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Post-Transfer Outcomes in Cultured Bovine Embryos Supplemented with Epidermal Growth Factor, Fibroblast Growth Factor 2, and Insulin-Like Growth Factor 1

McCauley T. Vailes

Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University

in partial fulfillment of the requirements for the degree of

Master of Science In

Animal and Poultry Sciences

Alan D. Ealy Vitor R. G. Mercadante

David A. Roper

May 5, 2017 Blacksburg, Virginia

Keywords: Embryogenesis, Pregnancy, Placenta

Post-Transfer Outcomes in Cultured Bovine Embryos Supplemented with Epidermal Growth Factor, Fibroblast Growth Factor 2, and Insulin-Like Growth Factor 1

McCauley T. Vailes

Abstract

The high incidence of pregnancy loss is a major issue facing the cattle industry.

Use of in vitro fertilized (IVF) bovine embryos has become increasingly popular to help

alleviate several of these reproductive issues and provide a means to enhance genetic

gain for production traits. An uterine paracrine factor cocktail containing epidermal

growth factor (EGF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1

(IGF1) (collectively termed EFI) was recently identified as a means for improving in vitro

derived bovine embryo development and trophectoderm cell numbers. The objectives of

this work were to determine if EFI treatment during in vitro bovine embryo culture

improves transferable embryo quality and post-transfer placental and fetal development.

For each replicate (3 total), slaughterhouse-derived bovine oocytes were matured and

fertilized in vitro. At day 4 post-fertilization, 8 cell embryos were harvested, pooled, and

exposed to either the EFI treatment (10ng/ml EGF, 10ng/ml FGF2, 50ng/ml IGF1) or

carrier only (1% Bovine Serum Albumin). At day 7, individual embryos were transferred

to estrous synchronized beef cattle. Artificial insemination (AI) was completed on a

subset of cows. The EFI treatment increased (P

Post-Transfer Outcomes in Cultured Bovine Embryos Supplemented with Epidermal Growth Factor, Fibroblast Growth Factor 2, and Insulin-Like Growth Factor 1

McCauley T. Vailes

General Audience Abstract

The high incidence of pregnancy loss is a major issue facing the cattle industry.

Use of in vitro fertilized (IVF) bovine embryos has become increasingly popular to help

alleviate several of these reproductive issues and provide a means to enhance genetic

gain for production traits. An uterine paracrine factor cocktail containing epidermal

growth factor (EGF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1

(IGF1) (collectively termed EFI) was recently identified as a means for improving in vitro

derived bovine embryo development and trophectoderm cell numbers. The objectives of

this work were to determine if EFI treatment during in vitro bovine embryo culture

improves transferable embryo quality and post-transfer placental and fetal development.

For each replicate, slaughterhouse-derived bovine oocytes were matured and fertilized

in vitro and at day 4 post-fertilization, embryos were exposed to either the EFI treatment

(10ng/ml EGF, 10ng/ml FGF2, 50ng/ml IGF1) or carrier only (1% Bovine Serum

Albumin). Artificial insemination (AI) was completed on a subset of cows and the

remaining cattle receive embryos at day 7. The EFI treatment increased (P

iv

Acknowledgements

I would first like to thank my parents and my sister for the endless support and

love and always letting me know how proud they are of my accomplishments. I would

also like to thank Jake Bailey for being there for me and being a shoulder to lean on. To

Erin Fitzgerald, thank you for knowing what to say and encouraging me throughout the

years. Also, to my lab mates and office mates, thank you for bringing laughter to my

days. Thank you to my awesome committee members, Dr. Vitor Mercadante and Dr.

David Roper, for your guidance and talent. Finally, to Dr. Alan Ealy, thank you for your

inspiration and teaching me how to think and write like a scientist.

v

Table of Contents

Acknowledgements ...................................................................................................... iv

List of Figures ............................................................................................................. vii

List of Abbreviations .................................................................................................. viii

Chapter 1 - Literature Review ....................................................................................... 1

Introduction ................................................................................................................ 1

Stages of Pregnancy Losses in Cattle ..................................................................... 2

Economic Impacts of Pregnancy Loss .................................................................... 4

Events Associated with Pregnancy Success in Cattle ........................................... 5

Estrous Synchronization and Detection ................................................................... 5

Oocyte Maturation .................................................................................................... 6

Fertilization ............................................................................................................... 7

Early Embryonic Development ................................................................................. 8

Maternal Recognition of Pregnancy ....................................................................... 10

Implantation ........................................................................................................... 12

Placental Development .......................................................................................... 12

Major Contributors to Pregnancy Loss in Cattle .................................................. 13

Oocyte Competency ............................................................................................... 13

Uterine Development and Function ........................................................................ 15

The Progesterone-Uterus Link ............................................................................... 16

Maternal Recognition of Pregnancy ....................................................................... 17

Implantation and Placental Failures ....................................................................... 18

Physiological and Environmental Contributors to Pregnancy Losses in Cattle 19

Metabolic Status ..................................................................................................... 20

Environmental Factors ........................................................................................... 21

Clinical/Subclinical Diseases .................................................................................. 23

Using Bovine In Vitro Fertilization to Study Early and Late Embryonic Losses 25

Epidermal Growth Factor ....................................................................................... 26

Fibroblast Growth Factor-2 .................................................................................... 27

Insulin-like Growth Factor 1 ................................................................................... 27

Multiple Uterine Factor Supplementation Schemes ............................................. 28

vi

Summary and Concluding Remarks ...................................................................... 29

Hypothesis and Objectives ..................................................................................... 30

Chapter 2 Post-transfer Developmental Effects of Bovine Embryos

Supplemented with Epidermal Growth Factor, Fibroblast Growth Factor 2, and

Insulin-Like Growth Factor 1 ...................................................................................... 31

Introduction .............................................................................................................. 31

Methods .................................................................................................................... 33

Estrous Synchronization ........................................................................................ 33

In vitro Bovine Embryo Production ......................................................................... 34

Embryo Transfer .................................................................................................... 34

Pregnancy Diagnosis ............................................................................................. 35

Pregnancy Associated Glycoproteins ..................................................................... 35

Crown-Rump Length Determination ....................................................................... 36

Replication of the Study ......................................................................................... 37

Statistical Analyses ................................................................................................. 37

Results ...