Fundamental Development of Reproduction Management Systems in Dairy Cattle: Insight into Hormonal and Molecular Controls of Embryo Development and Survival

Optimized systems for timed artificial insemination (TAI; Pregnancy per AI of 40 to 50%) entail controlling the period of follicle dominance, need to sustain progesterone exposure throughout the period of ovulatory follicle development, induction of complete luteolysis in lactating dairy cows, and finally the need to optimize timing of AI relative to induction of ovulation. Nevertheless, factors such as anovulation, lactation, and breed differences influence herd fertility. Objective of this presentation is to utilize the optimized TAI protocols as a platform to evaluate global gene expression of endometrial and conceptus tissues at 15 to 17 days of pregnancy, as to differences associated with: lactation (Exp. 1), ovulation of a 1st wave (FW) follicle (+ or - progesterone supplementation) versus a 2nd wave (SW) follicle (Exp. 2), and genetic/cyclic status groups (anovular Holstein [AH], cyclic Holstein [CH] and cyclic Jersey/ Holstein crossbred [CC]; Exp.3). Our hypothesis was that this analytical approach would unveil global changes in gene expression of endometrium and/or conceptus tissues that would provide potential candidate genes that may be regulated to improve fertility in lactating dairy cows. The foundation approach for all three experiments was to utilize experimental TAI protocols to program ovulation and fertilization with collection of target tissues at either 15 or 17 days after AI. RNA from conceptuses and endometrial tissues from the respective experiments were subjected to global gene expression (Affymetrix Gene Chip® Bovine Array). Microarray data were analyzed using Bioconductor software in R project. Data were preprocessed using Gene Chip Robust Multi-Array algorithm. Limma package was used to fit a linear model and adjust variances by empirical Bayes method. Moderate t-test and factorial ANOVA were performed for all appropriate comparisons, and P values were adjusted for multiple testing using the Benjamini and Hochberg false discovery rate. Adjusted P < 0.05 characterized minimum significant differences of 1.5 fold-change. Functional analyses were performed using David (http://david.abcc.ncifcrf.gov/home.jsp), Pathway Studio (http://www.ariadnegenomics.com), or Ingenuity Pathway Analysis (http://www.ingenuity.com/) software.

In Exp. 1. Endometrial FCGRT, IGHG1, IGLL and TRD genes were up-regulated by lactation, but not altered by pregnancy. These genes may reflect a possible increase in B-lymphocyte and γδ T cell activity that could affect immunological balance of the uterus necessary to accept the semi-allograph embryo at day 17. The cascade of the Wnt signalling pathway represented by WNT2, GSK3B, CTNNB1 and MSX1 was down regulated on d 17 of pregnancy, which indicates an important feature of the bovine uterine regulation in preparation to accommodate the conceptus. Furthermore, a pregnancy up regulation (almost 4 fold) of DKK1, a secreted inhibitor of the Wnt signalling may be the main force contributing to the down regulation of the canonical Wnt pathway. Lactation decreased expression of DKK1 as well as RELN, two genes also involved with embryonic neural and limb development. Thus these processes associated with Wnt signalling and embryonic development may be antagonized by lactation due to decreased expression of DKK1 and RELN. Up-regulation of PDK4 by lactation is indicative of a negative role on glucose homeostasis caused by lactation.