours. In addition, transzonal projections (TZP) will be evaluated at 9 and 18 h by denuding the oocytes prior to fixing and actin staining with phalloidin as another measure of oocyte development. At 12 and 22 hours, transcript profiling of cumulus and oocytes will be conducted with RNA-sequencing. After maturation, COCs will be fertilized and cultured. After 1 hour in culture, embryos will be evaluated for reactive oxygen species (ROS) level as previously described. Embryo development will be assessed to determine cleavage rates (d 3) and blastocyst rates (d 7-8). On day 8, embryos will be fixed and stained to determine inner cell mass (ICM) and trophectoderm (TE) cell number as previously described. Data will be analyzed using SAS 9.4 to detect any differences between MEL supplementated and non-MEL supplemented oocytegroups on embryodevelopment.