Ovarian hormones AMH and E2 in juvenile
gilts as markers of reproductive success
Alicia Steel
BAnVetBioSci (Hons)
A thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
The University of Sydney
Sydney School of Veterinary Science
Faculty of Science
2019
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Declaration
This thesis is submitted to The University of Sydney in fulfilment of the requirements for the
Degree of Doctor of Philosophy.
The work presented in this thesis is original except as acknowledged in the text. I hereby declare that I have not submitted this material, in either full or in part, for a degree at this or any other university.
Signature: Date: 28/02/19
Alicia Steel
BAnVetBioSci (Hons)
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Acknowledgements
Chris Grupen –You have made this PhD so enjoyable and easy-going. I really can’t tell you how much I appreciate your support and understanding. Thank you for your guidance and knowledgeable input into this project and thesis. I greatly appreciate every minute you took to help me through this! …. Annnd I’m sorry for all those times I pretended everything was no big deal at the highest pressure-points of my PhD journey. I know this stressed you out, but I couldn’t help myself hahahaha. Thank you for believing me, it means the world! Rebecca Athorn – Thank you for all the generosity you showed me throughout this project. You really made me feel at home in the little town of Corowa! I really appreciate the many times you put me at ease when I thought the sky was falling. I am forever grateful for your calm and collected nature and all of your industry knowledge! Peter Thomson – Thanks for always responding to my (many) emails with fiddly questions…especially the ones where I’d email you an hour later only to tell you “Nevermind! I have figured it out on my own!”. Thank you for always making time for the little guys. Even when you were swamped with your own work, your patience and kindness never wavered. Thank you for always encouraging me to expand my knowledge and get back into coding. I will never ever forget that time you PM’d me code a week before Christmas in your much- deserved holiday period!! You made me feel less alone in this PhD journey. I thoroughly enjoyed every meeting we had and I always left inspired to learn more! The scientific community needs more people like you and it would be an absolute honour to work with you again someday. Jenna Lowe – You made my honours so enjoyable I continued on to do a PhD…. You are to blame :p . Just kidding, but I do owe this largely to you! I am so thankful for your friendship and ongoing support, even from afar. Your passion is infectious. I hope your fire never goes out! Charley, Dom, Alysia, Louise, Jess, Alex, Ha, Harriet, Evan, Danni x2, … and all the rest of the PhD students I met along the way! Can’t forget about Ruby and Bandit also! You helped me keep my sanity. Thank you for your ongoing support, I love you all. Paula –You are so compassionate and bubbly and the breath of fresh air on the other side of a PhD dungeon. Your smile started my day off right every day for four years and I really miss this! You always do your best to lend a hand (even if way outside of your job description) and I admire you for this. I hope we remain great friends! Technical and farm staff – Thank you to Craig Kristo for always going over and above what is required of you. You got me out of strife a few times! Thank you to the staff at Wollondilly Abattoir, Rivalea and Sunpork, I could not have done this without your guidance and willingness to help!
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To my Mamma Rose, thanks for taking me to science camp and enduring my talks about the life cycles of insects I found when I was little. Without you I wouldn’t have even dared to try this career path, you’re the reason why this whole adventure started. I thank you from the bottom of my heart for giving me the courage to dream big. Your zest, excitement, and appreciation for the small wonders in life is so infectious and has contributed to my passion for the natural world – this is something we tend to lose as adults and you, unknowingly, always remind me how wonderful the world truly is. To my Pappa Scott, thanks for all those times throughout my childhood you cleaned out those caterpillar tanks when I was too scared to touch them when they turned into moths, instead of the butterflies I thought I was growing. Thanks for also supporting my young dreams of becoming a fish breeder. I appreciate, so much, all the time you took to really understand what I was doing in my PhD and your amazing ability to add perspective. You spent endless hours lending me a much-needed ear and this has been invaluable to me. You always ground me and have taught me to think critically and to never take anything at face value. Ever since I was little you cultivated my curiosity, teaching me to build things and showing me how stuff worked and that it's okay to be interested in things outside the realm of typical ‘girl’ activities. Most of all, I want to thank you for giving me my most valued trait, independence. To my dear little sister, Sarah, you are so special and inspiring to me. I hope to be as courageous and confident in my career as you are one day. May our agreement of unconditional support remain unsaid, just to forbid Mum the satisfaction of hearing it…. Oh, and I’m really sorry Mum made you go to science camp.
I am truly privileged to be surrounded by such great people,
Thank you all,
Alicia
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Abstract
Poor sow retention is a common inefficiency in piggeries. In Australia, only around
60% of gilts being retained to parity three (Plush et al., 2016). This is concerning as gilts do not reach optimal reproductive performance until parity three (Engblom et al., 2007).
Reproductive inadequacy is the main contributing factor for this premature culling (Plush et al., 2016). Thus, the traditional process for selecting breeding gilts is inadequate.
Previously, circulating E2, ovarian follicle populations and hormonal profiles in response to gonadotropin stimulation have been linked with reproductive potential (Laufer et al., 1986; Kondapalli et al., 2012). Anti-Müllerian hormone (AMH) is the best known endocrine marker for antral follicle counts and ovarian reserve in other livestock species (cattle:
Batista et al., 2014; sheep: Torres-Rovira et al., 2014; mares: Claes et al., 2014).
This thesis aimed to determine the serum E2 and AMH levels in juvenile gilts prior to and after gonadotrophin stimulation and assess their associations with fertility and reproductive performance in order to determine their use as markers for gilts with greater reproductive success.
Experiment One examined serum AMH and E2 levels in juvenile gilts 0, 2 and 4 days after gonadotropin administration and compared these measurements with mating, litter and culling information for three parities. Experiment Two assessed whether juvenile levels of E2 and
AMH were associated with ovarian and uterine properties at 160 days. Experiment three involved two geographically different farms to validate results of Experiment One. The final experiment was similar to Experiment two but was longitudinal and involved a more detailed ovarian assessment.
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To our knowledge, the quantification of AMH in juvenile gilts in this thesis was novel.
Results showed serum AMH was negatively associated with ovarian follicle numbers, but its association with uterine properties was inconsistent. A negative association between E2 and future litter numbers was also found. Whether serum AMH and E2 levels in juvenile gilts are associated with uterine traits requires further investigation. The results highlight the complexities of endocrinology, emphasising the difficulty of determining hormonal markers for reproductive potential in a production setting.
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Contents
Declaration ...... 2 Acknowledgements ...... 3 Abstract ...... 5 List of peer-reviewed publications...... 12 List of conference presentations ...... 13 List of grants ...... 15 Thesis style...... 16 Disclosure and author contributions ...... 17 Chapter Three ...... 18 Chapter Four ...... 19 Chapter Five ...... 20 Chapter Six ...... 21 List of Abbreviations ...... 22 List of tables ...... 25 List of Figures ...... 27 CHAPTER ONE: LITERATURE REVIEW ...... 29 Preface...... 29 LITERATURE REVIEW: PART ONE ...... 30 Introduction ...... 30 Management of female pigs ...... 33 Gilt selection ...... 33 Timing of first mating ...... 34 Culling ...... 35 Measures of poor reproductive performance ...... 36 Delayed onset of oestrus ...... 36 Reduced farrowing rate ...... 36 Prolonged weaning to oestrus interval ...... 37 Sow reproductive physiology ...... 38 The Oestrous cycle ...... 38 Establishment and maintenance of pregnancy ...... 39 Development of the female reproductive system ...... 41 Foetal development: The ovary and oogenesis ...... 41 Folliculogenesis ...... 42
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Ovulation ...... 45 Oocyte maturation ...... 46 The uterus ...... 47 Endocrinology: from birth until puberty ...... 48 Circulating gonadotropin levels in the prepubertal gilt ...... 48 Puberty: Establishment of the hypothalamic-pituitary-gonadal axis ...... 48 Gonadotropin receptors ...... 50 Oestradiol (E2) ...... 52 Synthesis and production ...... 52 Receptors and signalling ...... 52 Receptor expression ...... 56 Circulating E2 in juveniles ...... 57 Circulating E2 as a marker of reproductive potential ...... 58 Puberty ...... 58 Gonadotropin responsiveness and fertility ...... 58 LITERATURE REVIEW: PART TWO ...... 60 Introduction ...... 60 Anti-Müllerian hormone (AMH) ...... 62 Discovery ...... 62 The protein, it’s Synthesis and Activation ...... 62 Receptors and Smad signalling pathways ...... 64 Receptor expression ...... 68 AMH production and expression in the ovary ...... 68 The role of AMH within the ovary ...... 69 Regulation of AMH ...... 70 1.11 Circulating AMH in juvenile animals ...... 73 Intra- and inter-individual variation ...... 73 Establishment of the HPG axis ...... 75 1.12 Circulating AMH in adulthood ...... 77 Menstrual cycles ...... 77 Oestrus cycles ...... 78 Season ...... 79 Aging and ovarian reserve ...... 80 Disease ...... 80 1.13 Maternal factors that affect circulating AMH in offspring ...... 82 Gestational nutrition ...... 82 Polycystic ovary syndrome ...... 82
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Maternal Inflammation ...... 83 1.14 Circulating AMH as a marker of future reproductive performance ...... 84 Puberty ...... 84 Gonadotropin responsiveness ...... 85 Ovarian reserve and antral follicle populations ...... 88 Fertility ...... 89 1.15 Concluding remarks and objectives of the present study ...... 91 CHAPTER TWO: GENERAL METHODS ...... 92 2.1 Ethics ...... 92 2.2 Animals and farms ...... 92 2.3 Blood collection and storage ...... 93 2.4 Assays...... 93 AMH Assay ...... 93 E2 Assay – Experiments One and Two ...... 93 E2 Assay – Experiments Three and Four ...... 94 CHAPTER THREE: EXPERIMENT ONE ...... 96 Abstract ...... 96 3.1 Introduction ...... 97 3.2 Methods ...... 99 Animals and ethics ...... 99 Gonadotropin stimulation, blood collection and storage ...... 99 Hormonal assays ...... 99 Mating and parity measurements ...... 101 Statistics ...... 102 3.3 Results ...... 103 Serum AMH and E2 levels of 60, 80 and 100 day old gilts before and after PG600 administration ...... 103 Parity one to parity three mating, litter and culling data ...... 104 Intra-parity correlations ...... 106 Inter-parity correlations ...... 107 Number of piglets born alive ...... 109 Probability of stillbirth ...... 109 Gestation length ...... 110 Other breeding parameters ...... 111 3.4 Discussion ...... 112 3.5 Acknowledgements ...... 117 CHAPTER FOUR: EXPERIMENT TWO ...... 118
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Abstract ...... 118 4.1 Introduction ...... 120 4.2 Methods ...... 123 Animals and ethics ...... 123 Blood samples and assays ...... 123 Assessment of uterine and ovarian development ...... 123 Statistical analysis ...... 124 4.3 Results ...... 125 Serum hormone concentrations, carcass traits, ovarian and uterine measurements 125 Correlations between ovarian hormones and uterine and ovarian traits ...... 127 Intra-uterus correlations ...... 127 Intra-ovarian correlations ...... 128 Calculating uterine mass indices (UMIs)...... 128 Calculating surface antral follicle indices (SFIs) ...... 130
Association between AMH and E2 concentration and UMIshape ...... 132 Other body condition, uterine and ovarian parameters ...... 133 4.4 Discussion ...... 133 3.5 Acknowledgements ...... 139 CHAPTER FIVE: EXPERIMENT THREE ...... 140 Abstract ...... 140 5.1 Introduction ...... 141 5.2 Methods ...... 142 Animals and ethics ...... 142 Blood samples and assays ...... 143 Mating and parity data ...... 143 Statistics ...... 143 5.3 Results ...... 145 Summary of AMH and E2 levels at 80 days of age and mating and pregnancy data ...... 145 Association between E2 concentration at 80 days of age and litter size (total born) ...... 148 Other measurements ...... 149 5.4 Discussion ...... 149 5.5 Acknowledgements ...... 153 CHAPTER SIX: EXPERIMENT FOUR ...... 154 Abstract ...... 154 6.1 Introduction ...... 155
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6.2 Methods ...... 158 Animals and ethics ...... 158 Blood samples and assays ...... 158 Assessment of uterine and ovarian development ...... 159 Histological preparation and analysis ...... 160 Statistical analysis ...... 160 6.3 Results ...... 162 Serum hormone levels, carcass traits, ovarian properties ...... 162 Correlations between ovarian hormones and uterine and ovarian traits ...... 164 Intra- and inter-hormone correlations ...... 165 Intra-uterus correlations ...... 165 Uterine mass indices (UMIs) ...... 165 Intra-ovary correlations...... 168 Ovarian Follicle Indices (OFIs) ...... 168
Association between E2 levels and UMIsize ...... 170
Association between E2 levels and OFIprop ...... 171
Association between AMH levels and OFItot ...... 172 Other body condition, uterine and ovarian parameters ...... 173 6.4 Discussion ...... 174 6.5 Conclusions ...... 177 6.6 Acknowledgements ...... 178 CHAPTER SEVEN: GENERAL DISCUSSION ...... 179 7.1 Summary ...... 179 7.2 Discussion ...... 181 AMH ELISA Assay ...... 181 Quantifying serum AMH in juvenile gilts ...... 182 Studies examining serum AMH and E2 levels and production parameters ...... 182 Studies examining serum AMH and E2 levels and ovarian and uterine properties at slaughter ...... 184 Reproductive properties at slaughter vs. production outcomes ...... 187 7.3 Final Conclusions ...... 189 REFERENCES ...... 192
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List of peer-reviewed publications
Steel, A., Athorn, R.Z., Grupen, C.G., 2018. Anti-Müllerian hormone and Oestradiol as markers of future reproductive success in juvenile gilts. Animal Reproduction Science 195,
197-206.
Steel, A., Athorn, R.Z., Grupen, C.G., 2019. Serum Concentrations of AMH and E2 and
Ovarian and Uterine Traits in Gilts. Animals 9, 881.
These publications have been reformatted for the purpose of this thesis in Chapters Three and
Six.
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List of conference presentations
Australasian Pig Science Association, Melbourne 2017
Steel, A.N., Athorn, R.Z Grupen, C.G. (2017). Can serum levels of anti-Müllerian hormone and oestradiol in juvenile gilts be used to predict future reproductive performance?. Animal
Production Science 57, p. 2476.
NSW Reproduction Forum, Sydney 2017
Steel, A.N., Athorn, R.Z Grupen, C.G. (2017). PhD Thesis: AMH and E2 as predictors of reproductive performance in gilts. In “NSW Reproduction Forum 2017” Sydney, Australia.
World Congress of Reproductive Biology, Japan 2017
Steel, A.N., Athorn, R.Z., Grupen, C.G. (2017). Serum anti-Müllerian hormone and oestradiol in juvenile gilts can predict future reproductive performance parameters. In "4th World
Congress for Animal Reproduction " Okinawa, Japan.
Society of Reproductive Biology, Gold Coast 2016
Steel, A.N., Athorn, R.Z., Grupen, C.G. (2016). Are serum levels anti-Müllerian hormone and oestradiol in juvenile gilts predictive of the onset of puberty?. In "59th Society of Reproductive
Biology Annual Scientific Meeting" Gold Coast, QLD, Australia.
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Int’l Congress of Animal Reproduction, France 2016
Steel, A.N., Athorn, R.Z., Grupen, C.G. (2016). Serum anti-Müllerian hormone concentrations and ovarian response in juvenile gilts: potential predictors of reproductive performance. In “18th
International Congress of Animal Reproduction" Tours, France, p. 56-57.
Australasian Pig Science Association, Melbourne 2015
Steel, A.N., Lowe, J.L., Somfai, T., Grupen, C.G. (2015). The influence of cumulus cells on porcine oocyte maturation in the presence of L-carnitine. Animal Production Science 55, p.
1507.
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List of grants
This project was funded by Australian Pork Limited (Grant number: 2014/217).
The PhD scholarship was funded partly by Australian Pork Limited (Grant number: 2014/482) and partly by The University of Sydney
Society of Reproductive Biology (SRB) student travel subsidy 2016
Postgraduate Research Support Scheme (PRSS) Scholarship in 2016
Postgraduate Research Support Scheme (PRSS) Scholarship in 2017
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Thesis style
This thesis has been formatted in the style of the peer-reviewed journal Animal Production
Science.
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Disclosure and author contributions
Chapters Three and Six are published in a peer-reviewed journal. Chapters One – Part Two, and Five are to be submitted to a peer-reviewed journal.
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Chapter Three
I, Alicia Steel, was responsible for the study design, data collection, statistical analysis and the original preparation and editing of this manuscript.
Contributions to this manuscript made by all authors are as follows:
Task Author
Study design AS, RZA, CGG
Data collection AS, RZA
Statistical Analysis AS
Writing – original draft preparation AS
Writing – review and editing AS, RZA, CGG
As supervisor for the candidature upon which this thesis is based, I can confirm that the authorship attribution statements above are correct.
Signature: Date: 17/02/19
Assoc. Prof. Christopher Grupen
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Chapter Four
I, Alicia Steel, was responsible for the study design, data collection, statistical analysis and the original preparation and editing of this manuscript.
Contributions to this manuscript made by all authors are as follows:
Task Author
Study design AS, RZA, CGG
Data collection AS, RZA
Statistical Analysis AS
Writing – original draft preparation AS
Writing – review and editing AS, RZA, CGG
As supervisor for the candidature upon which this thesis is based, I can confirm that the authorship attribution statements above are correct.
Signature: Date: 17/02/19
Assoc. Prof. Christopher Grupen
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Chapter Five
I, Alicia Steel, was responsible for the study design, data collection, statistical analysis and the original preparation and editing of this manuscript.
Contributions to this manuscript made by all authors are as follows:
Task Author
Study design AS, RZA, CGG
Data collection AS, RZA
Statistical Analysis AS
Writing – original draft preparation AS
Writing – review and editing AS, RZA, CGG
As supervisor for the candidature upon which this thesis is based, I can confirm that the authorship attribution statements above are correct.
Signature: Date: 17/02/19
Assoc. Prof. Christopher Grupen
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Chapter Six
I, Alicia Steel, was responsible for the study design, data collection, statistical analysis and the original preparation and editing of this manuscript.
Contributions to this manuscript made by all authors are as follows:
Task Author
Study design AS, RZA, CGG
Data collection AS, RZA
Statistical Analysis AS
Writing – original draft preparation AS
Writing – review and editing AS, RZA, CGG
As supervisor for the candidature upon which this thesis is based, I can confirm that the authorship attribution statements above are correct.
Signature: Date: 17/02/19
Assoc. Prof. Christopher Grupen
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List of Abbreviations
ALK Activin receptor-like kinase
AFC Antral follicle count
AMH Anti-Müllerian hormone
AMH-N N-terminal homodimer of AMH
AMH-C C-terminal homodimer of AMH
AMH-N/C Non-covalent complex of AMH-C and AMH-N
AMHRII Anti-Müllerian hormone receptor type-II
BMP Bone morphenogenic protein cAMP Cyclic adenosine monophosphate
CA Corpus albicans
CL Corpora lutea
Co-Smads Coactivator Smad proteins
COC Cumulus-oocyte complex
CW Trimmed carcass weight
D Days of age
DNA Deoxyribonucleic acid
E Days of embryonic life
E2 Oestradiol
FSH Follicle stimulating hormone
FSHr Follicle stimulating hormone receptors
GDF Growth differentiation factor
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GnRH Gonadotropin releasing hormone
HPG Hypothalamic-pituitary-gonadal hCG Human chorionic gonadotropin
I-Smads Inhibitory Smad proteins
IVM In vitro maturation
IVP In vitro production
LD Long daylight-hours
LH Luteinising hormone
LHr Luteinising hormone receptors
MII Metaphase II mRNA messenger ribonucleic acid
NIP Not-in-pig
NPD Non-productive days
OFI Ovarian follicle index
OMI Oocyte maturation inhibitor
OPU Ovum pick-up
PC Princial component
PCA Principal component analysis
PCOS Polycystic ovary syndrome
PG Prostaglandin
PG600 Injectable solution made up of two parts PMSG and one part hCG
PMSG Pregnant mare serum gonadotropin
PP Postpartum
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PVN Paraventricular nucleus
R-Smads Pathway-regulated Smad proteins
RNA Ribonucleic acid
SD Short daylight-hours
SFI Surface follicle index
SM Secondary messengers
StAR Steroidogenic acute regulatory
TF Transcription factor
TGF Transforming growth factor
UMI Uterine mass index
WOI Weaning-to-oestrous interval
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List of tables
Table 1: Associations between serum anti-Müllerian hormone (AMH) levels and ovarian follicle populations and gonadotropin responsiveness in livestock...... 87
Table 2: Herd management conditions at the two farms ...... 95
Table 3: Dilutional linearity of two porcine serum samples containing varying AMH concentrations diluted with phosphate buffered saline...... 100
Table 4: Descriptive statistics for reproduction and culling parameters measured from parity one to three (mean ± SD, where applicable) ...... 105
Table 5: Reasons for culling according to parity number ...... 106
Table 6: Correlation coefficient matrix comparing parameters within parities ...... 107
Table 7: Correlation coefficient matrix comparing inter-parity parameters ...... 108
Table 8: Comparison between models fitted using E2 levels at 0, 2 or 4 days after PG600 injection to predict the number of piglets born alive...... 109
Table 9: Summary of serum concentrations of AMH and E2 in gilts at 80 days of age and carcass, uterine and ovarian traits at 160 days of age ...... 126
Table 10: Pearson’s correlation coefficients between ovarian hormones at 80 days of age and uterine and ovarian traits at 160 days of age...... 127
Table 11: Pearson’s correlation coefficients between uterine traits at 160 days of age...... 127
Table 12: Pearson’s correlation coefficients between surface antral follicle counts at 160 days of age...... 128
Table 13: Summary of mating, litter and culling data from parities one to three ...... 146
Table 14: Reasons for culling according to parity number ...... 147
Table 15: Relationships between serum E2 concentration at 80 days of age and litter size (total born) for each parity...... 149
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Table 16: Descriptive statistics for serum concentrations of AMH and E2 in gilts at 80 and 160 days of age and carcass, uterine and ovarian properties at 160 days of age ...... 163
Table 17 Pearson’s correlation coefficients between ovarian hormones at 80 and 160 days of age (D80 and D160, respectively) and uterine and ovarian traits at D160...... 164
Table 18: Pearson’s correlation coefficients between ovarian hormones at 80 and 160 days of age (D80 and D160, respective)...... 165
Table 19: Pearson’s correlation coefficients between uterine traits at 160 days of age...... 165
Table 20: Pearson’s correlation coefficients between ovarian follicle populations at 160 days of age...... 168
Table 21: Table of variable loadings from the principal component analysis ...... 170
Table 22: Proposed traits according to AMH and E2 hormone profiles at 80 days of age and the recommended selection preference ranking...... 190
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List of Figures
Figure 1: Three different oestradiol (E2) signalling pathways...... 55
Figure 2: Anti-Müllerian hormone (AMH) signalling pathway...... 67
Figure 3: Changes in plasma anti-Müllerian hormone (AMH) according to age ...... 74
Figure 4: Parallelism between dilution curves of two samples of porcine serum and the standard curve ...... 101
Figure 5: A) Serum anti-Müllerian hormone (AMH) and B) serum oestradiol (E2) levels in 60,
80 and 100-day old gilts, 0, 2 and 4 days after PG600 injection ...... 104
Figure 6: Serum oestradiol (E2) and number of piglets born alive per litter...... 108
Figure 7: Serum oestradiol (E2) and the probability of stillbirth...... 110
Figure 8: Serum anti-Müllerian hormone (AMH) and gestation length...... 111
Figure 9: Principal component analysis for uterine traits...... 129
Figure 10: Principal component analysis for surface antral follicle counts...... 131
Figure 11: Three-dimensional spline model showing the association between serum E2 concentration and serum anti-Müllerian hormone (AMH) concentration at 80 days of age (D80) and D160 UMIshape ...... 132
Figure 12: Associations between serum oestradiol (E2) concentration at 80 days of age and litter size (total born) in parities one to three...... 148
Figure 13: Principal component analysis for uterine traits...... 167
Figure 14: Principal component analysis for ovarian follicle counts...... 169
Figure 15: The relationship between serum oestradiol (E2) levels at 80 days of age (D80) and
Uterine Mass Index (UMI)size in gilts that were cycling at D160 ...... 170
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Figure 16: Smoothing spline showing the relationship between serum oestradiol (E2) levels at
80 days of age (D80) and Ovarian Follicle Index (OFI)prop values in non-cycling gilts at D160
...... 171
Figure 17: Three-dimensional smoothing spline model showing the (a) two-way interaction effect between serum anti-Mullerian hormone (AMH) levels at 80 (D80) and 160 days of age
(D160) on OFItot in non-cycling gilts at D160 ...... 172
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CHAPTER ONE: LITERATURE REVIEW
Preface
Part One of the literature review includes details of general pig reproductive management and physiology. Part Two of the literature review is presented as a separate manuscript submitted for publication and focuses on the role of AMH in reproductive processes.
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LITERATURE REVIEW: PART ONE
Introduction
Poor gilt retention within pig breeding herds is a common reproductive inefficiency amongst piggeries. In Australia, it is estimated that around 27% of replacement gilts are culled from Australian breeding herds prior to parity one and around 40% are culled prior to parity three (Serenius et al., 2006; Hughes et al., 2010; Plush et al., 2016). This premature culling has resulted in an average herd parity of just 2.7 (Plush et al., 2016). This is concerning considering a gilt only becomes profitable between parity three to six (Stalder et al., 2004). A lower herd age distribution severely impacts productivity as younger females tend to have lower pregnancy rates, smaller litter sizes, higher chances of savaging and greater non-productive days (D'Allaire and Drolet, 1999). Further loss is incurred due to the higher rearing costs involved per weaner pig bred from young females. It has been reported that costs per weaner are highest in a sow’s first litter and then decrease over the subsequent two litters (Kroes and
Van Male, 1979). Increasing the average herd parity by a single parity has been shown to be equivalent to a 0.5% increase in lean pork percentage at slaughter (Stalder et al., 2004). Thus, it would be in the best economic interest of producers to increase gilt retention rates.
It has been known for some time that the most significant factor contributing to the premature culling of replacement breeding females in Australian swine production systems is reproductive failure (Hughes et al., 2010). However, teat number, body conformation and dam performance are typically the only reproductive traits considered when gilts are selected to enter the breeding herd. Selection normally occurs at around 150 days of age and it is not until after this that a gilt’s reproductive potential becomes more evident. Maintaining unproductive
Page 30 of 213 females up to this point is costly. Hence, an early-age predictive marker for reproductive potential is required to aid with the selection process.
It is well known that ovarian characteristics at a young age, such as follicular populations and gonadotropin responsiveness, are linked with future fertility in mammalian females
(reviewed by Monget et al. (2012)). Previously, these parameters have been difficult to measure without performing an ultrasound and/or ovariectomy. In recent decades, numerous studies have investigated the relationship between circulating ovarian hormone concentrations and ovarian properties. Anti-Müllerian hormone (AMH) has been of particular interest as it has been identified as a highly dependable endocrine indicator of large follicular reserve and growing follicle populations (Humans: reviewed by Visser and Themmen (2005), Cattle: Rico et al. (2009), Sheep: Campbell et al. (2012), Mice: Kevenaar et al. (2006) Mares: Claes (2014)).
The hormone has also proved to be a good measure of gonadotropin responsiveness (Cattle:
Souza et al. (2015), Sheep: Campbell et al. (2012); Lahoz et al. (2012)). Furthermore, recent findings also unveiled that AMH may be the most reliable predictor for antral follicle populations (Cattle: Ireland et al. (2011); Monniaux et al. (2012); Batista et al. (2014)). Anti-
Müllerian hormone as an endocrine indicator for ovarian properties associated with reproductive potential is yet to be examined in female pigs (Di Clemente et al., 1994a;
McCoard et al., 2002; Almeida et al., 2012). Oestradiol (E2) is another hormone that has been used as a marker for gonadotropin responsiveness within the ovary at a young age. Ovaries from domestic gilts become responsive to gonadotropin stimulation from about 60 days of age, as indicated by the presence of tertiary follicles (McCoard et al., 2003). After this point, follicles are able to differentiate further in response to gonadotropins which results in an increase in the production of E2.
This project examined the relationship between circulating levels of AMH and E2 in juvenile gilts and reproductive characteristics including ovarian reserve, gonadotropin
Page 31 of 213 responsiveness, puberty attainment, uterine capacity, mating outcomes and litter characteristics. This review will provide a summary of the current gilt selection processes and management strategies for replacing gilts, the reproductive development of the gilt, particularly, folliculogenesis and endocrinology in the period leading up to the first ovulation, as well as an introduction to the hormones AMH and E2, discussing their synthesis, release, receptors and role in reproductive processes. The potential of AMH and E2 to act as markers of future reproductive success in young gilts will also be discussed.
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Management of female pigs
Gilt selection
The profitability of a pork production system is dependant on its ability to select gilts that are prolific and long-living within the breeding herd. The age at which gilt selection occurs is usually around 150 to 160 days and, traditionally, the selection process is quite rudimentary.
Other than bodyweight, the only reproductive selection criteria considered are teat number, teat and vulva conformation and performance of the dam. The number, size and placement of functional teats on a gilt is crucial for providing adequate nutrition for piglets and reducing the competition for teats, which results in reduced pre-weaning mortality (Andersen et al., 2011b).
It is recommended that selected gilts have ≥7 evenly spaced teats on each side and that gilts with blind, small or introverted teats be culled. The development and shape of the vulva are also important as infantile or small vulvas are indicative of underdeveloped reproductive tracts which may lead to birthing difficulties at farrowing. Vulvas with injury or a ‘fish hook’ appearance have an increased chance of infection and are also selected against. Along with teat number and vulva conformation, the performance of the dam is also considered and are traditionally the only reproductive parameters assessed at selection.
It is clear that this basic gilt selection process is inadequate as the average Australian piggery has low gilt retention rates beyond parity three, high sow replacement rates and a low average herd parity number (Plush et al., 2016). Identifying this, Plush et al. (2016) conducted a review of the Australian pork industry, comparing the management practices of some of the top-performing Australian piggeries to those that are underperforming to identify factors that contribute to low gilt retention rates. It was found that Australian farms are commonly observing reproductive failure of gilts and young sows and that inconsistencies in both first- mating and culling strategies used between farms were identified as main contributing factors.
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Timing of first mating
Although the average age at first service reported by Australian producers is within accepted targets, there is great variability in the age at first mating between farms, with many timing the first mating outside the recommended age limits (Plush et al., 2016). Differences proposed include farm-specific variability in other selection criteria, such as weight, backfat depth or oestrus number, which also affect the timing of mating. Presence of such confounding factors makes determining an optimal mating strategy complex, particularly with inconsistent findings and recommendations from previous literature. It has been documented that early- maturing gilts have greater reproductive output if bred at or after their second oestrus (Nelson et al., 1990), as delayed breeding of precocial gilts results in greater numbers of ovulated follicles and increased first litter size compared with breeding at their first oestrus (Kirkwood and Thacker, 1992; Holder et al., 1995). A number of studies suggest increased backfat depth and body weight contribute to the improved performance observed from delayed breeding
(reviewed by Kirkwood and Thacker (1992); Stalder et al. (2004)). For example, Young et al.
(1990) showed that breeding gilts at first oestrus, if they are at least 110 kg in body weight, results in equal reproductive output to gilts bred at second or third oestrus. Also, a significant increase in the number of piglets produced per lifetime was achieved when gilts were first mated with a backfat of >18mm compared with <16mm. However, these improvements were only documented in gilts that were on the leaner end of the normal curve in the aforementioned studies (Kirkwood and Thacker, 1992; Stalder et al., 2004). From this, it appears that there is indeed a minimum body condition threshold for selecting gilts, but otherwise, body weight and backfat has minimal effects on gilt performance and longevity. This is supported by three large- scale, retrospective studies showing there were no negative effects of mating gilts at a younger
Page 34 of 213 age but did show negative effects for mating gilts at older ages, which aligns with studies that show reduced fertility in gilts that reach sexual maturity late (Schukken et al., 1994; Culbertson and Mabry, 1995; Le Cozler et al., 1998). Similarly, Plush et al. (2016) showed a negative quadratic relationship between gilt age at first service and retention to parity three where reduced retention rates were observed if mated at <210 or >240 days of age. More emphasis on mating gilts within these ages could improve gilt retention, which would be assisted by the selection of gilts that reach sexual maturity and minimum weight and backfat requirements early.
Culling
The number one reason for the early culling of sows is reproductive failure (Hughes et al., 2010). This commonly includes the failure of first oestrus detection, post-pubertal anoestrus
(staleness), post-weaning anoestrus, return to service and failure to farrow. Gilts and young sows appear to be the most vulnerable due to reproductive inadequacy (D'Allaire and Drolet,
1999). However, the culling strategies between Australian farms appear to differ greatly with culling due to reproductive performance ranging from 0 to 50% and culling for not-in-pig or return to oestrus reasons ranging from 0 to 36% (Plush et al., 2016). This indicates that some farms do not cull according to reproductive performance and others cull vigilantly for reproductive shortcomings. In order to improve gilt retention and replacement, there is a need for more stringent selection strategies against gilts that show reduced reproductive performance.
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Measures of poor reproductive performance
Delayed onset of oestrus
The time at which gilts reach sexual maturity largely influences the timing of first mating, which in turn affects conception rates, farrowing rates and litter size. Ultimately, greater breeding herd entry-to-mating lengths result in greater costs per piglet produced. Gilts that reach puberty late are not only more costly to maintain, but also appear to have reduced fertility. Late maturing gilts have less regular cycling patterns, fewer litters, reduced litter sizes
(Nelson et al., 1990) and a greater rate of premature culling than early maturing gilts (Patterson et al., 2010). Moreover, progeny from late maturing gilts appear to have reduced growth rates and backfat depth (Nelson et al., 1990). Further studies have shown that when selected for early puberty, gilts show similar ovulation rates, litter sizes, birth weights, weaning weights, number of piglets weaned and lactation weight loss, and have improved percentage farrowing and improved weaning to oestrus intervals when compared to gilts that were not selected for early puberty Holder et al., 1995; Sterning et al., 1998; Patterson et al., 2010). This suggests that selecting early maturing gilts would improve reproductive output and reduce the number of non-productive days (NPD).
Reduced farrowing rate
Reduced farrowing rates also contribute to a greater number of NPD, increasing the costs per piglet produced and reducing the number of piglets produced per sow per year. The average farrowing rate in Australia is low, at just under 85% (Plush et al., 2016). Poor farrowing
Page 36 of 213 rates are attributed to females that fail to conceive and those that experience pregnancy loss. In
Australian herds, conception rates are commonly below the accepted target of 90% and the rate of culling due to pregnancy loss is about 14% (Plush et al., 2016). Factors affecting conception rates and pregnancy loss include the ability to detect heat accurately, age at first heat, mating strategies, oocyte quality, and ovulation interval. This puts further emphasis on the requirement for selecting gilts that display an early onset of oestrus.
Prolonged weaning to oestrus interval
Long weaning-to-oestrus intervals (WOI) are another source of economic loss in pork production systems. Prolonged WOIs also tend to result in a decreased duration of oestrus, leading to a reduced fertility window and reduced pregnancy rates and litter sizes due to incorrect timing of mating/insemination (Kemp and Soede, 1996). Long WOIs can be alleviated via the use of exogenous hormones, most commonly PG600 injection (2:1 pregnant mare serum gonadotropin (PMSG): human chorionic gonadotropin (hCG)) at weaning.
However, this treatment is costly and may result in reduced farrowing rate and litter sizes. In addition, the use of PG600 on the seventh day post-weaning in problem-sows is less effective in stimulating oestrus (reviewed by Plush et al. (2016)). Furthermore, long WOIs are exacerbated by season and age. Delayed oestrus or anoestrus after weaning occurs at a greater rate in sows at their first parity, particularly in the summer months during the seasonal infertility period (Kirkwood, 2003).
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Sow reproductive physiology
The Oestrous cycle
The first oestrus is usually detected by visual observation of physical and behavioural changes in the gilt, specifically swelling and reddening of the vulva and the standing reflex upon backpressure application. This may be coupled with attentive ears, increased vocalisation, arched posture, decreased appetite and an increased desire for physical contact. The porcine oestrus cycle is about 21 days in length (generally varying from 18 to 24 days) and can be broken down into a luteal phase and a follicular phase (Mariscal et al., 1998; Rátky and
Brüssow, 1998). Changes that occur within the reproductive system throughout the oestrous cycle are a result of alterations to the balance of chemical messengers, in particular, the gonadotropins follicle stimulating hormone (FSH) and leutinising hormone (LH) and the steroids progesterone and E2. Though both FSH and LH communicate via structurally similar receptors, using virtually identical cyclic adenosine monophosphate (cAMP) pathways, they have different functions. Early in the follicular phase of the oestrous cycle, increased FSH elicits proliferation of granulosa cells within follicles in the preantral stages via manipulation of cell regulatory genes. Whereas, pulsatile LH evokes terminal differentiation of granulosa cells, growth of advanced follicles to the point of exit from the cell cycle through action on genes responsible for ovulation and luteinisation (Conti, 2002).
A 21-day porcine oestrous cycle starts at ovulation (Day 0). This starts the luteal phase, a stage that lasts around 15 to 16 days. During this stage, CL are formed from ruptured post- ovulatory follicles which synthesise and secrete progesterone. Secretion of progesterone increases between Days 5-7 until reaching a plateau. Early in the luteal phase, the growing
Page 38 of 213 follicular pool is populated with roughly 40-60 follicles, the majority of which are small antral follicles, <3 mm in diameter (reviewed by Schwarz et al. (2008). Growing follicle populations increase to around 80 in the mid-luteal phase with the majority of them (~63%) still small and the rest being medium in size (3-6.9 mm). If pregnancy does not occur, luteolysis causes the
CL to degenerate into a corpus albicans (CA), resulting in a rapid decline in progesterone levels
(Rátky and Brüssow, 1998). No large antral follicles (>7 mm) are present in the luteal phase due to the negative-feedback mediated by progesterone (reviewed by Schwarz et al. (2008)).
At the start of the follicular phase (Day 15-16), the ovary may contain 40-100 small and medium growing antral follicles. During the follicular phase, follicles are selected from the growing pool of medium-sized follicles to develop to the pre-ovulatory stage (>7 mm), under the control of LH. However, not all medium-sized follicles undergo selection and around
70% enter atresia (reviewed by Schwarz et al. (2008)). Granulosa cells of growing follicles produce E2 at an increasing rate until the preovulatory LH surge. After this, E2 levels drop and ovulation occurs within 44-48 hours from the beginning of the LH surge, marking the beginning of another cycle and luteal phase.
Establishment and maintenance of pregnancy
The initial steps required for the establishment of pregnancy include maintaining the uterus in a quiescent state and preventing luteolysis through the interaction between the embryos and the uterine epithelium (Wojciechowicz et al., 2016). In mammals, progesterone is responsible for maintaining uterine quiescence, promoting uterine histotroph secretion and suppressing uterine immune function during early pregnancy (Fomin et al., 1999; Spencer and
Bazer, 2002; Arck et al., 2007). Within one week after mating, suppression of the progesterone receptor within the luminal epithelium and glandular epithelium (but not the endometrial stoma
Page 39 of 213 or myometrium) occurs and appears vital for pregnancy establishment. The downregulation of the progesterone receptors within the first week of pregnancy appears to be induced by progesterone itself, which steadily increases after ovulation (Mathew et al., 2010). In sows, progesterone receptor downregulation results in a reduction in mucin-1 within the luminal epithelium, which is thought to facilitate embryonic attachment (Bowen et al., 1996).
Communication between the conceptus and endometrium elicits the maternal recognition of pregnancy. In pigs, this predominantly involves the production of oestrogens (Geisert et al.,
1990), and to a lesser extent the production of interferons and interleukin-1β, by the conceptus
(Cencič and La Bonnardière, 2002; Ross et al., 2003). The porcine embryo starts producing oestrogens, mainly E2, on Days 11 and 12 of gestation (Geisert et al., 1982; Bazer et al., 1994).
Oestradiol causes changes to the capillary walls of the uterus, including poration of the face directly underneath the uterine epithelium, and the formation of caveolae in the opposite face, and discontinuous, multilayered basal laminae (Keys and King, 1995). As a result, the site of secretion of prostaglandin (PG)-F2α changes from the uterine vasculature to the uterine wall, where it is directed away from the CL (Bazer and Thatcher, 1977; Frank et al., 1977; Spencer et al., 2004). Both the endometrium and the myometrium express a unique set of genes on Days
12 and 13 of gestation in response to embryonic signalling (Wojciechowicz et al., 2016). These genes are regulated similarly within both tissues, indicating that both tissues are involved in pregnancy recognition and embryonic attachment in pigs. The endometrium supports pregnancy by providing enzymes, transport proteins, neuropeptides, growth factors, chemokines and cytokines (Bazer et al., 2012; Okrasa et al., 2014). In the pig, both the endometrium and myometrium produce PGs and steroid hormones (Akinlosotu et al., 1986;
Franczak et al., 2014).
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Development of the female reproductive system
Foetal development: The ovary and oogenesis
Ovarian development commences shortly after conception, just after blastocyst elongation. It starts with thickening of the mesothelial layer in the peritoneum, coupled with the aggregation of primordial germ cells that migrate from the yolk sac epithelium and through the gut mesentery from as early as E17 (E= days of embryonic/foetal life). By about E24-E25, this eventuates into a protrusion called the gonadal ridge, the precursor structure of the gonads
(Black and Erickson, 1968; Kanitz et al., 2001). In the pig, this structure is morphologically similar in both males and females until E26 when differentiation of the gonadal ridge occurs, becoming the ovary in females and the testes in males (Pelliniemi et al., 1979). In the female, germ cells are now referred to as oogonia and tend to cluster to form egg nests as part of the newly forming ovary (Picton, 2000). The oogonia undergo a finite number of mitotic divisions at a high frequency throughout gestation, up until shortly after birth. Previous studies have shown that at E20, the number of germ cells within the gilt foetus is about 5,000 and peaks at around 0.9 to 1.4 million, varying with breed (Erickson, 1967; Black and Erickson, 1968; Wise et al., 2001). The time at which the peak has been reported to occur varies considerably between studies (E50: Black and Erickson (1968); E90: McCoard et al. (2003); E105: (Wise et al.,
2001)). Some of the variation may be due to the differences in breeds and genetic sources between studies.
The peak germ cell number experienced prior to birth represents the maximum number of germ cells available throughout the lifetime of the gilt. After this point, mitotic division decreases and necrosis increases. The maximal number of germ cells in pigs is exceeded by
Page 41 of 213 that of cows (2.7m) and humans (7.0 m) but is much higher than that of rodents (50,000-75,000) and sheep (900,000) (reviewed by van den Hurk and Zhao (2005)). The rate of germ cell degeneration up until birth in rodents, sheep, cows and humans is much greater than that in pigs. Germ cell loss occurs at rates of 95% in heifers, 91% in ewe lambs, 90% in humans and
80% in rodents, but a rate of only 58% is seen in gilts (reviewed by van den Hurk and Zhao
(2005)). This results in oocyte numbers at birth being reduced to 700,000 in humans, 500,000 in pigs, 135,000 in cows, 82,000 in ewes and 10,000-15,000 in rodents (reviewed by van den
Hurk and Zhao (2005)). Germ cell degeneration continues after birth such that pigs have less than 30% of their maximal number (~300,000) by 25 days postpartum (PP) (Black and
Erickson, 1968; McCoard et al., 2003).
Folliculogenesis
At E50, gene expression for FSH and LH in the anterior pituitary first occurs (Ma et al., 1996). This coincides with the time when some oogonia commence meiosis, progressing from prophase through the preleptotene, leptotene, zygotene and pachytene stages, to finally arrest at the diplotene stage as primary oocytes (Black and Erickson, 1968). The transformation from oogonia to oocytes continues after birth, such that around 99% of oogonia will have made this change by 20 days PP (Black and Erickson, 1968). Upon reaching this final stage of prophase, oocytes become encapsulated individually by a single layer of 4 to 8 mesenchymal cells, surrounded by a thin basement membrane. These mesenchymal cells then differentiate into flattened granulosa cells, forming primordial follicles (Byskov et al., 1977; Fair, 2003;
Kezele and Skinner, 2003). Any oocytes that are not enclosed undergo apoptosis. Primordial follicles can first be seen at E68 and makeup around 80% of ovarian follicles from E95 up until around 90 days PP (Oxender et al., 1979). Porcine primordial follicles are quite small,
Page 42 of 213 averaging around 25.0 ± 8.2 μm in diameter, which is similar to that in sheep (36.9 ± 9.9 μm) and cattle (30.0 ± 7.6 μm) (Warren et al., 2015).
Follicles remain static in the primordial stage until stimulated to continue development in a process called recruitment. Once a primordial follicle is recruited it becomes part of the growing follicle pool as a primary follicle. Activation of the primordial follicles is marked by the proliferation of granulosa cells, the transformation of these cells from flat to cuboidal in shape, and an increase in the diameter of the oocytes enveloped within (Baker and Franchi,
1967). Although oocytes grow in diameter and volume, they do not resume meiotic development until ovulation is induced by the LH-surge (Black and Erickson, 1968). In pigs, recruitment of primordial follicles commences prenatally, with the first primary follicles observed at around E75, and continues throughout life or until the primordial store is depleted
(Oxender et al., 1979; McCoard et al., 2003). The transition of follicles from the primordial to primary stages varies in length and appears to take anywhere from a few days to a few decades in mammals (Hirshfield, 1991). The number of follicles in the recruitment stage at any given time is proportional to the population of primordial follicles in store, which decreases with age
(Erickson, 1967; Webb et al., 1999). Hence, the population of growing follicles is often used as an indicator of the size of the oocyte reservoir, commonly known as the follicular reserve.
The recruitment process is an irreversible one and once follicles enter this stage they do not stop growing until they undergo atresia or ovulation (Kezele and Skinner, 2003). Porcine primary follicles are very similar in diameter to those of sheep and cattle (Pig: 67.4 ± 13.2 μm;
Sheep: 66.0 ± 10.0 μm, Cattle: 68.4 ± 8.4 μm) (Warren et al., 2015).
Recruited follicles that continue growing develop into secondary (or preantral) follicles with granulosa cells proliferating to form two or more layers. In pigs, secondary follicles are occasionally observed from E90 (McCoard et al., 2003) but are more regularly observed at birth (Mauleon, 1964; Oxender et al., 1979; McCoard et al., 2003). After birth, the proportion
Page 43 of 213 of secondary follicles slowly increases and accounts for around 30% of all ovarian follicles present at around 90 days of age (Oxender et al., 1979). It is during the secondary follicle stages that ribonucleic acid (RNA) synthesis is first detected in the oocyte. Also during this stage, the oocyte manufactures a membrane, the zona pellucida, from three zona pellucida glycoproteins,
ZP1, ZP2 and ZP3. Moreover, in response to FSH stimulation, granulosa cells form protrusions called gap-junctions between each other and the oocyte (van den Hurk et al., 1997). Gap junctions from the granulosa cells penetrate the zona, abutting the oocyte cell membrane, providing a means of bi-directional communication. Porcine secondary follicles are similar in diameter to bovine secondary follicles (Pigs: 141.3 ± 18.8 μm; Cattle: 112.0 ± 19.9 μm) but are significantly larger than that of sheep (98.6 ± 16.0 μm) (Warren et al., 2015).
Follicles develop a fluid-filled cavity (antrum) and are now referred to as tertiary or antral follicles. Circumferential cells differentiate into two layers of theca cells, the theca externa and the theca interna (Driancourt, 1991). Further granulosa cell proliferation occurs, and the cells which surround the oocyte differentiate into cumulus cells, forming a cumulus- oocyte-complex (COC). The cells that line the inside of the developing follicle differentiate into stratum granulosa. The time at which tertiary follicles first become apparent in porcine ovaries varies with breed. In the highly prolific Meishan breed, tertiary follicles can be observed as early as 25 days PP (McCoard et al., 2003). In white composite breeds, tertiary follicles appear around 60 days PP (Mauleon, 1964; Oxender et al., 1979; McCoard et al.,
2003), but are present at low numbers, slowly increasing such that by 90 days of age they account for around 2-3% of the follicular population (Oxender et al., 1979). Tertiary follicles are sensitive to gonadotropins, thus, the presence of such is an indicator that ovaries are now more responsive to gonadotropin stimulation (reviewed by Schwarz et al. (2008)). Further development of antral follicles into preovulatory follicles is dependent on gonadotropin stimulation. However, prior to sexual maturation, gonadotropin signalling is inadequate as the
Page 44 of 213 hypothalamic-pituitary-gonadal endocrine axis (HPG) is not fully established (discussed in
Section 4 of this review). Thus, all recruited follicles become atretic until puberty.
Upon the establishment of the HPG, some of the growing follicles avoid the fate of atresia and undergo selection. This is a process in which 6 mm antral follicles develop into dominant or preovulatory follicles 7-8 mm in diameter. The granulosa cells within these follicles produce E2 and inhibin (reviewed by Fortune et al. (2001)), both of which have negative-feedback on FSH secretion by the anterior pituitary. The decrease in FSH inhibits the growth of smaller follicles, preventing more from entering the selection stage (Ireland, 1987;
Hunter et al., 2004). Simultaneously, the granulosa cells of dominant follicles begin expressing
LH receptors and inferior follicles become atretic (Campbell et al., 1995; Webb et al., 2003).
Now responding to LH, the granulosa cells of dominant follicles differentiate further in preparation for ovulation and luteinisation (Robker and Richards, 1998).
Ovulation
Once the preovulatory LH surge occurs, meiosis resumes in oocytes of selected follicles and ovulation follows. In domestic gilts, the first ovulations commonly occur between 130 to
220 days of age, marking sexual maturity (Rátky and Brüssow, 1998). Gilts usually ovulate around 12 to 18 oocytes, whereas sows may release anywhere from around 15 to 30 oocytes in one cycle (Rátky and Brüssow, 1998; Soede et al., 2011). The ruptured follicles develop into structures called CL which begin secreting progesterone in preparation for pregnancy. If no pregnancy occurs, the CLs degrade via the action of macrophages and are infiltrated by collagen produced by fibroblasts, resulting in whitening structures referred to as corpora albicantia. A new oestrous cycle will commence.
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Oocyte maturation
Oocytes remain in the diplotene stage of meiotic prophase until the encapsulating follicle undergoes selection and is stimulated by the preovulatory LH surge. Oocytes enter a growth period in preparation for meiotic resumption. During the growth phase, porcine oocytes will grow from around 40 µm to 120µm and many cytoplasmic changes, including the manufacture, edition or redistribution of organelles and proteins occur. High levels of RNA synthesis and increases in mitochondria and ribosome populations are seen within growing oocytes. Molecule transport across the oocyte membrane increases along with nutrient storage, as glycogen granules, protein, lipid droplets, membrane-bound vesicles and multi-vesicular bodies increase. Cytoplasmic maturation not only aids in the growth, development and maturation of the oocyte, it is also vital in protecting against polyspermy and for facilitating the formation of pronuclei at fertilisation (van den Hurk et al., 1997; Picton and Gosden, 1998;
Kanitz et al., 2001).
Up until fertilisation, the oocyte is maintained within the cumulus matrix, which is important for providing nutrients to the oocyte, aiding oocyte expulsion at ovulation and oocyte transport through the oviduct and providing a selective barrier for good quality sperm (van den
Hurk and Zhao, 2005). Along with indirect signalling, the cumulus cell matrix communicates directly with the oocyte via gap junctions that protrude through the zona, abutting the oocyte membrane. These gap junctions become disrupted in response to the LH surge, gradually releasing the oocyte from the cumulus cell matrix. This process is widely referred to as cumulus cell expansion, and involves the release of hyaluronan from cumulus cells and, in combination with intrafollicular and serum proteins, causes the cumulus cell matrix to become muco-elastic.
The degree of cumulus cell expansion that has occurred is often used as an indicator of oocyte
Page 46 of 213 maturity and quality (Nevoral et al., 2014). Together with cumulus cell expansion, the LH surge induces the resumption of meiosis. The oocyte continues nuclear maturation by undergoing germinal vesicle break down, and progressing through the metaphase-I, anaphase-I and telophase-I stages to the metaphase-II (MII) stage, where the first polar body is extruded. It is at this stage that the oocyte again arrests meiosis until fertilisation. Nuclear maturation from diplotene to MII takes around 44 h in pigs. The signalling factors responsible for the regulation of oocyte maturation are discussed in later sections of this review.
The uterus
In the embryo, the Wolffian and Müllerian ducts are anlagen to the reproductive tract. In females, the Wolffian ducts gradually atrophy while the Müllerian ducts persist, eventually growing into the uterus, fallopian tubes and fusion of the two ducts form part of the vagina
(Josso et al., 1998). Uterine growth is independent of the ovary until around 60 days PP in gilts
(Wu and Dziuk, 1988). Up until 70 days PP, there is a gradual increase in uterine weight and horn length (Dyck and Swierstra, 1983) as well as uterine wall thickness and uterine gland development (Erices and Schnurrbusch, 1979). The uterine wall thickness and development of glands increase for another 2-3 weeks until puberty. Increases in the growth rate of uterine horn length and weight continue to around 170 days PP, such that significant increases are observed by the time the gilt displays first oestrus (Dyck and Swierstra, 1983).
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Endocrinology: from birth until puberty
Circulating gonadotropin levels in the prepubertal gilt
Typically, neonatal animals have elevated concentrations of FSH and LH. From birth, levels of FSH remain relatively stable or slightly increase up to 50 days of age (Dyck, 1988).
From 50 to 150 days of age, FSH levels decline, despite the first tertiary follicles appearing from 60 to 90 days of age. Levels then plateau until the first oestrus. In contrast, LH levels decline immediately after birth in gilts (Deligeorgis et al., 1985), rise again during the mid-to- late stages of puberty onset, and become stable once puberty is attained (Foxcroft et al., 1988).
Oestrogen levels rise in accordance with LH release at this stage of development (Lutz et al.,
1984; Prunier et al., 1993; Patterson, 2001).
Puberty: Establishment of the hypothalamic-pituitary-gonadal axis
Reproductive cycles are regulated by positive and negative-feedback systems of hormones between the hypothalamus, pituitary and gonads. The hypothalamus is a part of the brain and consists of groups of nerve cell bodies. The clusters that directly influence reproduction are the paraventricular nucleus (PVN), the preoptic area (also known as the surge centre) and the arcuate nucleus (also known as the tonic centre) (Soede et al., 2011). Neurons in the surge and tonic regions are responsible for the production of gonadotropin-releasing hormone (GnRH) and, thus, are called GnRH neurons. As the nomenclature would suggest, the tonic centre is responsible for the basal levels of GnRH and the surge centre controls the high frequency, high amplitude pulsatile release of GnRH. These areas are connected to the pituitary
Page 48 of 213 by a capillary network which allows GnRH to stimulate the anterior pituitary causing the release of FSH and LH, which, via the circulation, act on the ovary to regulate follicular development and ovulation.
Prior to sexual maturation, low amplitude and low-frequency pulses of GnRH are released from the hypothalamus. The surge centre, though able to function at a young age if artificially stimulated, is relatively inactive prior to puberty. This is due to the two regions responding differently to E2. Oestradiol has a positive-feedback effect on the surge centre and a negative-feedback effect on the tonic centre. The surge centre is always highly sensitive to positive-feedback from E2 prior to birth. However, prepubertal ovaries are not able to produce sufficient levels of E2 for stimulation as the positive-feedback system of the surge centre is overridden by the high sensitivity of the tonic region to negative-feedback from E2. Puberty involves the reduction of the E2-sensitivity of the tonic region and the establishment of the positive-feedback loop within the surge centre (Andrews et al., 1981; Rapisarda et al., 1983).
The mechanisms by which this occurs are complex and largely unknown. However, research has identified several of the neural pathways involved, which are proposed to work in concert to initiate puberty (Herbison and Plant, 2018). The neural components of recent particular interest are those that produce kisspeptin. The neurons that express kisspeptin and their receptors are called Kiss1 neurons. There are Kiss1 populations in both the PVN and the tonic centre of the hypothalamus. There are many studies that suggest that the activation of Kiss1 neurons in the PVN may be involved in the development of the E2 positive-feedback system as they project into GnRH neurons located in the surge centre in response to E2 (Herbison and
Plant, 2018). The Kiss1 neurons within the tonic centre also project to GnRH neurons in the surge centre, but to a lesser extent (Herbison and Plant, 2018). However, tonic centre Kiss1 neurons negatively respond to E2, which causes the inhibition of Kiss1 gene expression in this cluster. In gilts, the development of these neural pathways, and therefore, puberty initiation,
Page 49 of 213 requires attainment of a threshold body size and composition and is impacted by certain environmental stimuli, such as stocking density or the presence of a boar (Mavrogenis and
Robison, 1976).
Once the positive-feedback system in the surge centre becomes more established,
GnRH secretion increases. This stimulates the anterior pituitary to produce LH and FSH which promote follicular growth in the ovary. As follicles mature, E2 production increases until levels are sufficient to stimulate the surge centre to produce rapid, high amplitude pulsatile release of
GnRH, resulting in the preovulatory LH surge. The health of dominant follicles is highly dependent on LH support and dysfunction of the surge or tonic centre results in ovulation failure. For example, reduced frequency in LH pulses has been shown to initiate atresia of dominant follicles (Savio et al., 1993; Bergfeld et al., 1996; Yuan et al., 1996).
Gonadotropin receptors
Most studies on the expression patterns of FSH receptor (FSHr) within the porcine ovary have focussed on pre-antral to antral follicles from peripubertal and mature females. The expression patterns within foetal pig ovaries are unknown. However, one study in newborn gilts reported FSHr expression in oocytes from oocyte nests, primordial follicles and primary follicles, suggesting that early folliculogenesis is influenced by FSH (Durlej et al., 2011). In mature female pigs, FSHr expression is high within the granulosa cells of follicles at the primary to medium antral stages (<3 mm), and decreases as follicles mature to the preovulatory stages (Yuan et al., 1996; Liu et al., 1998; Słomczyńska et al., 2001). Receptors for LH (LHr), on the other hand, are not expressed within primary follicles. Expression of LHr is restricted to theca cells within follicles at the preantral to medium antral stages. It is not until the late antral stages that LHr is expressed in granulosa cells (Yuan et al., 1996; Liu et al., 1998). The low
Page 50 of 213 expression of LHr within preantral follicles has been suggested to mark the commencement of
P4 synthesis, which then causes subsequent differentiation of theca interna cells. Within small antral follicles, the level of expression of both FSHr and LHr increases and results in increased synthesis of progesterone and E2. In late antral phases, LHr mediates the terminal differentiation of dominant follicles and further E2 release, leading to the LH-surge.
In the cycling female pig, the FSHr and LHr expression pattern directly reflect the period of follicular growth that is highly dependent on gonadotropin stimulation, that is, the antral and preovulatory stages. Follicular growth is primarily under FSH control before becoming reliant on LH stimulation. The changing dependence from FSH to LH is complete in large antral follicles (Yuan et al., 1996) and coincides with a decrease in circulating concentrations of FSH and an increase in the frequency of LH pulses (Flowers et al., 1991; Trout et al., 1992; Guthrie et al., 1995). The decline in FSHr observed in larger follicles is suggested to regulate the growth of subordinate follicles as larger follicles continue developing.
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Oestradiol (E2)
Synthesis and production
Oestrogens are particularly important for normal sexual and reproductive development and function (reviewed by Heldring et al. (2007)). In mammals, they elicit a large variety of biological processes in the cardiovascular, musculoskeletal, central nervous and immune systems. The 17β isomer of E2 is the most potent form of oestrogen and is the most prominent oestrogen produced in the ovary. It is an aromatized 18-carbon steroid hormone with a hydroxyl group in the 3β and 17β position and was first isolated by Edward Doisy and colleagues in
1936 from porcine ovaries and has been extensively studied since (Doisy, 1976).
Oestradiol is synthesised from cholesterol via numerous pathways. Within the ovary, ovarian steroid production is orchestrated by theca and granulosa cells and is dependant on LH and FSH. Theca cells are stimulated by LH and granulosa cells by FSH (and possibly LH) to produce the necessary enzymes for steroidogenesis (Raju et al., 2013). Lipoproteins in circulation and de novo biosynthesis gives rise to cholesterol in theca cells. Androgen production from cholesterol is facilitated by steroidogenic acute regulatory (StAR) protein.
Resulting androstenedione diffuses into granulosa cells and is converted into testosterone or estrone which both can be converted into E2 with aid of 17β-hydroxysteroid dehydrogenase and/or aromatase enzymes (Hillier et al., 1994; Raju et al., 2013).
Receptors and signalling
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Cellular signalling of oestrogens is mediated by two isoforms of nuclear protein oestrogen receptors (ER), ER-α and ER-β (Beazely and Watts, 2006). The receptors are located in numerous organs including the uterus, ovary, mammary glands, bones, heart, hypothalamus, pituitary gland, liver, lung, kidney and adipose tissue. Each ER consists of a central domain, a deoxyribonucleic acid (DNA)-binding domain and a ligand-binding region that is located at the C-terminus. There are two transcriptional activation sites, one at the N-terminus (AF1) and one at the C-terminal ligand-binding region (AF2). The most conserved region between the two ERs is the central domain, whereas there is high variability seen between the N-terminal regions. Despite this, both ERs have similar affinity for E2 and are able to bind to the same
DNA response components. E2-activated ER receptors can associate to form dimers, either
ER-α, ER-β homodimers or ER-αβ heterodimers. The activated E2-ER complexes translocate to the nucleus and bind to DNA, affecting transcription. The actions of E2 on transcription is cell-specific and dependent on the current transcriptional state of the cell and the type of cell it's acting on (Katzenellenbogen et al., 1996; Nilsson et al., 2001; Katzenellenbogen and
Katzenellenbogen, 2002). Unlike some other nuclear receptors, ERs ligand-binding cavity is quite large in comparison to E2, allowing it to have quite a non-specific binding site. This results in the receptor being susceptible to endocrine disruption.
There are different modes of estrogen receptor signalling including direct, tethered, and non-genomic (Figure 1; reviewed by Heldring et al. (2007)). Direct signalling involves the direct DNA binding of a ligand-ER complex to an estrogen response element for altering gene regulation. Tethered signalling is indirect and involves an intermediate protein interaction with the ligand-ER complex, which binds to the DNA. Non-genomic signalling is complex, has a rapid effect and is poorly understood. It involves either a classical ER in the cytoplasm or a classical ER, an isoform ER or a distinct receptor associated with the membrane. In response,
Page 53 of 213 signalling cascades are produced, involving secondary messengers (SM) that rapidly influence ion channels or increase cytoplasmic nitric oxide concentrations.
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Figure 1: Three different oestradiol (E2) signalling pathways. Direct signalling involves the direct DNA binding of a ligand-oestrogen receptor (ER) complex for altering gene regulation. Tethered signalling is indirect and involves an intermediate transcription factor (TF) interaction with the ligand-ER complex, which binds to the
DNA. Non-genomic signalling is complex, has a rapid effect and is poorly understood. It involves either a classical ER in the cytoplasm or a classical ER, an isoform ER or a distinct receptor associated with the membrane.
Signalling cascades are initiated in response, involving secondary messengers (SM) that rapidly influence ion channels or increase cytoplasmic nitric oxide concentrations.
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Receptor expression
Though ER-α has been found in the granulosa and theca cells of various species, including the pig (Billiar et al., 1992; Orimo et al., 1995; Tetsuka et al., 1998; Sar and Welsch,
1999; Slomczyńska et al., 2001), ER-β appears to have a more significant role in the development of ovarian follicles (Emmen et al., 2005). ER-β knockout mice experience a failure to ovulate or reduced litter sizes (Krege et al., 1998; Emmen et al., 2005). It is expressed in the granulosa, theca and luteal cells within the ovary in various species including rodents, pigs, cattle, sheep and humans (Kuiper et al., 1996; Byers et al., 1997; Enmark et al., 1997;
Fitzpatrick et al., 1999; Cardenas et al., 2001; Slomczyńska et al., 2001). In sows, ER-α may have a role within the ovary during pregnancy as ER-α is expressed in granulosa cells to a greater extent than ER-β at times throughout gestation (Knapczyk et al., 2008). During follicular development, the level of ER-β messenger RNA (mRNA) expression remains similar from the small to large antral follicle stages (1-10mm in diameter) and in the CL, but, reduces in the regressing CL within the cycling sow (LaVoie et al., 2002). The predominant ER in the uterus appears to be ER-α (reviewed by Srisuwatanasagul (2011)). After the initial development, ER-α plays an important part in the growth and maturation of the uterus. In general, the expression of ER is upregulated by oestrogens and down-regulated by progesterone
(Batra and Iosif, 1989; Ciesiółka et al., 2016). Concordantly, ER-α is expressed maximally in the sow uterus at a time when E2 levels peak during oestrus, before declining with the progesterone rise (Sukjumlong et al., 2003). However, E2 has also been shown to reduce the expression of ER-α mRNA in the endometrium of ovariectomised gilts (Sahlin et al., 1990).
Furthermore, in inseminated sows, uterine ER-α in the surface epithelium declines to lower levels 22h after ovulation compared with cyclic sows (Sukjumlong et al., 2003; Sukjumlong et
Page 56 of 213 al., 2004). This is thought to be a result of negative-feedback from oestrogens in boar semen
(Claus et al., 1987).
Circulating E2 in juveniles
Foetal oestrogen levels were found to be very high at 20 days of gestation in the pig
(Robertson et al., 1985). After this point, oestrogen levels decline until around 60 days of gestation and then increase towards the end of gestation to levels greater than at 20 days
(Robertson et al., 1985). After birth, circulating concentrations are quite low until around 20 days of age when E2 levels appear to spike upward. From 20 to 100 days of age, there is a rise in circulating E2 (Wise, 1982), coincident with a period when the first tertiary follicles form.
Stickney (1981) reported that increases in oestradiol occur from 150 to 210 days of age, around the time when gilts reach sexual maturity. This is likely due to gilts experiencing a rise in E2 six days before their first ovulation (Prunier et al., 1993). After puberty, female pigs experience the classical and cyclic rise and fall in circulating E2 according to the stage of their oestrus cycle, as discussed previously.
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Circulating E2 as a marker of reproductive potential
Puberty
It is clear that E2 has direct involvement in the establishment of puberty in mammals.
There are a few studies in species other than the pig that have investigated the differences in circulating E2 levels between females that experience precocial puberty and their normal counterparts. Girls displaying precocial puberty have circulating E2 levels that are three times that of normal levels in prepubescent females (Bidlingmaier et al., 1977). Similarly, precocial heifers (<300 days of age) generally have greater E2 levels compared to late maturing heifers, regardless of weaning or dietary treatment (Gasser et al., 2006). It should be noted that subjects in the aforementioned studies were compared according to calendar age, and one study that grouped girls according to their stage of sexual maturity showed that precocial girls appear to have lower E2 levels than normal-maturing girls at the same developmental stage
(Bidlingmaier et al., 1977). However, another study showed that when comparing precocial individuals only, girls displaying two or three signs of puberty had greater circulating levels of
E2 compared to those that only showed one sign of puberty (Giabicani et al., 2013). These results may reflect increased receptor sensitivity (Bidlingmaier et al., 1977) or different ER polymorphisms (Lee et al., 2013; Luo et al., 2015) in precocial individuals, but this is yet to be proven (Soares-Jr et al., 2018).
Gonadotropin responsiveness and fertility
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There is great variation in how females respond to gonadotropin stimulation, and circulating hormone response profiles have been linked with reproductive potential. The association between circulating E2 levels and gonadotropin responsiveness and reproductive outcomes has mainly been studied in women undergoing assisted reproductive treatment for infertility, with only a few studies investigating this in livestock. The relationship between E2 levels and reproductive output appears to vary with the time of measurement within the stimulation cycle. In women, lower circulating E2 levels three days after ovarian stimulation are predictive of increased gonadotropin response, a greater number of oocytes retrieved and higher pregnancy rates per cycle (Licciardi et al., 1995; Smotrich et al., 1995; Evers et al.,
1998). Higher E2 levels more than four days after stimulation have been linked with good follicular development in sheep (Valasi et al., 2007), improved pregnancy outcomes in women
(Pena et al., 2002; El Maghraby et al., 2009) and greater numbers of viable embryos in cattle
(Gradela et al., 1994). However, longitudinal studies in women showed that higher peak E2 levels during stimulation cycles were associated with adverse placental outcomes (Royster et al., 2016). Furthermore, not all females respond similarly to gonadotropin stimulation, resulting in different hormonal profiles of E2, that is, some women experience blood E2 concentrations that decline while others have levels that plateau or increase. Women who displayed a plateau in E2 were found to have fewer oocytes recovered than women with other E2 response profiles, and women who experienced a plateau or decrease in E2 had reduced ovarian reserve and implantation rates compared to those who had an increase in E2 (Kondapalli et al., 2012).
Further, resultant embryos from women with a declining E2 profile gave rise to lower live birth rates compared to embryos produced from women that exhibited a plateau or increase in circulating E2 (Laufer et al., 1986; Kondapalli et al., 2012).
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LITERATURE REVIEW: PART TWO
Anti-Müllerian hormone (AMH) as a marker for improving
livestock production
Introduction
Anti-Müllerian hormone is a glycosylated, cysteine-linked homodimer protein that is part of the transforming growth factor-beta (TGFβ) superfamily. It is mainly produced by granulosa cells within the ovary and has regulatory function, having inhibitory action on the rate of primordial follicle recruitment in rodents, humans and cows (Durlinger et al., 1999a; Gigli et al., 2005; Campbell et al., 2012; Yang et al., 2017) and on the transition of early antral follicles into the gonadotropin-dependant antral stages in sheep (Campbell et al., 2012). The AMH type-
II receptor (AMHRII) is expressed in the endometrium of adult rats and humans (Renaud et al., 2005), and in rats, the receptor is upregulated in the endometrium during pregnancy
(Renaud et al., 2005). Furthermore, transcripts for the AMRII have been found in the hypothalamus, motor and cortical neurons, and the pituitary glands in mice (Bédécarrats et al.,
2003; Wang et al., 2005; Lebeurrier et al., 2008). Thus, it is expected to have further regulatory effects on the HPG axis and uterus.
In humans, circulating AMH is a sensitive indicator of healthy follicle populations and, therefore, ovarian reserve (La Marca et al., 2006a). Plasma AMH is commonly measured in humans and in livestock to estimate reproductive potential for clinical or breeding applications
(reviewed by Ireland et al. (2011)). The potential for AMH to be used as a marker of
Page 60 of 213 reproductive performance in pigs will be discussed in this review, with particular focus on the variations in circulating levels in juvenile and adult animals, factors that contribute to different levels of circulating AMH, and its use as a marker for reproductive performance in livestock with some mention of humans and rodents.
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Anti-Müllerian hormone (AMH)
Discovery
In 1947, AMH was discovered as the factor responsible for the regression of the
Müllerian ducts in the male foetus. If absent in males, the Müllerian ducts persist and proceed to develop into female reproductive organs (reviewed by Josso et al. (1998)). The hormone was first thought to be produced solely by the Sertoli cells within testes (Jost, 1948). It was not until much later, while examining the foetal ovaries of fowls, that AMH was discovered to also be expressed in females (Hutson et al., 1981). Subsequently, studies in rats revealed the site of secretion to be the granulosa cells (Vigier et al., 1984). Since then, there have been numerous studies in various species unveiling the structure and function of AMH and the mechanisms by which it acts upon female reproductive processes.
The protein, it’s Synthesis and Activation
Anti-Müllerian hormone is part of the TGF-β superfamily and is synthesized as a large, glycosylated, cysteine-linked precursor homodimer protein, made up of two monomers known as pre-proAMH. Pre-pro-AMH possesses a cleavage site 109 amino acids away from the C- terminal. In pigs, this precursor form of AMH is 577-amino acids in length (GenBank
Accession No: NP_999475.2) and is coded by a gene located on chromosome two (Lahbib-
Mansais et al., 1997). Porcine pre-proAMH has high sequence similarity with the pre-proAMH of other species (80% in cattle, 79% in sheep, 78% in horses, 74% in humans, 69% in mice and
68% in rats) (GenBank Accession No: NP_776315.1, NP_001295528.1, AEA11205.1,
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NP_000470.2, NP_031471.2, NP_037034.1, respectively). The majority of the differences observed are seen at the N-terminal region and the aforementioned species all share ≥89% similarity with the pig at the C-terminal domain. The pre-proAMH peptide contains a signal sequence, which is 22 amino acids in length in the pig. The signal sequence is lost during synthesis, giving rise to proAMH.
Like many other members of the TGF- β family, proAMH must undergo proteolytic cleavage to become activated (Nachtigal and Ingraham, 1996). Cleavage of proAMH gives rise to an N-terminal homo-dimer (AMH-N) and a bioactive C-terminal homo-dimer (AMH-C).
After cleavage, AMH-C and AMH-N remain associated in a non-covalent complex (AMH-
N/C) (Pepinsky et al., 1988). Commonly, an association between the N- and C-termini after cleavage of TGF-β members results in inactivity and it is only when the mature C-terminus is released that they are able to bind with receptors (Gentry and Nash, 1990; Massague, 1990;
Roberts et al., 1990). However, this is not the case with AMH. Though AMH-C can elicit some response, it is much less efficient and bioactive than the AMH-N/C complex (Wilson et al.,
1993).
Generally, the amount of cleaved AMH available in the body is low. In calf testes, only a very small proportion of cleaved AMH has been detected (Pepinsky et al., 1988; di Clemente et al., 1994b), and in Chinese hamster ovary cells only 5 to 20% of isolated recombinant human
AMH was cleaved efficiently (Pepinsky et al., 1988). This is also the case in humans as the vast majority of AMH in human blood is also found to be in the unprocessed form (Pankhurst and McLennan, 2013). It has been proposed that the function of AMH may be regulated by cleavage within the target tissue. It is important to note that the occurrence of proAMH in the blood may potentially lead to biased diagnostic results as many commercially available assays do not discriminate between the two forms of AMH (Picard and Josso, 1984; Pepinsky et al.,
1988; Pankhurst and McLennan, 2013).
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The mechanisms by which proAMH is processed into the active AMH-C/N are poorly understood. Total processing has been achieved experimentally using the blood enzyme plasmin, though this has not been shown in vivo (Pepinsky et al., 1988; Wilson et al., 1993).
The protein PC5, and to a lesser extent furin, have been shown to successfully cleave AMH in transfected mammalian cells, and thus, are nominated as potential activation enzymes that may be responsible for cleavage in vivo (Nachtigal and Ingraham, 1996).
Receptors and Smad signalling pathways
Members of the TGF-β superfamily communicate by binding and assembling transmembrane type-I and type-II receptor serine/threonine kinases on the target cell (reviewed by Shi and Massagué (2003)). Activation occurs as the formation of this complex enables the type-II receptor to phosphorylate the kinase region of the type-I receptor. The receptor type-I and type-II serine/threonine kinases are roughly 500 amino acids in length and consist of an N- terminal extracellular binding region, a transmembrane region and a C-terminal serine/threonine kinase region (Manning et al., 2002). Activation of type-I receptors causes phosphorylation of signal transducer proteins called Smads. There are three types of Smad proteins, the pathway-regulated Smads (R-Smads), coactivator Smads (Co-Smads) and inhibitory Smads (I-Smads). R-Smads bind with activated type-I receptors, and once phosphorylated dissociate from the receptor complex and associate with Smad4, the only mammalian Co-Smad (Clarke et al., 2001). This facilitates translocation into the nucleus of the target cell and, with other factors, regulation of gene transcription (Massague and Wotton,
2000). I-Smads are inhibitory proteins and block signalling pathways by binding to active type-
I receptors or active R-Smads (Massague, 1998). The signalling pathways that I-Smads inhibit tend to also induce the production of the protein as I-Smad promotors are activated by R-
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Smad/Smad4 complexes, forming a negative-feedback system (Nagarajan et al., 1999; Ishida et al., 2000). Thus, the intensity and length of the intracellular response depend on the concentration and continual presence of the ligand for signalling (Nakao et al., 1997; Afrakhte et al., 1998; Hata et al., 1998).
The TGF-β superfamily is subdivided into two subfamilies according to their communication pathways and structure. These two groups include the TGF-β/Nodal/Activin subgroup and the bone morphogenetic protein (BMP)/growth differentiation factor
(GDF)/AMH subgroup. The interaction between the various ligands and their associated type-
I receptors appears to be quite similar for all the TGF-β family members. The majority of the variation between activation pathways is attributed to the type-II receptors and their relevant ligands, and this is the basis of how the ligands are sorted into their subfamilies (Gray et al.,
2000; Hart et al., 2002). All ligands of the subgroup that includes AMH activate the R-Smads-
1, -5 and -8 via the binding of type-I receptors (Gouedard et al., 2000; Clarke et al., 2001;
Visser et al., 2001).
There are three type-I receptors that are known to bind with AMH, activin receptor-like kinase (ALK)-2, ALK3 and ALK6. Both ALK2 and ALK3 are also capable of binding with
BMP (Figure 2) (Macias-Silva et al., 1998; Clarke et al., 2001; Visser et al., 2001; Jamin et al.,
2002; Orvis et al., 2008; di Clemente et al., 2010). The type-I receptor kinases are phosphorylated via AMHRII, which is unique as is the only TGF-β receptor that binds to one ligand (Imhoff et al., 2013). It has been suggested that a third unique binding interface may be involved in the AMH and AMHR-II interaction process as well (Greenwald et al., 2003).
Once activated, the AMH receptor complex goes on to activate (phosphorylate) the 1,
5 and 8 R-Smads. The R-Smads form heteromeric complexes with the Co-Smad, Smad4 and
Page 65 of 213 are translocated into the nucleus, along with other nuclear cofactors, to regulate the transcription of hundreds of target genes (reviewed by Shi and Massagué (2003)).
Along with inducing the Smad 1-5-8 pathway, AMH upregulates the production of I-
Smad 6 proteins and, to a lesser extent, I-Smad7 proteins (Clarke et al., 2001). These inhibitory proteins negatively feedback on TGF-β and BMP signalling by competing with the R-Smads for receptor-binding (or Co-Smad binding) and via the receptors that promote degradation
(reviewed by Shi and Massagué (2003) and Yan et al. (2009)).
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Figure 2: Anti-Müllerian hormone (AMH) signalling pathway. The AMH protein binds with AMH type-II receptor (AMHRII). Type-I activin receptor kinases, (ALK)-2, ALK3 or ALK6, are then phosphorylated via the activated AMHRII. A third unique binding interface may also be involved. The AMH receptor complex goes on to phosphorylate the 1, 5 and 8 R-Smads. The R-Smads form heteromeric complexes with the Co-Smad, Smad4 and are translocated into the nucleus, along with other nuclear cofactors, to regulate the transcription of target genes
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Receptor expression
The AMH-specific AMHRII receptor is expressed in the gonads of foetuses of both sexes during sexual differentiation (Rey et al., 2003). In adult rats, both AMH and AMHRII, are expressed within granulosa cells of growing follicles, with negligible expression in CL and large antral follicles and none in oocytes and primordial follicles (Baarends et al., 1995). In cattle, AMHRII expression decreases from small to large antral follicle stages (Poole et al.,
2016). Expression of AMHRII in the oocyte appears to be species-specific as it is expressed in mice oocytes, but not in rats or sheep (Zhang et al., 2014). Recently, AMHRII was discovered in the endometrium of adult rats and humans and studies in rats showed that the receptor is upregulated in the endometrium during pregnancy (Renaud et al., 2005). Furthermore, transcripts for the AMRII have been discovered in the hypothalamus, motor and cortical neurons, and the pituitary glands, suggesting that AMH has undiscovered actions within the female body (Bédécarrats et al., 2003; Wang et al., 2005; Lebeurrier et al., 2008).
AMH production and expression in the ovary
There have been many studies investigating AMH expression in the ovary in rodents, humans, cattle, sheep and horses (reviewed by La Marca et al. (2009a)), but very few have examined this in pigs. Secretion starts in granulosa cells of recruited primordial follicles in rats, mice, humans and cattle, and in granulosa cells of secondary follicles in sheep (Campbell et al., 2012). In most species, intrafollicular concentrations increase until the small antral follicle stage and generally diminish to the preovulatory stages (Hirobe et al., 1992; Baarends et al.,
1995; Weenen et al., 2004; Monniaux et al., 2008; Rico et al., 2009; Rico et al., 2011; Campbell et al., 2012). In women, low concentrations of AMH have been detected in preovulatory follicles (Seifer et al., 1993; Fanchin et al., 2005a; Andersen and Byskov, 2006a; Eilso Nielsen Page 68 of 213 et al., 2010). In cattle and sheep, differences in AMH protein expression between granulosa cell layers have been observed, with production becoming more and more restricted to the cumulus cells in larger antral follicles (Rico et al., 2011; Campbell et al., 2012). Within atretic follicles of cattle, expression of AMH protein in granulosa cells ceases completely and only occurs within cumulus cells (Rico et al., 2011).
Similar to other species, expression of AMH commences in granulosa cells of recruited follicles, in pigs (Almeida et al., 2018). In contrast to other species, AMH expression appears to remain stable through to the large antral follicle stage (Monniaux et al., 2012; Almeida et al., 2018). Recently, Almeida et al. (2018) showed that sow ovaries uniquely express AMH protein strongly in the theca cells of preovulatory follicles, and in luteinised granulosa and theca cells within the CL of non-pregnant and pregnant sows, at least until 30 days of gestation.
The role of AMH within the ovary
Upon investigation into the role of AMH in sexual differentiation in the foetus, it was discovered that exposure of foetal ovaries to AMH causes endocrine sex reversal by downregulating the production of E2 from testosterone (Vigier et al., 1989). This led to the finding that AMH is produced postnatally in granulosa cells (Bezard et al., 1987). In immature porcine granulosa cells, AMH downregulates E2 production by inhibiting FSH-induced aromatase activity and LHr expression (Di Clemente et al., 1994a). These studies sparked great interest in how AMH influences folliculogenesis.
In species other than the pig, AMH has since been shown to be necessary for regulating follicular development. It has an inhibitory action on primordial follicle recruitment in rodents, humans and cows (Durlinger et al., 1999a; Gigli et al., 2005; Campbell et al., 2012; Yang et
Page 69 of 213 al., 2017) and inhibits granulosa cell differentiation and proliferation in cattle (Poole et al.,
2016). In sheep, AMH appears to have no effect on primordial follicle recruitment but regulates the rate of follicular development from the preantral to the gonadotropin-dependant antral stages, throughout which AMH expression is maximised (Campbell et al., 2012).
Without AMH, follicles develop and mature in an uncontrolled manner causing premature ovarian exhaustion (Durlinger et al., 1999a).
Though not yet investigated in pigs (to our knowledge), the unusual expression patterns of AMH within the porcine ovary implies a unique role for AMH in this species. The increased expression in preovulatory follicles (Almeida et al., 2018) and the inhibitory action on LH receptors in porcine granulosa cells (Di Clemente et al., 1994a) suggests AMH has a role in dominant follicle selection in pigs. Further, the presence of AMH in the CL suggests AMH involvement during pregnancy, a hypothesis supported by the finding that endometrial
AMHRII is upregulated during pregnancy in rats (Renaud et al., 2005).
Recently, Detti et al. (2018) demonstrated that treatment of human ovarian cortex cells with AMH reduced the expression of not only FSHr and LHr, but also inhibin B and insulin- like growth factor-I receptor-I, both of which are involved in androgen production. This suggests that the inhibitory action of AMH is not limited to gonadotropin sensitivity and is much more complex than first thought.
Regulation of AMH
The mechanisms that regulate AMH expression and production are poorly understood.
There seems to be a complicated and indirect two-way relationship between AMH and E2 that involves FSH. Not only does AMH have inhibitory effects on FSH-induced E2 production as
Page 70 of 213 discussed previously, but E2 also appears to have indirect inhibitory effects on AMH production. In women undergoing ovarian stimulation for IVF cycles, there is a negative relationship between AMH and E2 in the circulation (Fanchin et al., 2003; La Marca et al.,
2004; Lee et al., 2010; Weintraub et al., 2014) and within the follicular fluid of small antral follicles (Andersen and Byskov, 2006b; Dumesic et al., 2009). However, in natural ovulation cycles, some studies have indicated that the negative relationship between AMH and E2 does not exist (La Marca et al., 2004). Recent studies, using a more sensitive AMH assay, showed a decline in AMH just prior to and until a few days after ovulation, coincident with the increase in E2 levels that occur at this time (Gnoth et al., 2015). Some of the variation between studies may be due to the different actions that E2 has on AMH through its different receptors. Through
ER-α, E2 action on AMH is stimulatory, while through ER-β, the receptor predominantly expressed until luteinisation, E2 action on AMH is inhibitory (Couse et al., 2005; Grynberg et al., 2012).
AMH inhibits follicle sensitivity to FSH, however, whether FSH stimulates or inhibits
AMH production is unclear. A reduction in circulating AMH levels can be observed in FSH- stimulated rats (Baarends et al., 1995) and women (Fanchin et al., 2003; La Marca et al., 2004;
Weintraub et al., 2014). Also, in girls, a slight reduction in circulating AMH levels is observed during puberty, at a time coincident with a rise in circulating FSH and LH levels (Kelsey et al.,
2011; Hagen et al., 2012a; Lashen et al., 2013). This has also been demonstrated at a cellular level in cows, where FSH administration reduced AMH secretion by around 50% in granulosa cells of preovulatory follicles (Rico et al., 2011). However, resultant changes in other confounding factors such as testosterone or E2 may be causing changes in AMH production.
Also, opposing responses of AMH to FSH have been shown in primates (Thomas et al., 2007) and woman (Anderson et al., 2006; Hagen et al., 2012b; Chan and Liu, 2014).
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It is well known that the oocyte itself communicates with granulosa and cumulus cells to orchestrate the release of factors that are beneficial for its own growth and development. It is evident that the oocyte also regulates AMH production within granulosa cells in this way as
AMH production is upregulated in granulosa cells when cultured in close proximity with oocytes in mice (Salmon et al., 2004). It appears that the oocyte does this via oocyte-secreted factors, such as growth differentiation factor 9 (GDF9) and BMPs, which are important for granulosa cell differentiation and are essential for oocyte developmental competence (Gilchrist et al., 2008). Treatment with BMPs has been shown to simulate AMH mRNA expression and
AMH protein production in sheep, human, cow and hen granulosa cells in vitro (Shi et al.,
2009; Rico et al., 2011; Monniaux et al., 2012; Ocon-Grove et al., 2012). Furthermore, a study in ovulatory women undergoing fertility treatment showed that AMH mRNA expression was greater in cumulus cells of preovulatory follicles containing immature oocytes or atretic oocytes compared with those of preovulatory follicles containing mature, healthy oocytes
(Kedem-Dickman et al., 2012). These findings support previous studies in mice that indicate
AMH may have a role in inhibiting meiosis (Takahashi et al., 1986; Ueno et al., 1988). From this, it is hypothesised that AMH also has a role in inhibiting the development of oocytes and follicles that are of suboptimal quality.
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1.11 Circulating AMH in juvenile animals
Intra- and inter-individual variation
Studies measuring AMH over time on a per-individual basis are very limited. This is likely due to the high cost of AMH assays and difficulties in following individuals for extended periods of time. Most that are longitudinal in nature are in humans, with only a few focusing on non-childbearing years. One such study showed that levels of AMH increase substantially in girls from birth until three months of age (Hagen et al., 2010). Another in girls from age seven to 11 years, showed a 20% increase in circulating AMH levels from ages seven to nine, followed by a decrease by a similar amount by 11 years of age (Lashen et al., 2013). However, whether girls started cycling during the study and, if so, at what stage of the cycle the blood was collected was not reported. Subsequently, Jeffery et al. (2015) conducted a study involving a large cohort of girls from the age of five to 14 years and considered age in terms of both their stage of reproductive development as well as calendar age. The results confirmed those of
Lashen et al. (2013) and, when considering age in terms of sexual maturity, showed that girls experience a slight prepubertal peak in circulating AMH between three and five years prior to menstruation. Though there seemed to be a great variation of circulating AMH levels between individuals, there was great intra-individual tracking in these studies (Lashen et al., 2013).
Though scarce, there are a few longitudinal studies in livestock species that indicate the circulating AMH patterns are similar to those in humans throughout the prepubertal ages.
Sheep and cattle both appear to experience a prepubertal peak in circulating AMH concentrations followed by a decline until around the age of expected puberty. This has been demonstrated in Holstein heifers (Mossa et al., 2017), Nelore heifers (Mossa et al., 2013),
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Japanese black heifers (Hirayama et al., 2017) and Rasa Aragonesa lambs (Lahoz et al., 2014).
In all four studies, circulating AMH concentrations were shown to initially increase, peaking at around three months in dairy cattle, four months in beef cattle and 4.5 months in lambs.
Levels then declined to six months in the ewe lambs and Japanese black heifers and to eight months in Holstein and Nelore heifers before becoming relatively stable in adulthood (Figure
3). In sheep, no intra-individual correlation of AMH levels between pubertal (3, 4.5 and 6 months) and adult ages (19 months) was apparent (Lahoz et al., 2014). This may be due to individuals experiencing their prepubertal peak in AMH at different times, as the intra- individual repeatability increased when considering only lambs that experienced their peak at
4.5 months of age.
Figure 3: Changes in plasma anti-Müllerian hormone (AMH) according to age in Nelore beef calves, Holstein dairy calves, and Rasa Aragonesa lambs. Modified from (Mossa et al., 2017) and (Lahoz et al., 2014).
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When comparing these studies with those examining fluctuations in adulthood it appears that individual animals experience greater variability in AMH during prepubertal stages. Prepubertal animals experience a drastic decline in AMH prior to puberty, such that in adulthood circulating AMH levels are roughly 2.4 times lower in ewes, 3.5 times lower in
Nelore heifers and around seven times lower in Holstein heifers during the prepubertal peak compared to levels in mature animals (Baruselli et al., 2015). These differences are much greater than the fluctuations experienced throughout the oestrous cycles (discussed in latter sections)
Similarly, in the longitudinal livestock studies (Lahoz et al., 2014; Hirayama et al.,
2017; Mossa et al., 2017), the inter-individual variation in AMH concentrations was also greater at the prepubertal stages compared to that during adulthood (Figure 3). It could be that, though individuals are in the same age bracket, they have varying degrees of HPG axis and ovarian development, contributing to more variable levels of circulating AMH. There appear to be no reports of prepubertal AMH profiles in pigs and none in any livestock species that show circulating levels according to their stage of reproductive development.
Establishment of the HPG axis
The discovery of the expression of AMHRII and AMH in the hypothalamus and pituitary led scientists to believe that AMH could influence the HPG axis (Bédécarrats et al.,
2003; Wang et al., 2005; Lebeurrier et al., 2008). During prepubertal years, the HPG axis is relatively dormant and its activation stimulates the onset of puberty in mammals. The mechanisms by which the HPG axis is activated are not well understood. Very recently, though some suggest AMH and FSH have inhibitory actions on each other in the ovary, exogenous
AMH administration was found to increase FSH secretion in the rat pituitary without affecting
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LH secretion or E2 production (Garrel et al., 2016). Moreover, AMH receptivity in the pituitary was found to be reduced in rats treated with a GnRH agonist (Garrel et al., 2016). These results support the finding that circulating levels of FSH are reduced in AMH knockout adult mice
(Durlinger et al., 1999a). Garrel et al. (2016) also showed that pituitary cells express pro- protein convertases that are known to cleave AMH into its active form and that both AMH and
AMHRII mRNA are expressed there. This led to the proposal that AMH could be synthesised and activated locally and may participate in the activation of the HPG axis during sexual maturation. Such involvement may be reflected by the slight spike in AMH levels just prior to puberty, as demonstrated in longitudinal studies in cattle, sheep and humans (Lahoz et al.,
2014; Jeffery et al., 2015; Mossa et al., 2017), and contribute to the elevated FSH levels commonly observed in prepubertal mammals.
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1.12 Circulating AMH in adulthood
Menstrual cycles
In humans, there are two studies that have looked at the intermenstrual variation extensively. Both found that 89% of the variation in circulating AMH levels is due to inter- individual variation and only 11% is attributed to intra-individual cycle variation (Fanchin et al., 2005b; van Disseldorp et al., 2010). When compared with other reproductive hormones,
AMH has a greater inter-cycle correlation (r=0.88) than testosterone (r=0.84) , FSH (r=0.63),
E2 (r=0.54), inhibin B (r=0.53) and LH (r=0.56) in women (Andersen et al., 2011a). Whether
AMH varies with a woman's cycle is highly argued (reviewed by La Marca et al. (2013)). Some studies indicate that AMH levels fluctuate to a small extent (Hehenkamp et al., 2006; La Marca et al., 2006b; Tsepelidis et al., 2007; van Disseldorp et al., 2010). It has been proposed that the lack of dramatic cyclic changes can be attributed to the minimal expression of AMH in dominant follicles and the small variation is due to the change in AMH-producing growing follicle populations throughout the menstrual cycle. However, other studies have demonstrated more dramatic cyclic fluctuations of AMH (Wunder et al., 2008; Sowers et al., 2010; Hadlow et al., 2013). One study, though very small (n=12), found an intra-individual variation to be as high as 80%, with levels significantly greater in the follicular phase, following a similar profile to that of FSH (Hadlow et al., 2013). Another small study (n=20) showed fluctuations in AMH to be random throughout the menstrual cycle and found that women with greater basal AMH levels experienced greater fluctuations than those with lower levels. From this, it was suggested that younger women experience greater intra-cycle variations than do older women (Sowers et al., 2010). In support of this, Overbeek et al. (2012) reported that the magnitude of changes in
Page 77 of 213 serum AMH levels throughout one cycle was negatively associated with age in women aged
25-46 years.
Oestrus cycles
The inter- and intra-cycle variability of AMH has not been researched extensively in livestock species, and the few studies on the topic involve low numbers of animals. One study in cows showed no change in AMH over eight days leading up to ovulation (Ireland et al.,
2008). In contrast, Rico et al. (2011) found that after ovulation occurs in cows, AMH levels decline slightly (by about 37%) in the follicular phase, reach minimal concentrations after the first follicular wave (Days four to eight), and increase again to the next ovulation (Rico et al.,
2011). This pattern aligns with those found in women (Wunder et al., 2008; Sowers et al.,
2010). Though the number of follicular waves can vary between cows, the endocrine profile of
AMH during the oestrus cycle appears to be independent of the number of follicular waves that occur (Rico et al., 2011). Furthermore, though there are slight fluctuations in AMH over the oestrus cycle in the cow, a single measurement in an individual on any day in the cycle is very highly correlated with the average AMH concentration for the duration of the cycle (Ireland et al., 2011). In mares, the AMH concentration repeatability and correlation within and between oestrous cycles was also found to be high (Claes and Ball, 2016). In both mares and cattle,
AMH has a biological half-life of around two days and may contribute to some of the day-to- day repeatability of AMH concentrations in blood (Vigier et al., 1983; Almeida et al., 2011).
However, more research is required to clarify whether serum AMH levels vary within and between oestrous cycles in livestock species.
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Season
Short-day photoperiods inhibit reproductive physiology and development in Siberian hamsters, but the involvement of AMH in seasonal cyclicity is unclear. In the ovaries of adult females exposed to short daylight-hours (SD), AMH expression was most notable in granulosa cells of primary and secondary follicles and was reduced in tertiary follicles, which were rarely found (Kabithe and Place, 2008). Despite having reduced numbers of small secondary follicles,
SD hamsters had intra-ovarian AMH concentrations that were three times higher, than that in similarly-aged females exposed to long daylight-hours (LD) (Kabithe and Place, 2008).
Interestingly, circulating AMH concentrations were not correlated with intra-ovarian AMH concentrations and, though SD hamsters had higher ovarian AMH levels, they had lower circulating AMH levels compared to LD hamsters (Kabithe and Place, 2008). This disparity between circulating and ovarian levels of AMH supports the notion that AMH may have different actions and be involved in different feedback loops within the brain and ovary.
Findings of a study conducted in goats also suggest that circulating AMH may be affected by season. Serum AMH levels were measured over two seven-week periods, one in spring, when the transition to the non-breeding season occurs, and one in autumn, when the transition back into the breeding season occurs (Monniaux et al., 2011). During the seven-week period in spring, circulating AMH concentrations in goats increased linearly by around 50% over the testing period, while FSH did not change. During autumn, a slight decline was observed in the last two weeks of the testing period, while FSH, again, remained stable
(Monniaux et al., 2011).
Seasonal differences in AMH levels have also been observed in mares (Gharagozlou et al., 2014). Similar to female hamsters and goats, breeding season conditions appear to result in elevated concentrations of circulating AMH in mares. During the breeding season, cycling
Page 79 of 213 mares had three-fold higher AMH levels than seasonally anoestrous mares (Gharagozlou et al.,
2014).
Female pigs experience seasonal periods of reduced fertility in the summer months, with reduced farrowing rates, fewer ovulations, poorer oocyte quality and decreased follicular fluid progesterone concentrations having been identified in sows (Bertoldo et al., 2010;
Bertoldo et al., 2011). These findings indicate that there are seasonal changes in the ovarian follicle populations. Therefore, AMH levels may also be affected by season in pigs, but this is yet to be investigated.
Aging and ovarian reserve
As mammals age, ovarian reserve and the population of growing follicles that produce
AMH decline. As such, circulating AMH levels are relatively stable in sexually mature women, until around 25 years of age when levels start to decline, becoming negligible around the age of 45 when ovarian exhaustion occurs (Hagen et al., 2010). Ovarian reserve exhaustion is not often exhibited in production animals due to their artificially shortened lifespan. However, it is possible that premature exhaustion at a young age may occur in a small number of individuals.
Disease
A recent study in women has shown that women with occluded fallopian tubes due to chronic pelvic inflammation have 22% lower AMH levels than healthy women (Cui et al.,
2016). These results suggest that ovarian follicle populations are disrupted in response to chronic inflammation. Furthermore, reductions in circulating AMH levels appear to be
Page 80 of 213 associated with haematological malignancies in adults (Lawrenz et al., 2012) and cancer in newly diagnosed young girls (van Dorp et al., 2014). Inflammation is proposed to play a significant part in the reduced AMH levels observed in cancer patients, but the underlying mechanisms involved are unknown.
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1.13 Maternal factors that affect circulating AMH in offspring
Gestational nutrition
In various species, nutrition of the mother during gestation significantly impacts ovarian qualities in their offspring. Heifers born from cows that endure nutritional deficits during the first trimester have been shown to have lower levels of AMH, lower AFC, and elevated circulating FSH; all of which are indicative of reduced ovarian reserve and, therefore, reproductive potential (Mossa et al., 2013). Elevated dietary protein imposed during the second trimester has also been shown to result in compromised antral follicle populations in cattle offspring (Sullivan et al., 2010). In rats, high-fat diets during gestation and lactation compromise the ovaries of daughters, marked by reduced numbers of oocytes at 20 days after conception, reduced circulating AMH concentrations at birth and increased follicular atresia at the prepubertal and adult stages (Tsoulis et al., 2016). In rats, excess nutrition (ad libitum feed) throughout gestation results in offspring with decreased AMH expression, ovarian reserve and gonadotropin release at ovulation (Sominsky et al., 2016). Similarly, excess nutrition leads to decreased follicular reserve and increased numbers of recruited follicles in cattle fetuses
(Weller et al., 2016).
Polycystic ovary syndrome
Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women of reproductive age and is prevalent in pigs and other livestock animals (Beek et al., 2011). It is a cause of ovulatory dysfunction and affects fertility (Ryu, et al. 2019), often resulting in
Page 82 of 213 hyperandrogenism (Garg and Tal, 2016; Szulanczyk-Mencel et al., 2010). It has been shown to affect the reproductive potential of offspring as prenatal exposure of excess testosterone results in negative changes in ovarian morphology and function later in life and this may also result in PCOS (Rosenfield, 1997; West et al., 2001; Franks et al., 2008; Smith et al., 2009;
Padmanabhan and Veiga-Lopez, 2011). Excess testosterone in the maternal environment reduces AMH expression in the preantral and antral follicles and increases AMH expression in the blood of female offspring in adulthood (Veiga-Lopez et al., 2012). Further, the inhibitory action of AMH on aromatase production is attributed to the hyperandrogenism seen in PCOS and is used to predict PCOS treatment response (Garg and Tal, 2016). The involvement of
AMH with PCOS is complex and an in-depth discussion is outside the scope of this review.
Maternal Inflammation
It is clear that the compromised health of a mother during gestation can have negative effects on offspring. It is suspected that inflammation not only has direct effects on the mother’s ovaries but could also affect the developing fetus. One study in cattle provides evidence to support this theory, showing that dairy cows with a high frequency of milk somatic cell counts of greater than 200,000 have daughters with lower AMH concentrations in adulthood (12 months of age) (Ireland et al., 2011).
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1.14 Circulating AMH as a marker of future reproductive
performance
Puberty
Anti-Müllerian hormone was only very recently suspected to have effects on the HPG axis, therefore its involvement in puberty attainment is a relatively novel research topic. Thus far, one cross-sectional study in women showed that levels of circulating AMH at eight years of age, around the time that the prepubertal peak in AMH concentration occurs, are negatively associated with age at puberty. Girls with lower AMH at this age were much more likely to have precocial puberty (Lashen et al., 2013). Conversely, in a ten-year longitudinal study by
Jeffery et al. (2015), earlier developing girls had higher mean circulating AMH concentrations over the ten years when compared to those that developed later. It is proposed that earlier maturing girls have higher mean AMH levels throughout their childhood but also may experience their prepubertal AMH peak earlier and levels would then start to decline earlier.
Thus, resulting in precocial girls having lower concentrations of AMH when compared to non- precocial girls in a single blood measurement at eight years of age. Though it has not been tested in livestock, it is hypothesised that because cattle and sheep experience a prepubertal peak followed by a decline in AMH levels, like women do, AMH may also be a useful indicator of individuals that attain puberty early in these species. An examination into whether this is also the case in pigs would be of great commercial benefit as reducing the timeframe from when gilts enter the breeding herd to when they display their first oestrus would significantly reduce the mean number of NPDs.
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Gonadotropin responsiveness
Assisted reproductive technologies in humans and livestock species usually involve exogenous gonadotropin stimulation to induce superovulation. Individual responses vary greatly and are associated with ovarian reserve and antral follicle count (AFC), factors directly influencing circulating AMH, and are affected by maturity status and age. The resultant embryos from individuals exhibiting poor gonadotropin responsiveness are typically poor- quality (reviewed by Monget et al. (2012)). Over the last decade, there has been great focus on
AMH as a marker for individuals that respond well to gonadotropin stimulation, both in livestock for production purposes, and in women undergoing fertility treatments. A summary of the key findings in livestock is displayed in (Table 1)
Numerous studies in cattle have shown that a single measurement of circulating AMH in adult cows before or during lactation may be used to predict the superovulatory response.
Serum AMH levels were positively associated with the number of follicles aspirated and oocytes recovered, and the number of CL on the day of flushing in several breeds of cattle
(Rico et al., 2009; Hirayama et al., 2012; Rico et al., 2012; Guerreiro et al., 2014; Souza et al.,
2015; Vernunft et al., 2015; Ghanem et al., 2016; Hirayama et al., 2017). There was also a positive relationship between circulating AMH levels and the number of embryos obtained per donor cow (Hirayama et al., 2012; Rico et al., 2012; Guerreiro et al., 2014; Souza et al., 2015;
Ghanem et al., 2016; Hirayama et al., 2017). Of the studies that found AMH to be related to the number of embryos produced, the majority found no association with the proportions of oocytes that were fertilised, the proportion of embryos that reached the blastocyst stage, or the proportion of blastocysts that were of transferable grade (Hirayama et al., 2012; Guerreiro et al., 2014; Souza et al., 2015). This suggests that AMH is predictive of increased ovulation rates in response to gonadotropin stimulation, but not the quality of the ovulated oocyte. Findings
Page 85 of 213 from a study in goats are similar to those in cattle. Basal AMH levels in adult Saanen goats aged 10 months, before FSH treatment, were positively associated with the number of ovulations and the total number of embryos produced per donor doe (Monniaux et al., 2011).
Like adult cows, AMH levels are predictive of gonadotropin responsiveness in prepubertal heifers at eight to 10 months of age, an age at which circulating AMH levels are either declining or plateauing (Guerreiro et al., 2014). In sheep, the relationship between prepubertal levels of AMH and ovarian response to gonadotropins in adulthood have been inconsistent. Torres-Rovira et al. (2014) showed a positive association between circulating
AMH levels and AFC prior to stimulation in 40-day old lambs. There was also a positive correlation between plasma AMH levels and AFC in response to gonadotropin stimulation.
Furthermore, AFC was positively associated with greater numbers of oocytes obtained and embryos produced per donor ewe (Torres-Rovira et al., 2014). Lahoz et al. (2012) found ewes that ovulate in response to superovulation stimulation had significantly higher AMH levels at
3.6 months of age compared to those that did not ovulate. However, a subsequent study by the same researchers did not find an association between prepubertal AMH levels and AFC after stimulation or in natural cycles (Lahoz et al., 2014). No known studies have examined the relationship between AMH levels and gonadotropin responsiveness in pigs.
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Table 1: Associations between serum anti-Müllerian hormone (AMH) levels and ovarian follicle populations and gonadotropin responsiveness in livestock.
Natural cycle Ovarian stimulation cycle Stage when OPU IVM IVP AI Species Breed AMH was Ovarian Reference AFC AFC / Oocyte No. of No. of measured reserve No. of CL Oocytes Quality Embryos Embryos Ireland et al 2008, Batista Mix Adulthood + et al 2014 Holstein Adulthood + + Rico et al 2009
Holstein Adulthood + + + Rico et al 2012
Japanese Black Adulthood + + + + Hirayama et al 2012 Prepubertal & Cows Holstein & Nelore + + + Geurreiro et al 2014 Adult Holstein Adulthood + x x Vernunft et al 2015
Holstein Adulthood + + Souza et al 2015
Korean Hanwoo Adulthood + + Ghanem et al 2016 Prepubertal & Japanese Black + + Hirayama et al 2017 Adult Rasa Aragonesa Prepubertal +/x* Lahoz et al 2012 Prepubertal x x Sheep Rasa Aragonesa Lahoz et al 2014 Adulthood + + Sarda Prepubertal + + + + Torres-Rovira et al 2014
Goats Saanen Adulthood + + Minnaux et al 2011 Mares Light-horse, Mix Adulthood + Claes et al 2014
“+” = positive association , “x” = no association ; * Correlation with ovulation occurance but not number of ovulations; AFC = antral follicle count; OPU = Oocyte pick- up; IVM = In vitro maturation; IVP = in vitro embryo production; AI = Artificial Insemination; CL = corpora lutea;
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Ovarian reserve and antral follicle populations
In humans, reduced numbers of primordial ovarian follicles (ovarian reserve) are associated with reduced reproductive lifespan (Broer et al., 2014). Ovarian ultrasonography in the cow has revealed that low AFCs have multiple characteristics associated with infertility, including smaller ovaries, reduced overall follicle counts and lower oocyte numbers, poorer response to superovulation and chronically heightened gonadotrophin secretion (reviewed by
Ireland et al. (2011)). Currently, AFC is routinely assessed in estimating the reproductive potential for clinical or breeding applications (reviewed by Ireland et al. (2011)). There have been numerous studies in rodents and humans that show a positive association between circulating levels of AMH and ovarian reserve and AFC (reviewed by Broer et al. (2014)).
Anti-Müllerian hormone is the best known endocrine indicator of ovarian reserve in women and is used to identify women who are experiencing premature ovarian exhaustion or are approaching menopause (ASRM, 2012; Jamil et al., 2016). Though plasma AMH has been shown to be a good indicator of antral follicle count in superovulated cows, sheep and goats, there are few livestock studies that have investigated its effectiveness as a marker of follicle populations in animals that have not received gonadotropin stimulation (Table 1).
In cattle, both Ireland et al. (2008) and Hirayama et al. (2012) found AMH to be a good marker of ovarian reserve and AFC. Plasma AMH concentrations were particularly reflective of the numbers of small antral follicles but were also associated with total follicle counts
(Hirayama et al., 2012). Average AMH levels over the six to nine days leading up to ovulation were highly correlated with average daily AFC and average peak AFC during follicular waves
(Ireland et al., 2008).
Similarly, studies in sheep have shown levels of plasma AMH to be positively correlated with AFC and ovarian reserve (Torres-Rovira et al., 2014). When measured at 19
Page 88 of 213 months of age, circulating AMH levels were positively correlated with AFC in both natural and superovulatory cycles, although the association with AFC was much weaker in natural cycles (Lahoz et al., 2014). A 100 pg/mL increase in circulating AMH concentrations was associated with an increase of 5.1 follicles in superovulatory cycles, and an increase of only
1.4 follicles in natural conditions. This suggests that AMH may be a better indicator of gonadotropin responsiveness than of AFC in sheep.
Claes et al. (2014) examined the relationship between AMH and AFC in young (3-8 years), middle-aged (9-18 years), and old (19-27 years) mares. Plasma AMH levels were positively associated with large AFC (6-20 mm) but not with small AFC (2-5 mm). These results are interesting considering that AMH is expressed within small antral follicles to a much greater extent than in large antral follicles in normal mare ovaries (Ball et al., 2008). When comparing age groups, AMH levels were most strongly linked with AFC in old mares (r =
0.86), less so in middle-aged mares (r = 0.60), and not at all in young mares (r = 0.40). Whether these findings are unique to horses is unknown, as similar age comparisons have not been conducted in other livestock species.
Fertility
In addition to being a sensitive indicator of both ovarian reserve and AFC, some evidence suggests that AMH may also be a marker of fertility and oocyte quality. High AFC was associated with higher pregnancy rates and lower calving intervals in cattle (Cushman et al., 2009; Mossa et al., 2012). Also, high AFC was associated with greater blastocyst cell numbers in sheep (Torres-Rovira et al., 2014). Circulating AMH levels have been shown to reflect this in sheep, as higher AMH levels were associated with higher pregnancy rates at first mating (Lahoz et al., 2012). However, there are conflicting results in cattle. Ribeiro et al. (2014)
Page 89 of 213 found that AMH levels were indicative of pregnancy rates and foetal loss from 30 to 65 days after conception. Conversely, (Jimenez-Krassel et al., 2015) found there were no differences in pregnancy rates, calving interval or number of matings during the first, second or third lactation when heifers were assigned quartiles according to AMH levels. Cows in the lowest quartile were at a greater risk of culling after the first parity, and had greater removal rates due to reproductive inadequacy compared to all other groups (Jimenez-Krassel et al., 2015). In horses, the potential of AMH to predict fertility is also unclear. Suppressed levels of AMH identified mares that had hemorrhagic anovulatory ovaries during the breeding season
(Gharagozlou et al., 2014). However, elevated levels identified mares with granulosa cell tumours, which impair fertility (Almeida et al., 2011).
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1.15 Concluding remarks and objectives of the present study
In Australia, the current gilt selection process is inadequate, resulting in poor retention rates and a low average herd parity number. Useful markers of reproductive potential in young gilts are needed to better select gilts for entry into the breeding herd. Gilts are often culled for delayed onset of oestrus, failure to conceive, pregnancy loss or prolonged WOIs. Thus, finding markers of these traits would be ideal. It is proposed that AMH and E2 could be candidate hormone markers of reproductive potential. Age at sexual maturity has been previously associated with circulating levels of E2 in humans (Bidlingmaier et al., 1977; Gentry and Nash,
1990) and cattle (Gasser et al., 2006) and with circulating AMH levels in humans (Lashen et al., 2013). Circulating levels of E2 in response to ovarian stimulation has also been shown to be a marker of fertility in women (Laufer et al., 1986; Licciardi et al., 1995; Smotrich et al.,
1995; Evers et al., 1998; Pena et al., 2002; El Maghraby et al., 2009; Kondapalli et al., 2012;
Royster et al., 2016) and in sheep (Valasi et al., 2007). In humans, cattle, sheep, goats and mares AMH is a good marker of fertility in stimulated cycles as well as ovarian reserve and antral follicle populations in naturally-ovulating individuals (Table 1). However, there is a lack of information regarding the relationships between E2 and AMH levels in young gilts and their future reproductive properties and potential. The objectives of the current project were to determine serum concentrations of E2 and AMH in gilts before and after gonadotropin stimulation and whether those levels are associated with uterine and ovarian properties as well as fertility and reproductive performance up in the first three parities.
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CHAPTER TWO: GENERAL METHODS
2.1 Ethics
All animal procedures were conducted with prior institutional ethical approval by the
University of Sydney Animal Ethics Committee, CHM Alliance Animal Ethics committee
(Experiment Three only) and the Rivalea Australia Animal Ethics Committee, and under the requirements of the NSW Prevention of Cruelty to Animals Act 1985, in accordance with the
National Health and Medical Research Council/Commonwealth Scientific and Industrial
Research Organisation/Australian Animal Commission Code of Practice for the Care and Use of Animals for Scientific Purposes.
2.2 Animals and farms
Experiments were conducted across two Australian piggeries, one located in Southern New
South Wales (Farm A) and the other located in Southern Queensland (Farm B). All four experiments were conducted at Farm A, involving multiplier gilts (F1: Large White x Landrace,
PrimeGroTM Genetics, Corowa, NSW). The third experiment was also conducted at Farm B involving gilts from 80 days of age (PIC AustraliaTM Genetics, Grong Grong, NSW). Farm B gilts were separated into two herds according to their genetic lines. One herd contained nucleus
(F0: Large White) and multiplier gilts (F1: Large White x Duroc) and the other was made up of commercial gilts (F2: Large White x Duroc x Landrace). At both farms, gilts were selected conventionally, according to live weight; body, vulva and udder conformation; teat number; and the absence of any physical defects such as hernias or lameness. Details of the housing and breeding management conditions at each farm are shown in Table 2.
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2.3 Blood collection and storage
Blood collections commenced in gilts at either 60, 80 or 100 days of age across the four experiments. This period was chosen because this is a critical window for ovarian development in pigs (Schwarz et al., 2008). Blood was collected into serum separator tubes using 18 g x 25 mm vacutainer needles and left to clot for 2h at room temperature. The tubes were then centrifuged at 1000 RCF for twenty minutes and sera separated and stored at -80˚C for less than two months.
2.4 Assays
AMH Assay
After thawing, serum samples were diluted 1:2 in PBS, AMH was quantified using a competitive inhibition ELISA kit (CEA228Po: Cloud-Clone Corp, TX, USA) using monoclonal antibodies specific for porcine AMH. In-house validation of this kit was performed as described in Experiment One (Steel et al., 2018). The minimum detectable dose for the assay kit was 135.8 pg/mL. The intra- and inter-assay precision was <11.4% and
<12.9%, respectively.
E2 Assay – Experiments One and Two
Oestradiol was measured using competitive immunoassays using an anti-oestradiol sheep monoclonal antibody (ADVIA Centaur® eE2: Siemens Healthcare Diagnostics, NY
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USA). The minimum detectable dose for E2 was 43.6 pmol/L and the intra- and inter-assay precision was < 5% and < 8.1%, respectively.
E2 Assay – Experiments Three and Four
Oestradiol was measured using a competitive inhibition ELISA kit (CEA461Ge: Cloud-
Clone Corp, TX, USA) using a monoclonal antibody specific to E2. The minimum detectable dose for E2 was 46.2 pmol/L and the intra- and inter-assay precision was <2.9 % and <14.5 %, respectively.
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Table 2: Herd management conditions at the two farms
Farm A Farm B Weaner Age 28 - 70 days 24 - 70 days Flooring Conventional weaner pens DIG Dutchman plastic weaner flooring Space 45 pigs/ pen 230 pigs / pen (0.20 m2/pig) Diet Standard commercial weaner diet Standard commercial weaner diet Feeding Ad libitum Ad libitum Grower Age 70 - 130 days 70 - 150 days Flooring Conventional concrete slatting Concrete slatting and solid centre Space 200 gilts/pen (1.16 m2/pig) 39-40 gilts/pen (0.87 m2/pig) Diet Standard commercial grower diet Standard commercial grower diet Feed Ad libitum Ad libitum Selection Age 130 days to 190 days 150 days* until age of first heat Selection 160 days, >70kg 150 days, >60 kg Flooring Concrete slatted and solid (50:50) Concrete slatting and solid centre Space 50 gilts/pen (1.16 m2/pig) 15-20 gilts/pen (1.16-1.21 m2/pig) Diet Standard gilt developer Standard gilt developer Feeding Ad libitum Ad libitum
Mating Age 190 days* - first mating First heat - mating Flooring Concrete slatted Concrete slatting and solid centre Space 40 gilts/pen (1.5 m2/pig) 15 gilts/pen (1.76 m2/pig) Diet Formulated gilt developer Standard gilt developer Feeding Ad libitum Ad libitum Mating Artificial insemination (2x) Artificial insemination (2x) Mating criteria Est. Live weight: Outcome: Age at first heat: Outcome: <100 kg ; >150kg Cull <210 days Mate at next cycle 101-135 kg Mate at next cycle 210-235 days Mate 136-150 kg Mate >235 days Cull Gestation Flooring Concrete slatted and solid pad Solid concrete with slatting in centre Density 40-45 pigs/pen (1.8 m2/pig) 15-16 pigs/pen (1.65m2/gilt; 1.77 m2/sow) Diet Commercial gestation diet Commercial gestation diet Feeding 2.3-2.5 kg/day 2.3 kg/day Feeders Electronic feeders, two per pen Herd 1: Floor fed in shoulder stalls; Herd 2: Electronic feeders Farrowing Housing Farrowing crate Farrowing crate Space Outer: 2.00 x 1.66 m; Inner: 2.00 x 0.60 Outer: 2.02 x 1.56 m; Inner: 2.02 x 0.51 Diet Commercial lactation diet Commercial lactation diet Feeding Step-up for four days then Ad libitum Step-up for four days then Ad libitum
*Age that daily fence line (nose-to-nose) boar exposure and observation for first oestrus commenced
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CHAPTER THREE: EXPERIMENT ONE
Anti-Müllerian hormone and oestradiol as markers of future
reproductive success in juvenile gilts
Abstract
There is a need for an early marker for reproductive success in gilts as the traditional process for selecting breeding females is inefficient. There is evidence that circulating AMH is indicative of ovarian reserve, antral follicle populations, gonadotropin responsiveness and fertility in various species other than the pig. Additionally, oestradiol (E2) has been shown to mark antral follicle populations in cattle and pregnancy outcomes in women, after gonadotropin treatment. The aims of this study were to determine whether 1) serum levels of AMH or E2, prior to or after gonadotropin injection at 60, 80 or 100 days of age, and 2) hormonal changes in response to gonadotropin stimulation (i.e. declining, plateauing or increasing hormone levels), are associated with future reproductive success in juvenile gilts. Serum samples were obtained at 0, 2 and 4 days after injection and mating and litter data were collected until parity three. Results showed that, regardless of age group and parity, Day 0 E2 levels were positively associated with the probability of stillbirth (P = 0.035) and E2 levels on Day 0 (P=0.032), Day
2 (P=0.045) and Day 4 (P=0.019) were negatively associated with the number of piglets born alive. Further, both a single measurement of serum AMH levels at Day 2 (P=0.048) and the
AMH response type were associated with gestation length (P = 0.012). These findings suggest that serum AMH and E2 levels can be used to inform the selection of gilts for the breeding herd.
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3.1 Introduction
Poor sow retention rates are a major source of economic loss for pork producers. In
Australia, it is estimated that only around 60% of female pigs are retained to parity three (Plush et al., 2016). This is concerning as female pigs do not reach optimal reproductive performance until parity three (reviewed by (Engblom et al., 2007)). Moreover, gilt progeny have lower growth and sale weights, higher fat deposition and higher pre-weaning mortality rates than sow progeny (Smits and Collins, 2009). Poor reproductive performance is the most prevalent contributing factor for removal of breeding females prior to parity three and gilts are the most vulnerable age group to culling for this reason (Stalder et al., 2004; Hughes et al., 2010; Plush et al., 2016). This indicates that the traditional process for selecting replacement gilts (based on body conformation, number of teats and dam performance) is inadequate.
Ovarian characteristics such as follicular populations and gonadotropin responsiveness are positively linked with future fertility in females. However, up until recent decades, these parameters could not be measured without the use of ultrasound or surgery. Research shows that circulating AMH is a good marker for ovarian reserve and antral follicle populations in cattle (Hirayama et al., 2012; Batista et al., 2014), sheep (Torres-Rovira et al., 2014); mares
(Claes et al., 2014), humans (reviewed by La Marca et al. (2009a)) and mice (Kevenaar et al.,
2006) as well as a strong indicator of responsiveness to gonadotropins in cattle (Guerreiro et al., 2014; Souza et al., 2015; Ghanem et al., 2016; Hirayama et al., 2017), sheep (Lahoz et al.,
2014; Torres-Rovira et al., 2014), goats (Monniaux et al., 2011) and humans (reviewed by La
Marca et al. (2009a))
Oestradiol (E2) has also been shown to be associated with reproductive qualities in cattle and humans. The hormone is responsible for the growth and development of reproductive organs including ovarian follicles and the uterus. Clinical studies show that women with
Page 97 of 213 elevated basal E2 levels have poor reproductive capacity (Royster et al., 2016). Cattle with low antral follicle counts have concentrations of E2 that are twice as high in follicular fluid compared with cattle with high antral follicle counts (Ireland et al., 2008). Furthermore, E2 hormonal profiles (declining, plateau or increasing) in response to gonadotropin administration has been linked with reproductive potential in women. Previous studies have shown that embryos produced in vitro from women who showed a declining E2 profile after gonadotropin administration resulted in lower pregnancy and live birth rates when compared to those that have a plateau or increase in circulating E2 (Laufer et al., 1986; Kondapalli et al., 2012).
Furthermore, women who displayed a plateau in E2 had fewer oocytes recovered and women who experienced a plateau or decrease in E2 had reduced ovarian reserve and implantation rates (Kondapalli et al., 2012).
This study aimed to quantify circulating AMH in juvenile gilts and determine whether a single measurement of serum AMH and/or E2 in juvenile gilts (prior to, or after exogenous gonadotropin stimulation) or different hormonal profiles in response to exogenous gonadotropin can be used to predict future breeding characteristics.
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3.2 Methods
Animals and ethics
All animal procedures were conducted with prior institutional ethical approval as per
Chapter Two. Eighty-five multiplier gilts (F1: Large White x Landrace, PrimeGroTM Genetics,
Corowa, NSW) aged between 60 and 100 days of age were included in the study (D60: n=16;
D80: n=37; D100: n=32). Details of the housing and breeding management are shown in Table
2 (Farm A).
Gonadotropin stimulation, blood collection and storage
Juvenile gilts aged 60, 80 or 100 days (Rep 1: n=64; Rep 2: n=21) were injected with a low dose (200 IU) of PG600 (2:1 PMSG:hCG; Intervet, Holland). Collection and storage of blood samples were performed as described in Chapter Two.
Hormonal assays
Serum AMH was quantified using a competitive inhibition ELISA kit (CEA228Po:
Cloud-Clone Corp, TX, USA) using monoclonal antibodies specific for porcine AMH.
Samples were diluted 1:2 in PBS and tested in duplicate. To verify dilutional linearity, serial dilutions of two QC samples spiked with the standard were analysed using the kit. For each diluted sample, the back-calculated concentrations were within 20% of the expected values
(Table 3).
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Table 3: Dilutional linearity of two porcine serum samples containing varying AMH concentrations diluted with phosphate-buffered saline.
Dilution factor 0 1:2 1:4 1:8 Sample 1
Expected Conc. (ng/mL) 12.8 6.4 3.2 1.6 Observed Conc. (ng/mL) - 5.8 3.0 1.7 Recovery - 91% 95% 109% Sample 2 Expected Conc. (ng/mL) 21.8 10.9 5.5 2.7 Observed Conc. (ng/mL) - 8.7 5.3 2.6 Recovery - 80% 97% 96%
To assess parallelism, a standard and two serum samples were diluted serially, and the absorbance curves compared (Figure 4). The standard curve followed a linear-by-linear, rectangular hyperbola model: y=A+B/(1+D*x). Non-linear regression analysis showed that the constant parameter, A, was significantly different between the standard and sample curves
(Standard: A=0.20; Sample 1: A=0.17; Sample 2: A=0.11; P=0.002), but constants B and D, were the same (B=1.11, P=0.680 and D=17.56, P=0.097; over-all fit: R2=98.6; P<0.001).
Therefore, both serum sample dilution curves were parallel with the standard curve. Ten blank samples were assayed and the minimum detectable dose for the assay kit was determined as
354.9 pg/mL, the value two standard deviations below the mean optical density reading. To test the precision of the assay we tested two serum samples across five different plates, each run on different days. The intra- and inter-assay precision was <11.4% and <12.9%, respectively.
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Figure 4: Parallelism between dilution curves of two samples of porcine serum and the standard curve of the anti-
Müllerian hormone (AMH) ELISA kit.
Oestradiol was quantified in undiluted serum using competitive immunoassays using an anti-E2 sheep monoclonal antibody (ADVIA Centaur® eE2: Siemens Healthcare
Diagnostics, NY USA). The minimum detectable dose for E2 was 43.6 pmol/L and the intra- and inter-assay precision was <5.0% and <8.1%, respectively.
Mating and parity measurements
Mating and pregnancy information was received for only some of the gilts sampled
(D60: n=16; D80: n=25; D100: n=31). Mating outcome, gestation length, total number of piglets born, number of piglets born alive, mummified and stillborn, and weaning to oestrus intervals (WOI) and were recorded for parities one to three.
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Statistics
R (version 3.3.3) was used to assess correlations between ovarian hormones and intra- and inter-parity parameters via the rcorr function in the package Hmisc. Subsequently, restricted maximum likelihood analysis was conducted with the lme function from the nlme package to assess whether AMH and E2 levels, before and after PG600 administration, were associated with gestation lengths, number of piglets born alive, total number of piglets born and WOI. Binomial generalised linear mixed model analysis via the glmer function from the lme4 package was carried out to assess whether AMH and E2 were associated with pregnancy outcome, the proportion of piglets that were stillborn and proportion mummified and whether gilts were culled prior to parity three. Along with the ovarian hormones, parity and age groups were considered as predictors in the model. Two-way interactions were considered, and non- significant interaction terms were removed from the final model. Random variables considered in the analyses included pig ID nested within repetition. Missing values were omitted from calculation.
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3.3 Results
Serum AMH and E2 levels of 60, 80 and 100 day old gilts before and
after PG600 administration
Serum AMH values in juvenile gilts ranged from 9.5 to 30.9 ng/mL. The relationship between serum AMH concentration and time after the gonadotropin injection varied with age group (Figure 5A, P<0.001). There were no significant differences in basal AMH levels between 60, 80 and 100-day old gilts. Overall, the AMH concentrations of the 80-day old gilts decreased two days after PG600 injection, while both 60 and 100-day old gilts showed a dramatic increase in AMH two and four days after PG600 injection, respectively. Serum E2 levels decreased between Days 0 to 4 across all age groups (Figure 5B, P<0.001). Between age groups, E2 concentrations varied significantly with 80-day old gilts having the highest level, followed by 100-day old gilts and then 60-day old gilts (P<0.001). Results showed that
47% of gilts had a declining E2 profile (≥ 10% decline) over the test period, while 32% had a plateauing E2 profile and 21% had an increasing E2 profile (≥ 10% rise). Similarly, 52% of gilts had a declining AMH profile, while 15 % plateaued and 33 % had an increasing AMH profile. There was no association between serum AMH and E2 levels (P>0.05).
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Figure 5: A) Serum anti-Müllerian hormone (AMH) and B) serum oestradiol (E2) levels in 60, 80 and 100-day old gilts, 0, 2 and 4 days after PG600 injection (mean ± SEM). Different letters indicate significant differences between values (α=0.05).
Parity one to parity three mating, litter and culling data
A summary of the recorded mating, litter and culling data can be found in Table 4. On average, conception and farrowing rates and litter numbers met or exceeded industry standards
(Plush et al., 2016). However, only 58.3% of sows were retained to parity three. Table 5 shows the reasons for culling over the three parities. Overall, 63.3% of culls were due to reproductive inadequacy. Nulliparous gilts were the most vulnerable to culling for reproductive reasons
(87.5%) followed by parity one (61.5%) and two sows (44.4%). The most prominent reproductive reason for culling was due to not-in-pig (NIP) status and/or aborting after a positive ultrasound pregnancy test at 28 days (40% of total culls). There were no significant differences in age and weights at first mating between the 60, 80 and 100-day age groups
(Weight: 141.5 ± 2.9 kg; Age: 217.3 ± 5.2 days; P>0.05).
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Table 4: Descriptive statistics for reproduction and culling parameters measured from parity one to three (mean
± SD, where applicable)
Australian Parity 1 Parity 2 Parity 3 Targets*
Mating (n=72)
3 - - Number stale Number mated 69 58 46
% Conceived at first mating 97.1 91.4 91.3 >90 % Conceived 97.1 94.8 97.8
% Farrowed 91.3 87.9 91.3 >85
Reproduction
Gestation (days) 116.4 ± 1.5 116.3 ± 1.5 116.2 ± 2.0
Total born 11.7 ± 2.8 12.5 ± 3.2 13.5 ± 3.2 >13 Born alive 10.9 ± 2.6 11.5 ± 3.0 12.6 ± 2.9 >12
% Stillborn 5.2 ± 8.3 6.0 ± 10.8 6.0 ± 7.8 < 6
% Mummies 1.1 ± 3.3 1.1 ± 3.0 0.3 ± 1.2 < 2
WOI (days) 7.7 ± 8.3 6.5 ± 5.7 - < 8 Removal (prior to parity)
Number culled 8 13 9 % Culled 11.1 18.1 12.5 % Culled (cumulative) 11.1 29.2 41.7 ≤ 30 WOI= Weaning-to-oestrus interval; *Plush (2016).
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Table 5: Reasons for culling according to parity number
Removal reason: Parity 0 Parity 1 Parity 2 Overall % Stale 37.5 (3/8) - - 10.0 (3/30)
% Negative pregnancy test* 12.5 (1/8) 15.4 (2/13) - 10.0 (3/30)
% NIP/ Aborted 37.5 (3/8) 46.2 (6/13) 33.3 (3/9) 40.0 (12/30)
% Litter size - - 11.1 (1/9) 3.3 (1/30)
% Non- reproductive 12.5 (1/8) 38.5 (5/13) 55.6 (5/9) 36.7 (11/30) *Determined by ultrasound 28 days after mating; NIP = Not in pig
Intra-parity correlations
Table 6 shows the correlation coefficients for comparing different parameters within each parity. Gestation length was not correlated with the total number of piglets born per litter
(Total born) or the percentage of piglets born mummified (%Mummies) or stillborn
(%Stillborn) for parities one to three, nor was it correlated with WOI (P>0.05). There was a strong, positive, linear association between Total born and the number of piglets born alive
(Born alive) in all parities (P<0.001). Total born was correlated with %Stillborn and
%Mummies only in parity two (P<0.01) and parity three (P<0.05). In parity one and two, litter size and piglet survival parameters were not correlated with WOI (P>0.05). As expected, weight and age at first mating were highly correlated (P<0.001).
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Table 6: Correlation coefficient matrix comparing parameters within parities
Variables within the same parity Gest. length Born alive %Stillborn %Mummies Total born WOI Age1 Parity one (n=66)
Born alive 0.02 % Stillborn -0.18 -0.19
% Mummies -0.04 0.01 0.04
Total born -0.05 0.91 ‡ 0.19 0.19
WOI 0.10 0.17 -0.08 -0.11 0.11
Age1 -0.01 0.01 0.16 0.18 0.1 0.14 Weight1 0.08 0.19 -0.13 -0.05 0.14 0.1 0.51 ‡
Parity two (n=51)
Born alive 0.16
% Stillborn -0.19 -0.11
% Mummies 0.08 0.17 0.20
Total born 0.09 0.90 ‡ 0.32 * 0.39 † WOI -0.16 0.01 0.09 * 0.03 0.05 Parity three (n=42)
Born alive -0.07
% Stillborn -0.15 -0.03
% Mummies 0.09 0.18 0.32 * Total born -0.12 0.92 ‡ 0.35 * 0.35 * 1 At first mating; WOI = Weaning-to-oestrus interval; *P<0.05; † P<0.01, ‡ P<0.001;
Inter-parity correlations
The correlation coefficients for comparing parameters between each parity are shown in Table 7. Gestation lengths were significantly correlated across the three parities (P<0.05).
Parity two and three Total born were correlated with parity one Total born (P<0.01), but not with each other (P>0.05). Moreover, the number of piglets born alive and the proportion stillborn in the first two litters were correlated (P<0.05).
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Table 7: Correlation coefficient matrix comparing inter-parity parameters
Parity (P) Gestation length Born alive % Stillborn % Mummies Total born WOI P1 vs. P2 (n=50) 0.76 ‡ 0.43 † 0.35 * -0.08 0.37 † 0.14 P1 vs. P3 (n=42) 0.34 * 0.30 0.12 0.20 0.45 † P2 vs. P3 (n=42) 0.45 † 0.25 0.12 0.22 0.27 WOI = Weaning-to-oestrus interval; *P<0.05; † P<0.01, ‡ P<0.001
Figure 6: Serum oestradiol (E2) and number of piglets born alive per litter.A) There was an association between serum E2 levels 0, 2 and 4 days after PG600 injection in juvenile gilts aged 60, 80 and 100 days and number of piglets born alive per litter in the first three parities (Day 0: P=0.032; Day 2: P=0.045; Day 4:
P=0.019). B) There was no association between E2 profile type in response to PG600 and the number of piglets born alive (P>0.05). Different symbols denote significant differences between profile types (α=0.05).
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Number of piglets born alive
Serum levels of E2 at Day 0, 2 and 4 were negatively associated with the number of piglets born alive, regardless of age group and parity (Figure 6A). However, serum E2 profile type in response to PG600 was not (Figure 6B, P>0.05). When compared, Day 4 E2 levels were a better predictor of number of piglets born alive compared to serum E2 levels on Day 0 or Day
2 (Table 8). Serum AMH levels before or after PG600 injection were not associated with the number of piglets born alive (P>0.05).
Table 8: Comparison between models fitted using E2 levels at 0, 2 or 4 days after PG600 injection to predict the number of piglets born alive.
Predictor AIC BIC Log-Likelihood Predictor Coef. Predictor Coef. SE P-Value Day 0 E2 770.19 797.28 -376.09 -0.012 0.006 0.032* Day 2 E2 770.48 797.57 -376.24 -0.011 0.006 0.045* Day 4 E2 726.64 753.18 -354.32 -0.015 0.006 0.019* AIC = Akaike's information criterion; BIC = Bayesian information criterion; Coef. = Coefficient; SE=Standard error; * Denotes statistical significance
Probability of stillbirth
Basal Serum E2 concentrations were positively associated with the probability of stillbirth (Figure 7A, OR=1.007; P=0.035), across all age groups and parities (P>0.05).
However, E2 hormonal response type was not associated with stillbirths (Figure 7B, P>0.05), nor were AMH levels before or after PG600 injection (P>0.05).
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Figure 7: Serum oestradiol (E2) and the probability of stillbirth. A) Basal serum oestradiol (E2) levels in juvenile gilts aged 60, 80 and 100 days were associated with the probability of stillbirth in the first three parities (P=0.035).
B) There was no association between E2 profile type in response to PG600 and the probability of stillbirth
(P>0.05). Different symbols denote significant differences between profile types (α=0.05).
Gestation length
Day 2 AMH levels were positively associated with gestation length, irrespective of age group and parity (Figure 8A: gradient=0.05 ± 0.02, P=0.048). When gilts were grouped according to hormonal profile in response to PG600, it was found that gilts that displayed a decline in AMH had significantly shorter gestation lengths (Figure 8B; 115.88 ± 0.24 days) compared to gilts that had a plateauing or increasing AMH profile in response to PG600
(Plateau: 116.56 ± 0.44 days; Increasing: 116.83 ± 0.32 days; P=0.012), regardless of age or parity.
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Figure 8: Serum anti-Müllerian hormone (AMH) and gestation length. Both A) serum AMH levels two days after
PG600 and B) AMH profile type in response to PG600 in juvenile gilts aged 60, 80 and 100 days were associated with gestation length for the first three parities. Different symbols denote significant differences between profile types (α=0.05).
Other breeding parameters
Total born, WOIs, the proportion of piglets mummified, pregnancy outcomes after first mating attempt, or whether gilts were culled and the reason for culling were not associated with either single measurements of AMH or E2 before and after PG600, or hormone profile types in response to PG600 (P>0.05). These parameters will not be discussed further in this paper.
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3.4 Discussion
Results showed that a single measurement of basal serum E2 levels in juvenile gilts was associated with the number of piglets born alive and the probability of stillbirth. Specifically, lower basal levels of E2 in juvenile gilts were associated with increased litter numbers and a reduced probability of stillbirth from parities one to three. Though 80-day old gilts had 40% higher mean basal E2 levels compared to 60 and 100-day old gilts, the relationship between E2 levels and the number of piglets born alive or the proportion of piglets stillborn per litter was the same between age groups. The higher E2 levels in 80 day old gilts may be explained by the upregulation of follicular recruitment that occurs at this time, with tertiary follicles becoming more regularly observed until they account for around 2-3% of the ovarian follicles at 90 days of age (Oxender et al., 1979; McCoard et al., 2003).
Previous studies in humans have reported similar results, showing that oocytes retrieved from women with elevated basal E2 levels give rise to poor pregnancy rates (Licciardi et al.,
1995; Smotrich et al., 1995; Evers et al., 1998). The current study showed no relationship between E2 hormonal profile type in response to gonadotropin stimulation and litter qualities.
This contrasted the findings of Kondapalli et al. (2012) which showed that women who express a plateau in E2 in response to exogenous gonadotropin during IVF treatment have a >25% reduction in livebirth rates compared to women with an increase or decrease in E2 levels.
However, earlier studies also showed no difference in reproductive outcomes among women with different post-gonadotropin E2 profiles (Meyer et al., 1999; Chiasson et al., 2007). Aside from species, the major difference between the current study and the previous studies in humans is that at the time of blood sampling the women were all sexually mature whereas, all of the gilts in this study were not. The initiation of puberty involves the increased functionality of the reproductive neuron clusters in the surge centre of the hypothalamus as well as the
Page 112 of 213 increased E2 production by tertiary follicles within the ovary (reviewed by Sisk and Foster
(2004). This change causes the positive-feedback system between E2 and the surge centre to become dominant over the negative-feedback loop that exists between E2 and the tonic centre within the hypothalamus (Andrews et al., 1981; Rapisarda et al., 1983; Barb et al., 2010). The different E2 feedback systems in juvenile versus mature stages may explain some of the variation in results observed between porcine and human studies. It is proposed that the gilts in this study with lower basal E2 experience less inhibition from E2 on gonadotropin release, promoting earlier establishment of the HPG axis and follicular growth and sexual maturation.
Precocial sexual maturation has been associated with more regular cycling patterns, increased number of piglets born alive, increased growth rates and weaning weights of progeny and higher parity three retention rates (Nelson et al., 1990; Patterson et al., 2010). Studies investigating the relationship between circulating E2 levels and the timing of the first oestrus are currently underway.
Juvenile E2 levels may also be indicative of E2 mechanisms involved during birth. The incidence of stillborn piglets is commonly caused by asphyxia from dystocia and increases when parturition exceeds 20 minutes. Stillborn occurrence increases along with litter size and, in gilts, is more frequent in the first two piglets born (Randall and Penny, 1970; Hermesch,
2000; Canario et al., 2006; Dron et al., 2014).
The results of this study showed stillborn rates were positively associated with litter size in the first three parities. Though associated with stillbirth rates and number of piglets born alive, basal E2 levels were not associated with the proportion of piglets mummified or culling of gilts due to early- to mid-gestation foetal losses (which contributed to 50% and 62% of total culls prior to parity one and two, respectively). This suggests that juvenile E2 levels are associated with foetal loss during birth but not throughout gestation. The involvement of E2 during parturition is complex. It has involvement in the coordination of uterine contractions
Page 113 of 213 and cervical dilation as well as the activation of the foetal HPG axis, processes that are all essential in the birth of live offspring (reviewed by Kota et al. (2013). It is clear that E2 is vital for healthy parturition, but the mechanisms by which juvenile E2 levels influence the birthing process in adulthood warrants further investigation.
To our knowledge, this is the first study to quantify serum AMH in juvenile gilts.
Overall, serum AMH levels ranged from 9.5 to 30.9 ng/mL. Longitudinal studies in girls
(Lashen et al., 2013; Jeffery et al., 2015), heifers (Hirayama et al., 2012; Mossa et al., 2013;
Mossa et al., 2017) and lamb ewes (Lahoz et al., 2014) indicate that a peak in AMH levels occurs which is then followed by a dramatic decline until puberty onset. However, in the present study, results showed that there were no significant differences in basal AMH levels between 60, 80 and 100-day old gilts. Whether this is truly representative of a typical AMH hormonal profile over 60 to 100-day age range in gilts cannot be determined from these results.
It is possible that gilts do experience a juvenile peak in AMH levels but that it was masked due to the cross-sectional design of this study.
When comparing across species, juvenile gilts appear to have much greater AMH than mean levels reported in juvenile cattle (0.3-3.5 ng/mL), sheep (0.1-0.9 ng/mL) and, to a lesser extent, humans (3.0-4.7 ng/mL) (Kelsey et al., 2011; Lashen et al., 2013; Guerreiro et al., 2014;
Lahoz et al., 2014; Torres-Rovira et al., 2014; Baruselli et al., 2015). The higher AMH levels observed in pigs could be attributed to this species being polyovular and having a relatively large follicular reserve and antral follicle count (Warren et al., 2015). Some of the variation observed between studies could also be due to the different methods used to quantify AMH.
The present findings contradict those of Monniaux et al. (2012) that showed pigs had drastically lower AMH concentrations in antral follicles (where AMH expression is maximal in other species) compared with goats, sheep and cattle. However, Monniaux et al. (2012) suggest, these low levels may be due to porcine AMH having poor affinity to the antibodies used in the
Page 114 of 213 detection assay. It is also possible that follicles other than those at the antral stage could have a greater contribution to circulating AMH within the pig, resulting in the comparatively high circulating levels observed in the present study.
In this study, juvenile AMH levels in response to gonadotropin stimulation were associated with gestation length. Gilts with lower AMH levels determined by a single serum measurement, two days after PG600 injection or gilts with a declining AMH profile in response to PG600 had significantly lower gestation lengths than gilts with higher AMH levels two days after injection or gilts with plateauing or increasing AMH profiles, respectively. Type-II receptors specific for AMH (AMHRII) have been localised to the endometrium of adult rats and humans (Renaud et al., 2005). Moreover, some evidence suggests that AMH could have a role during pregnancy as uterine AMHRII is upregulated in pregnant rats, but the effects of
AMH on the uterus are unknown (Renaud et al., 2005).
The significance of gestation length as a reproductive parameter in sows varies between different studies. Some have reported gestation length to be negatively correlated with litter size and litter weight (Omtvedt et al., 1965; Martin et al., 1977) while others have found no relationship between the two (Cox, 1964). Further, Martineau and Badouard (2009) indicate that increases in gestation length by as little as two days may be associated with dramatic increases in colostral IgG, which is important for piglet immunity, growth and survival. In the present study, gestation lengths across parities were correlated and did not correlate with litter size (litter weight was not examined). The reason for the varying results between studies is unclear. It may be that the relationship between gestation length and litter size and piglet survival is circumstantial, depending on a multitude of genetic and environmental factors.
Serum AMH and E2 levels were not associated within the juvenile gilts examined.
These results contrast those of in vitro studies that showed AMH treatment had negative effects
Page 115 of 213 on E2 expression in foetal ovarian cells of rats, rabbits and sheep (Vigier et al., 1989). Anti-
Müllerian hormone acts by reducing biosynthesis of aromatase within the ovary, therefore preventing the conversion of testosterone to E2 (Vigier et al., 1989). Similar effects have also been demonstrated postnatally as AMH blocked the stimulatory effects of FSH and cyclic adenosine monophosphate (cAMP) on steroidogenesis in rat and porcine granulosa cells cultured in vitro, also causing a reduction in LH receptor expression in porcine granulosa cells
(Di Clemente et al., 1994a). Aromatase, cAMP, FSH and LH receptor expression have major involvement in promoting granulosa cell differentiation and follicular growth. The inhibitory action of AMH on these components is indicative that AMH has a regulatory function in folliculogenesis. However, AMH appears to have opposing effects on the pituitary of immature rats. Garrel et al. (2016) showed that AMH administration stimulated the secretion of FSH in the pituitary of juvenile rats and did not change the E2 or testosterone levels as it did in foetal ovaries. Furthermore, expression of AMHII in the pituitary decreases as rats approach pubertal ages. Our results support those of Garrel et al. (2016) as AMH and E2 levels were not dependent on each other, suggesting that AMH may have different effects on gonadotropins depending on the target tissue (pituitary versus ovary) and on the maturational age (juvenile versus pubertal).
In this study, we were able to detect and quantify serum AMH in juvenile gilts. The main findings of this study were that basal E2 levels at 60, 80 or 100 days of age were associated with the probability of stillbirth and that E2 levels before and after gonadotropin stimulation were associated with the number of piglets born alive. Furthermore, serum AMH levels after gonadotropin treatment were associated with gestation length. The influences of porcine AMH on various reproductive processes remains enigmatic and further research is required to determine its effectiveness as a marker of oocyte, ovarian and uterine qualities. We
Page 116 of 213 propose that measuring basal E2 levels in juvenile gilts could aid in selecting gilts for improved litter size and piglet survival rate.
3.5 Acknowledgements
This project was funded by Australian Pork Limited (Grant number: 2014/217). The authors thank C. Sjoblom, P. Spokes, A. Cruz and the staff of Westmead Fertility Centre for performing the serum E2 assays.
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CHAPTER FOUR: EXPERIMENT TWO
The relationship between serum AMH and E2 at 80 days of age
and uterine measurements and surface antral follicle counts at
160 days of age in gilts
Abstract
Premature culling of gilts is a common source of great economic inefficiency in piggeries.
The greatest contributing factor to this premature culling is reproductive failure. It is clear that traditional gilt selection processes are falling short and it would be beneficial to identify a marker of future reproductive success. We previously investigated whether AMH and E2 in juvenile gilts were associated with future reproductive output within the first three parities
(Experiment One) (Steel et al., 2018). It was demonstrated that basal concentrations of circulating oestradiol (E2) in prepubertal gilts aged 60, 80 or 100 days of age were positively associated with the probability of stillbirth and negatively associated with the number of piglets born alive in parities one to three (Steel et al., 2018). The aim of this study was to determine whether basal concentrations of circulating AMH and E2 in gilts 80 days of age (D80) were associated with uterine and ovarian properties at slaughter. Blood samples were collected from
48 multiplier gilts at D80. Sera AMH and E2 were measured via competitive inhibition ELISA.
At slaughter at 160 days of age (D160), uterine weight, horn diameter, horn length, ovarian weight and ovarian surface antral follicle counts (small < 3 mm, medium = 3-6 mm, large >6
Page 118 of 213 mm) were recorded. Principal component analysis showed that lower AMH and E2 concentrations at D80, equated to heavier, longer, thinner uterine horns at D160, irrespective of puberty status (P=0.024, Adj-R2 = 0.23), though puberty had a significant effect on uterine mass. Surface follicle counts were not associated with AMH, E2, CW or P2 measurements
(P>0.05). Ovarian hormones at D80 were also not associated with CW, P2 fat levels, and pubertal status at D160 (P>0.05). These findings extend those of Experiment 1 and provide further insight into the potential for E2 and AMH to act as markers of future reproductive potential in pigs.
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4.1 Introduction
Globally, a common cause for inefficiency within pig breeding herds is a poor sow retention rate. In Australia, it is estimated that an average of 40% of replacement females are culled prior to parity three (Plush et al., 2016). This is a source of huge economic loss as maintaining unproductive pigs is costly. Premature culling also shifts the average herd age distribution lower, further impacting productivity and profitability due to young females having lower pregnancy rates, smaller litters, reduced piglet sizes, and more NPD. It is well known that the number one factor contributing to this premature culling is reproductive failure. This includes failure to come into oestrus, long entry-to-weaning intervals, failure to conceive, early pregnancy loss and small litter sizes. Yet, teat number, body conformation and dam performance are traditionally the only reproductive traits assessed at selection. There is a great need for an efficient marker capable of predicting reproductive potential at a young age to improve the gilt selection process.
We previously demonstrated that basal concentration of circulating E2 in prepubertal gilts aged 60, 80 or 100 days of age were positively associated with the probability of stillbirth and negatively associated with the number of piglets born alive in parities one to three (Steel et al., 2018). In humans, girls that experience sexual maturity early appear to have lower serum
E2 concentration than normal-maturing girls, when compared at the same sexual developmental stage (Bidlingmaier et al., 1977). Given that E2 exerts a negative-feedback effect on the HPG axis, lower E2 concentration may promote earlier establishment of the HPG axis. In gilts, this may result in regular cycling patterns being established earlier, leading to increased number of piglets born alive, and higher retention rates to parity three (Nelson et al.,
1990; Patterson et al., 2010).
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The number of primordial follicles constitutes ovarian reserve and is an important determinant for the reproductive lifespan of an individual (Monniaux et al., 2014). The population of gonadotropin-responsive, growing small antral follicles forms a dynamic reserve for ovulation that is functionally related to the ovarian reserve (Monniaux et al., 2014). Antral follicle counts are often used to estimate ovarian reserve and are currently regularly assessed as a marker of reproductive potential in women undergoing infertility treatments (Gnoth et al.,
2015). Further, a longitudinal investigation in gilts aged around 110 to 150 days showed that
AFCs during follicular waves appear to increase in progressive waves (Schwarz et al., 2013), suggesting that AFCs could be associated with the time of puberty onset.
In species other than the pig, AMH has been found to be a good endocrine marker of both ovarian reserve and AFC (Humans: La Marca et al. (2009b); Loh and Maheshwari (2011);
Cattle: Rico et al. (2009); Ireland et al. (2011); Batista et al. (2014); Horses: Claes et al. (2014)
; Goats: Monniaux et al. (2011); Sheep: Lahoz et al. (2014). Anti-Müllerian hormone is a member of the TGF-β family of glycoproteins and was initially known for its role in sex differentiation. It is now known to be a key regulator of follicular growth, being produced by granulosa cells of ovarian follicles (Vigier et al., 1984). Ovarian production of AMH inhibits the recruitment of primordial follicles in humans, mice (Durlinger et al., 1999b; Gigli et al.,
2005; Park et al., 2011) and cattle (Gigli et al., 2005; Rico et al., 2011); and the transition of follicles to gonadotropin-dependent stages in sheep (Campbell et al., 2012). Expression of
AMH has been shown to commence in granulosa cells of recruited follicles in pigs and other species (Monniaux et al., 2012; Almeida et al., 2018). In pigs, AMH expression levels remain unchanged through to the large antral stage (Almeida et al., 2018). This contrasts with the AMH expression patterns observed in other species, where levels diminish from the small antral to preovulatory stages (Hirobe et al., 1992; Baarends et al., 1995; Weenen et al., 2004; Monniaux et al., 2008; Rico et al., 2009; Rico et al., 2011; Campbell et al., 2012). To our knowledge,
Page 121 of 213 there are no studies that have investigated whether circulating concentrations of AMH are reflective of antral follicle populations in gilts.
Along with age at puberty and antral follicle populations, uterine dimensions are also associated with reproductive efficiency as uterine crowding affects embryonic survival and litter numbers and piglet birth weights (Foxcroft et al., 2009). Uterine measurements at a prepubertal age, particularly uterine weight and horn length, have been genetically linked with uterine capacity in pigs (Young et al., 1996; Lents et al., 2014).
Thus, the aim of this study was to determine whether D80 concentrations of circulating
AMH and E2 in gilts are associated with ovarian and uterine properties at slaughter. It was hypothesised that the circulating concentrations of AMH and E2 in juvenile gilts are indicative of subsequent ovarian and uterine development.
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4.2 Methods
Animals and ethics
All animal procedures were conducted with prior institutional ethical approval as per
Chapter Two. Forty-eight multiplier gilts (F1: Large White x Landrace, PrimeGroTM Genetics,
Corowa, NSW) aged 80 ± 4 days were used in this study. There were five pairs of gilts and one trio of gilts that were from the same litter. Gilts of similar body condition were selected for this experiment, any gilts that had extreme body scores or gilts with any physical ailments were omitted. The management conditions for these gilts are outlined in Table 2 (Farm A).
Blood samples and assays
Blood samples were collected over consecutive weeks from two groups of gilts at 80 days of age (Group 1: N = 23, Group 2: N=25). Collection, storage and assaying of blood samples for AMH and E2 are described in Chapter Two.
Assessment of uterine and ovarian development
At 160 days of age, gilts were slaughtered at an on-site abattoir. Trimmed carcass weight (CW) and P2 back-fat scores were recorded and reproductive tracts were recovered.
The uteri and ovaries from each animal were weighed and the length and diameter of each uterine horn were recorded. Uterine diameter measurements were taken at the tubal, middle, and cervical ends of each uterine horn and averaged. Surface AFCs of each ovary were also
Page 123 of 213 recorded. Follicles were categorized as small (1-3 mm), medium (3-6 mm) and large (>6 mm) antral follicles, CL and CA.
Statistical analysis
Statistical analysis was conducted using the software package R (ver. 3.2.5). A significance determination threshold of α=0.05 was used for all statistical analyses in this study.
Replicate, sire and dam were considered as random factors. Paired T-tests were performed to assess differences between right and left ovarian and uterine traits. Principal component analysis (PCA) was performed using the stats::prcomp function in R to combine uterine weight, length and diameter parameters into summary variables (principal components; PCs) that could best summarise the variation between uteri, and to combine small, medium and large antral follicle counts into PCs that best describe the variation in AFC between gilts. Data were scaled prior to PCA. The gamm::mgcv function was used to fit multi-dimensional spline models to assess interactions between continuous variables and non-linear relationships. Linear relationships were assessed using lme4::lmer and lme4::glmer for continuous and binary outcome variables. Carcass weight and P2 fat scores and, where applicable, attainment of puberty at D160 (the presence of CL and/or CA) were considered as predictor variables.
Interactions were assessed and removed if not significant. There were no missing values in the dataset.
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4.3 Results
Serum hormone concentrations, carcass traits, ovarian and uterine
measurements
Serum hormone concentrations at 80 days of age and carcass, ovarian and uterine measurements at D160 are summarised in Table 9. Both AMH and E2 were within the limit of detection in the serum samples of all gilts studied. Right and left uterine horns did not differ in diameter or length (P>0.05). Left ovaries were heavier (4.6 ± 1.7 g) than right ovaries (3.9 ±
1.7 g, t = 3.29, DF = 47 P = 0.002), but the two were linearly related (b1 = 0.649, t (46) = 5.68,
P < 0.001, R2 = 0.40). Right and left uterine dimensions were averaged while the sum of the left and right ovarian traits was used for subsequent analysis. Medium antral follicles were present on ovaries from all but one gilt and 30 of the 48 gilts had large antral follicles visible.
Nine gilts were pubertal at D160, as indicated by the presence of CL, and one of these had cycled at least twice, as indicated by the presence of CA.
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Table 9: Summary of serum concentrations of AMH and E2 in gilts at 80 days of age and carcass, uterine and ovarian traits at 160 days of age
Age Variables: Median Mean SD Range (days) Serum hormone concentrations (n=48): AMH (ng/mL) 80 19.1 18.8 5.6 (8.4 - 36.3) E2 (pmol/L) 80 227.5 242.7 69.0 (130.0 - 488.0) Carcass traits (n=48): CW (kg) 160 83.7 82.3 9.1 (57.1 - 101.6) P2 Fat Depth (mm) 160 12.6 12.7 2.3 (8.0 - 18.4) Uterine trait (n=48): Weight (g) 160 185.3 195.0 112.0 (51.0 - 585.5)
Average horn diameter (mm) 160 13.2 13.4 3.1 (8.8 - 22.0)
Average horn length (mm) 160 600.0 669.1 272.7 (290.0 - 1495.0) Ovarian weight (n=48): Left Ovary (g) 160 4.0 4.6 1.7 (2.6-10.3) Right Ovary (g) 160 3.5 3.9 1.7 (1.4-10.4) Total (g) 160 7.3 8.5 3.0 (4.8-18.2) Antral follicle counts per gilt: Small antral (1-3 mm) 160 88.0 111.1 69.3 (31 - 288) Medium antral (3-6 mm) 160 19.0 20.1 14.8 (0-78) Large antral (> 6 mm) 160 1.5 6.3 6.9 (0-19) Total AFC 160 119.0 137.9 66.3 (48 - 297) Ovulations: First cycle* (n=9) 160 16.0 14.9 2.5 (11 - 18) Second cycle** (n=1) 160 20 20 - - AMH: Anti-Müllerian hormone; E2: Oestradiol, CW: Trimmed carcass weight; AFC: Antral follicle count; *Number of corpora lutea (CL) present. **Number of CL on ovaries that also had corpora albicantia (CA) present.
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Correlations between ovarian hormones and uterine and ovarian traits
AMH concentrations at D80 were positively correlated with uterine diameter at D160 but not with uterine weight or length, or surface antral follicle counts (Table 10). E2 concentrations at D80 were not correlated with AMH, uterine or ovarian traits.
Table 10: Pearson’s correlation coefficients between ovarian hormones at 80 days of age and uterine and ovarian traits at 160 days of age.
AMH E2
AMH
E2 0.17 Uterine Weight -0.03 -0.13 Horn Diamter 0.38 † 0.26 Horn Length -0.12 -0.25 Ovarian weight -0.18 -0.19 Surface Antral Follicle Counts: Small (1-3mm) 0.08 0.00 Medium (3-6mm) -0.16 -0.07 Large (>6mm) 0.09 0.25 Total 0.06 0.01 AMH = Anti-Müllerian hormone; E2 = Oestradiol; CW = Trimmed carcass weight; P2 = P2 fat depth; † p<0.01
Intra-uterus correlations
Uterine weight was significantly positively correlated with horn diameter and horn length (P <
0.001) (Table 11)
Table 11: Pearson’s correlation coefficients between uterine traits at 160 days of age.
Uterine Horn Horn Weight diameter Length
Uterine Weight
Horn diameter 0.51* Horn Length 0.76 * 0.22 * P<0.001;
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Intra-ovarian correlations
Large surface antral follicle counts were negatively correlated with small (P < 0.05) and medium surface antral follicle counts (P < 0.001; Table 12)
Table 12: Pearson’s correlation coefficients between surface antral follicle counts at 160 days of age.
Small Medium Large
Small
Medium -0.04 Large -0.30* -0.66‡ * P<0.001; ‡ P <0.001
Calculating uterine mass indices (UMIs)
The PCA revealed two PC values that accounted for the significant variation in uterine traits (67.7% and 26.5%, respectively). The first PC characterises the overall size of the uterus as it is proportionate to the uterine weight (loading = 1.66), horn length (loading = 0.59) and horn diameter (loading = 0.46) and is referred to as Uterine Mass Index (UMI)size. The second
PC describes uterine shape as it is proportionate to horn length (loading = 0.55) and to weak extent uterine weight (loading = 0.09) and strongly inversely proportionate to horn diameter
(loading = -0.83). Thus, it is referred to as UMIshape. A visual summary of UMI values for each uterus is shown in Figure 9.
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Figure 9: Principal component analysis for uterine traits. Two variables, or Uterine Mass Indices (UMIs), UMIsize and UMIshape, were created that summarised uterine weight, horn length and horn diameter measurements for each gilt, cumulatively accounting for 94.2% of the variation observed between uteri. Higher UMIsize values correspond to uteri with greater uterine weight, horn length and horn diameter, while, higher UMIshape values correspond to uteri with greater horn length and uterine weight and lesser uterine diameter. Each point on this graph represents a uterus, plotted according to their two UMI values. Points distributed towards the arrowheads of the red vectors have greater measurements of the corresponding uterine trait (labelled in red) than points located towards the tail- end of the vectors. Positively correlated traits have vectors that point in the same direction, negatively correlated traits have vectors that point in opposite directions and traits that have no correlation have vectors that are perpendicular to each other.
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Calculating surface antral follicle indices (SFIs)
The PCA showed two PC values that accounted for significant variation in surface antral follicle counts (52.9% and 36.6%, respectively). The first PC is proportionate to large surface AFC (loading = 0.73), inversely proportionate to medium surface AFC (loading = -
0.65) and very weakly inversely proportionate to small surface AFC (loading = -0.23). Thus, this PC has been named Surface Follicle Index (SFI)Lrg. The second PC was named SFIsml as it is strongly proportionate to small surface AFC (loading = 0.89), inversely proportionate to medium surface AFC (loading = -0.45) and very weakly proportionate to large surface AFC
(loading = -0.11). A visual representation of the PCA is shown in Figure 10.
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Figure 10: Principal component analysis for surface antral follicle counts. Two variables, or Surface Follicle
Indices (SFIs), SFILrg and SFISml, were created to summarise the surface small, medium and large antral follicle counts for each gilt. The SFILrg variable accounted for 52.9% of the variation in surface antral follicle populations observed between gilts, and SFISml accounted for 36.6% of the variation. Each point represents an individual gilt, plotted according to her two SFI values. Gilts with higher SFILrg values have greater large antral follicle counts
(AFC) and lesser medium and slightly lesser small AFC. Higher SFISml values have greater small and lesser medium, and slightly lesser large AFC. Points distributed towards the arrowheads of the red vectors have greater counts of the corresponding ovarian follicle type (labelled in red) than points located towards the tail-end of the vectors. Positively correlated traits have vectors that point in the same direction, negatively correlated traits have vectors that point in opposite directions and traits that have no correlation have vectors that are perpendicular to each other.
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Association between AMH and E2 concentration and UMIshape
There was a significant interaction effect between D80 AMH and E2 concentration on
UMIshape at D160 (Figure 11; P = 0.024, Adj-R2 = 0.23). Gilts with lower AMH and lower E2 concentration at 80 days of age had heavier uteri with longer, thinner horns compared with gilts that had higher AMH and/or E2 concentrations. Further, gilts that were pubertal at D160 had significantly greater UMIshape values (ie. heavier uteri with longer, thinner horns) compared to immature gilts (P<0.001). However, UMIshape values were independent of CW or P2 fat levels at D160 (P>0.05).
Figure 11: Three-dimensional spline model showing the association between serum E2 concentration and serum anti-Müllerian hormone (AMH) concentration at 80 days of age (D80) and D160 UMIshape (P=0.024, Adj-R2 =
0.23). Gilts with lower AMH and E2 concentration at D80 had greater UMIshape values. UMIshape values correspond to uteri that were heavier in weight with longer, thinner uterine horns.
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Other body condition, uterine and ovarian parameters
Much of the variation in UMIsize, SFISml and ovarian weight between gilts could be attributed to pubertal status (P<0.001) and none were associated with AMH, E2, CW or P2 measurements
(P>0.05). Ovarian hormones at D80 were also not associated with SFILrg, CW, P2 fat levels, and pubertal status at D160 (P>0.05).
4.4 Discussion
Most of the variation in uterine measurements between gilts at D160 was due to differences in overall uterine size (as summarised by UMIsize) or uterine shape, that is, heavier uteri with longer, thinner horns or light uteri with short, thick horns (as summarised by
UMIshape). Both D160 uterine weight and horn length have been positively correlated with uterine capacity in gilts (Young et al., 1996; Lents et al., 2014). Thus, gilts with greater UMI values will more likely have greater uterine capacity. Increasing uterine capacity alleviates problems associated with overcrowding, including early foetal losses (Wu and Dziuk, 1995), low birth weights and reduced litter size (Foxcroft et al., 2009). Uterine capacity is very difficult to accurately measure, requiring uni-lateral-hysterectomy and subsequent measurement of litter sizes. Thus, the ability to select for greater uterine capacity at a young age would improve the gilt selection process.
The results show that there was an interaction between D80 AMH and E2 concentration and D160 UMIshape. Gilts with a combination of both lower AMH and lower E2 concentrations at 80 days of age had greater UMIshape, and therefore uterine capacity, at the age that selection normally occurs. Previously, D80 AMH concentration was not associated with any mating, litter or culling outcomes (Steel et al., 2018). However, the negative relationship between D80
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E2 concentration and uterine development are in line with previous findings, suggesting that lower E2 concentrations are associated with more advanced uterine development, which in turn may reduce the frequency of stillbirth and increase the number of piglets born alive.
In juvenile gilts, a dramatic increase in plasma E2 concentration is observed around 60-
65 days of age, which is followed by a gradual decline until puberty commences (Wise, 1982).
This influx of E2 is coincident with an increase in ovarian activity, with tertiary follicles first becoming apparent on the ovaries from around 60 days of age, and accounting for 2-3% of the surface antral follicles by 90 days of age (Oxender et al., 1979; McCoard et al., 2003). Prior to puberty and the establishment of the HPG axis, E2 predominantly exerts negative-feedback on the hypothalamus. Thus, gilts with lower D80 E2 may experience less inhibition from E2 on gonadotropin release, promoting earlier establishment of the HPG axis. Though this could explain the greater uterine development observed in gilts with lower E2, the corresponding ovarian properties did not reflect this, as there were no associations between D80 E2 concentration and SFI values or the presence of CL by 160 days of age. Recently, Warren et al. (2015) found that, unlike in humans and cattle, AFC is not proportionate to primordial follicle counts (ovarian reserve) in pigs. Primary follicles were the only follicle type correlated with ovarian reserve in pigs (Warren et al., 2015). Though, higher ovulation rates have been negatively associated with small AFC in female pigs (Kelly et al., 1988). Thus, future research should include histological evaluation of follicle counts rather than surface follicle counts alone to estimate the ovarian reserve and reproductive potential.
In the foetus, AMH has inhibitory effects on the development of organs that are anlagen to the uterus. Interestingly, AMHRII receptor expression has been observed in the endometrium of mature rats and humans and appears to be upregulated in pregnant rats
(Renaud et al., 2005). The present findings indicate that AMH influences uterine development
Page 134 of 213 in gilts; however, the mechanism by which AMH affects uterine capacity remains unclear and deserves further investigation.
As with E2 concentration, longitudinal studies in humans, cattle and sheep indicate that juvenile females experience a peak in AMH concentration, followed by a gradual decline to sexual maturation (Lashen et al., 2013; Mossa et al., 2013; Lahoz et al., 2014; Jeffery et al.,
2015; Hirayama et al., 2017; Mossa et al., 2017). Though AMH has been proposed to be an indicator of early puberty in humans (Lashen et al., 2013), a relationship between circulating
D80 AMH concentration and puberty attainment at D160 was not apparent in the current study.
The current study was cross-sectional in design and a more detailed longitudinal assessment is required to better assess the relationship between AMH and early puberty attainment in gilts.
Contrary to other species, prepubertal AMH concentrations were not indicative of AFC in gilts, both immature and pubertal, at D160. In the pig, AMH is first expressed in recruited primordial follicles, similarly to most species, however, AMH continues to be expressed at similar levels to the antral stages and intensifies in preovulatory follicles, with theca cells also expressing the hormone (Almeida et al., 2018). Uniquely, AMH is also expressed in the CL of pigs (Almeida et al., 2018). The lack of relationship between AMH and AFC in the present study is expected to be due to the difference in timing between initial blood test and the measurement of AFC, particularly as previous findings show that AMH expression is most intense in dominant follicles if pigs, which would only develop as gilts become sexually mature. It is also likely that other factors affecting circulating AMH concentration may be at play, or that the larger populations of smaller follicle types contribute to circulating concentrations to a greater extent than antral follicles do.
The relationship between AMH and E2 is complex and largely involves FSH. Many studies in women going through ovarian stimulation cycles for IVF report that, on the day of
Page 135 of 213 hCG trigger, a decline in circulating AMH is observed, along with a negative association between serum AMH and E2 concentrations (reviewed by Dewailly et al. (2016)). Moreover, a negative relationship between the two hormones has also been observed in the follicular fluid of small antral follicles (Andersen and Byskov, 2006b; Dumesic et al., 2009). However, the current study and that by La Marca et al. (2004) showed no relationship between AMH and E2 in spontaneous ovulatory cycles. Granulosa cells produce E2 through the action of aromatase, which catalyses the conversion of androgens to oestrogens. From birth, aromatase expression levels are FSH-dependent (Gray et al., 1995). It appears that AMH attenuates aromatase activity by suppressing the stimulatory actions of FSH on the aromatase gene, CYP19 (Prapa et al., 2015; Sacchi et al., 2016). Curiously, AMH indirectly affects E2 production, which in turn suppresses AMH production. Though some studies have not been able to detect cyclic variation in circulating AMH in spontaneous ovulation cycles (Liberty et al., 2010), a recent longitudinal study in women using a relatively sensitive AMH assay showed that AMH concentration declined in the period spanning five days prior to ovulation to two days after ovulation, coincident with the cyclic rise of E2 concentration (Gnoth et al., 2015). Interestingly,
Grynberg et al. (2012) reported that E2 has both stimulatory and inhibitory effects on AMH via different receptors. While E2 had a stimulating effect on AMH via ER-α, it had an inhibitory effect through the ER-β. Granulosa cells predominantly express ER-β, but upon luteinisation, they predominantly express ER-α (Couse et al., 2005).
The absence of a negative relationship between serum concentrations of E2 and AMH observed in the present study may be attributed to the inability of circulating E2 concentration to rise in juvenile animals due to insufficient enzymatic apparatus in the theca cells and granulosa cells. Optimal steroidogenesis does not occur until puberty attainment, when the expression of aromatase increases in large antral and preovulatory follicles (Turner et al., 2002;
Guigon et al., 2003; Stocco, 2008). Thus, prior to puberty, any changes in aromatase activity
Page 136 of 213 caused by AMH may not be reflected by changes in circulating E2. Though AMH appears to oppose the effects of FSH within the ovary, recent studies have found that it stimulates FSH production in the pituitary of prepubertal rats (Garrel et al., 2016). Consistent with our findings, treatment with AMH did not evoke a change in serum E2 concentration within these immature rats (Garrel et al., 2016).
The carcass weights, uterine weights, uterine horn dimensions, ovarian weights and antral follicle counts recorded in this study were slightly greater than those previously reported for gilts slaughtered at D160 (Lents et al., 2014), and ovarian hormone concentrations were slightly lower than the previous experiment, likely due to genetic differences between herds used in the studies. However, the follicle counts were consistent with those from (Ferre, 2016).
The left ovaries of gilts were significantly heavier than the right ovaries and no other bilateral differences were observed. These results are consistent with previous studies in both sexually mature and immature gilts (Kunavongkrit et al., 1988). Bi-lateral disparities of the reproductive system have also been observed in other species including mice, rats, sheep and cattle (Casida et al., 1966; Reimers et al., 1973; Buchanan, 1974; McDonald, 1980; Wiebold and Becker, 1987). The reason for the left-right difference between ovaries, but not uterine horns is unknown. Some variation may be due to differences in blood flow through the left and right ovarian arteries resulting in differences in the level of gonadotropin stimulation received by each ovary (McDonald, 1980).
In this study, from a single blood sample taken at D80, we found that E2 and AMH concentration may be used to select gilts with different uterine properties. Using both D80
AMH and E2 concentration to select breeding herd replacement gilts could provide a way for producers to identify a larger proportion of gilts that have greater uterine capacity.Such selection would be expected to improve the number of piglets born alive per sow per year.
There was no relationship found between either ovarian hormone at a juvenile age and surface
Page 137 of 213 antral follicle counts at the age of selection. A more detailed assessment of ovarian follicle populations would be useful to confirm these results. Future longitudinal studies into the factors that influence E2 and AMH concentration throughout development and growth, and whether these can be used to manipulate uterine capacity and predict mating and pregnancy outcomes is needed.
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3.5 Acknowledgements
This project was funded by Australian Pork Limited. The authors thank C. Sjoblom and the staff of Westmead Fertility Centre for performing the serum oestradiol assays, Assoc. Prof.
P. Thomson for his guidance in statistical analysis and the farm staff for their assistance.
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CHAPTER FIVE: EXPERIMENT THREE
Relationship between serum concentrations of AMH and E2 at 80 days of
age and future reproductive potential
Abstract
In Australia, around 40% of gilts are culled prior to parity three. This contributes to huge economic wastage as replacement gilts are not profitable until parity three. The predominant reason for premature culling of gilts is reproductive failure. We previously identified circulating serum AMH and E2 concentration at an early age as potential markers of future reproductive success, demonstrating an association between basal levels of circulating E2 and the probability of stillbirth and the number of piglets born alive in the first three parities (Steel et al., 2018). Furthermore, AMH and E2 levels in juvenile gilts were related to uterine capacity at the time gilts are selected to enter the breeding herd (Experiment 2). The aim of this study was to extend these studies using a larger number of animals from different genetic and environmental sources. Blood samples were obtained from Landrace x Large White gilts at
D80 from two Australian commercial piggeries. Sera AMH and E2 concentration were measured via competitive inhibition ELISA. Age at first heat, first mating outcomes, gestation length, number of mummified, stillborn, and live piglets from the first litter and any culling information were recorded. A negative relationship between D80 E2 concentration and total number of piglets born at parity three was found. These results are consistent with those of
Experiment One and may be explained by the negative relationship between D80 E2 levels and
D160 uterine capacity observed in Experiment Two. As gilts are at their highest risk of culling prior to parity three, the use of E2 as a marker of future reproductive potential may be limited.
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5.1 Introduction
Poor sow retention rates are a problem that pork producers commonly experience. Sows are not profitable until their third litter (reviewed by Engblom et al. (2007)), and in Australia, an average of 40% of selected females are culled prior to this point (Plush et al., 2016).
Reproductive failure, including no heat, prolonged entry-to-oestrus intervals, failure to conceive, return to heat, small litter numbers and prolonged WOIs, is the predominant reason for premature culling. Gilts are particularly susceptible to reproductive failure (Stalder et al.,
2004; Hughes et al., 2010; Plush et al., 2016). It is clear that the traditional methods for selecting breeding gilts are not adequate and the industry would benefit from identifying an early marker of future reproductive success in gilts.
Anti-Müllerian hormone is a glycoprotein that is a member of the TGF-β family. It is produced by granulosa cells within the ovary and has an important role in regulating the rate of follicle recruitment. AMH-null mice exhibit premature ovarian exhaustion (Durlinger et al.,
1999a), and serum concentration of AMH have been found to be indicative of the ovarian reserve, antral follicle counts and superovulation responsiveness in species other than the pig
(Humans: (La Marca et al., 2009b; Loh and Maheshwari, 2011); Cattle: (Rico et al., 2009;
Ireland et al., 2011; Batista et al., 2014); Horses: (Claes et al., 2014) ; Goats: (Monniaux et al.,
2011); Sheep (Lahoz et al., 2014)). Recent studies suggest that AMH is also involved in the establishment of the hypothalamic-pituitary-gonadal axis during sexual maturation (Garrel et al., 2016). Thus, it is hypothesised that AMH may be associated with production parameters that have been previously linked with age at maturation such as regularity in cycling patterns, number of litters per year and litter size (Nelson et al., 1990)
Oestradiol is another ovarian hormone found to be a marker of fertility in several species. The hormone is produced in granulosa cells of the ovary and, to a lesser extent, many
Page 141 of 213 other tissues in the body. It plays a vital role during the development of the reproductive tract and regulation of ovarian cycles. In women, elevated serum E2 concentrations are indicative of poor reproductive capacity (Licciardi et al., 1995; Smotrich et al., 1995; Evers et al., 1998).
Furthermore, ovarian E2 concentrations are significantly higher in cows with low antral follicle counts (Ireland et al., 2008). This suggests that E2 may be a good marker of antral follicle growth and uterine development and, therefore, reproductive potential.
We previously examined the relationship between serum E2 and AMH concentrations in juvenile gilts and their future reproductive potential. Serum E2 concentrations were positively associated with the incidence of stillbirth and negatively associated with the number of piglets born alive. In addition, lower concentrations of serum E2 and AMH at 80 days of age were associated with greater uterine capacity at 160 days of age. The aim of this study was to extend these findings by conducting a larger-scale assessment of the relationship between
Day 80 serum E2 and AMH concentration and subsequent mating and pregnancy outcomes over three parities at two separate commercial farms.
5.2 Methods
Animals and ethics
All animal procedures were conducted with prior institutional ethical approval as per
Chapter Two. This study was conducted at two commercial farms, one located in Southern
New South Wales, Australia (Farm A) and one located in Southern Queensland, Australia
(Farm B). At Farm A, 178 multiplier gilts (F1: Large White x Landrace, PrimeGroTM Genetics,
Corowa, NSW) were used. Of these gilts, there were 30 pairs, 7 groups of three and one group of four from the same litter. At Farm B, 103 gilts (PIC AustraliaTM Genetics, Grong Grong,
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NSW) were used. Dam and sire information was obtained from 32 of the gilts, of these, there were six pairs of gilts from the same litter. Farm B gilts were separated into two herds according to their genetic lines. One herd contained nucleus (F0: Large White N=10) and multiplier gilts
(F1: Large White x Duroc, N=23) and the other herd was made up of commercial gilts (F2:
Large White x Duroc x Landrace, N=73). At both farms, gilts were selected conventionally, according to live weight; body, vulva and udder conformation; teat number; and absence of physical defects such as hernias or lameness. Details of the housing and breeding management at each farm are shown in Table 2.
Blood samples and assays
At 80 days of age, blood samples were collected over consecutive weeks from five groups of gilts from Farm A and two groups of gilts from Farm B. Collection, storage and assaying of blood samples for AMH and E2 are described in Chapter Two.
Mating and parity data
Parameters measured included age at first heat (Farm B only, n=57), mating outcome, gestation length, total number of piglets born, number of piglets born alive and the proportion of piglets mummified and stillborn at birth, WOI and whether gilts were culled, for each of the first three parities
Statistics
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The statistical package R (version 3.3.3) was used for all statistical analyses. The gamm::mgcv function was used to fit multi-dimensional spline models to assess interactions between continuous variables and non-linear relationships. Linear relationships were assessed using lme4::lmer for continuous outcome variables (gestation length, total born, piglets born alive) and lme4::glmer for binary (pregnancy at first mating, farrowing after mating) and proportional outcome variables (proportion of piglets stillborn and proportion mummified; binomial totals = Total born). Cumulative outcomes were also assessed for gilts that gave birth to three litters. Farm, herd, line, sire, dam and sow ID were considered as random effects to account for genetic, in utero and environmental differences and repeated measures on each sow. Whether fixed variables were significant to the model was evaluated via backwards stepwise elimination using the drop1 function. A significance level of 0.05 was used for all analyses and missing values were omitted from the calculations.
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5.3 Results
Summary of AMH and E2 levels at 80 days of age and mating and
pregnancy data
The mean (± SD) serum concentrations of AMH and E2 did not differ between the farms. The serum AMH concentrations were 9.6 ± 2.3 ng/mL at Farm A and 10.4 ± 2.0 ng/mL at Farm B. The serum E2 concentrations were 306.9 ± 61.9 ng/mL at Farm A and 329.2 ± 102.3 ng/mL at Farm B. Table 13 shows a summary of the recorded mating, litter and culling data at each farm. On average, conception and farrowing rates and litter numbers met or exceeded industry standards (Plush et al., 2016). However, the percentage of gilts culled by parity three at both farms exceeded the national targets of less than 30% (Farm A: 34.4%; Farm B: 40.2%).
Table 14 shows the reasons for culling over the three parities per farm. Overall, 42.2% of culls were due to reproductive inadequacy. Nulliparous gilts and uniparous sows were the most vulnerable to culling for reproductive reasons (43%) followed by parity two sows (39%). Not- in-pig (14% of total culls), staleness (12%) and failure to return to oestrus (10%) were the biggest contributors to culling for reproductive reasons. The age at first mating was 207 ± 18 days for Farm A and 228 ± 12 days for Farm B.
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Table 13: Summary of mating, litter and culling data from parities one to three
Parity 1 Parity 2 Parity 3*** Accepted Farm A Farm B Farm A Farm B Farm A Farm B Targets1
Mating
Number stale 17 * 3 3 0 0 Number mated 178 103 145 84 120 63 % Conceived 1st mating 78.1 93.2 68.3 81.0 93.3 98.4 >90 % Conceived 86.0 94.2 87.6 81.0 93.3 98.4 % Farrowed 86.0 89.3 87.6 79.8 *** 98.4 >85
Reproduction
Gestation (days) 115.7 ± 1.5 115.6 ± 1.5 116.3 ± 1.4 115.9 ± 1.5 115.7 ± 1.4 115.4 ± 1.8 Total born 12.4 ± 8.7 11.4 ± 2.2 12.9 ± 2.9 11.8 ± 3.1 14.3 ± 3.2 13.1 ± 2.9 >13 Born alive 10.9 ± 2.4 10.7 ± 2.2 12.0 ± 2.8 11.1 ± 2.9 13.2 ± 2.9 12.4 ± 2.7 >12 % Stillborn 4.2 ± 6.8 3.8 ± 7.6 5.0 ± 9.1 7.0 ± 25.1 5.6 ± 6.5 0.4 ± 0.6 < 6 % Mummies 2.7 ± 7.3 1.6 ± 3.8 1.5 ± 3.2 2.2 ± 12.4 1.6 ± 3.6 0.2 ± 0.5 < 2 WOI (days) 9.1 ± 11.2 8.1 ± 8.8 6.6 ± 8.2 5.1 ± 3.5 - - < 8 Removal (prior to parity) Number culled 32 14 25 25 7 4 % culled 17.2 13.1 13.4 23.4 3.8 3.7 % culled (cumulative) 17.2 13.1 30.6 36.4 34.4 40.2 < 30% ** WOI = weaning to oestrous interval; Values of reproduction parameters are displayed as mean ± SD; 1Plush (2016); * Only cycling gilts were selected into the breeding herd; **at parity three *** 44 of 112 sows that conceived were still gestating by the end of the testing period
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Table 14: Reasons for culling according to parity number
Parity 0 Parity 1 Parity 2
Removal Reason Farm A Farm B Farm A Farm B Farm A Farm B
% Stale 53.1 (17/32) ** 12.0 (3/25) 12.0 (3/25) - - % Return 3.1 (1/32) 35.7 (5/14) 28.0 (7/25) 16.0 (4/25) 14.3 (1/7) -
% Negative pregnancy test* 6.3 (2/32) 28.6 (4/14) 20.0 (5/25) 48.0 (12/25) 14.3 (1/7) 25.0 (1/4) % NIP/Aborted 12.5 (4/32) 7.1 (1/14) 4.0 (1/25) 8.0 (2/25) 14.3 (1/7) -
% Poor litter - - - - - 75.0 (3/4) % Non-reproductive 25.0 (8/32) 28.6 (4/14) 36.0 (9/25) 16.0 (4/25) 57.1 (4/7) -
* Determined by ultrasound 28 days after mating; **Stale gilts were not selected into the breeding herd; NIP = Not in pig
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Association between E2 concentration at 80 days of age and litter size
(total born)
There was an association between D80 E2 concentration and litter size (Total born) that differed according to parity number (Figure 12; Total born: χ2(2)=16.7, P <0.001). Table 15 shows the confidence intervals of the gradients for the linear relationships observed between
E2 concentration and total born across parities. The gradients at parities one and two were not significantly different to a gradient of zero and there was a negative relationship between D80
E2 concentration and total born at parity three. There was no variation between farms.
Figure 12: Associations between serum oestradiol (E2) concentration at 80 days of age and litter size (total born) in parities one to three. Significant relationships are denoted by an asterisks (χ2(2)=16.7, P <0.001).
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Table 15: Relationships between serum E2 concentration at 80 days of age and litter size (total born) for each parity.
Litter size (total) Parity Slope CI (lower) CI (upper)
One 0.005 -0.003 0.013
Two 0.005 -0.001 0.012 Three* -0.015 -0.025 -0.006
* denotes a slope significantly different to zero
Other measurements
There was no association between D80 AMH concentration and the total number of piglets born alive. Neither AMH or E2 serum concentration at D80 were associated with the number of piglets born alive, %stillborn, %mummified, WOI, age at first heat (Farm B only), pregnancy at first mating, whether mating resulted in a farrowing, whether gilts were culled, reasons for culling and days remaining within the breeding herd (P>0.05). In gilts that remained in the breeding herd until their third parity, concentrations of AMH and E2 were not associated with cumulative gestation length, total number of piglets born alive, total number of piglets born,
%stillborn or %mummified (P>0.05).
5.4 Discussion
The findings of this study extend those of our previous, smaller-scale experiments, providing evidence that serum concentrations of E2 at D80 are associated with some aspects of gilt reproductive potential. Lower concentrations of serum E2 were associated with greater total numbers of piglets born at parity three only. In Experiment One, lower concentrations of serum E2 at 60, 80 and 100 days of age were associated with greater numbers of piglets born
Page 149 of 213 alive and lesser proportions of piglets stillborn in the first three parities. Some of the inconsistencies between experiments may be attributed to in utero or genetic effects as dam and sire data were not obtained in Experiment One. Both results indicate that prepubertal E2 concentrations are related to future litter numbers. However, the number of gilts that were culled for poor litter qualities prior to parity three was low. Over both experiments, only three gilts after their second parity were culled for this reason at Farm B. Furthermore, E2 was not associated with cumulative litter size and survival for parities one to three. This indicates that using D80 E2 concentration to mark gilts that have greater reproductive capacity may not translate into greater gilt retention in the first three parities.
The relationship between D80 E2 concentration and future fecundity may be explained by the relationship observed between D80 E2 concentration and future uterine capacity observed in Experiment Two. Previous studies have shown increased uterine capacity to be associated with reduced early foetal loss and improved litter size (Wu and Dziuk, 1995;
Foxcroft et al., 2009). The relationship between E2 and the uterus is complex, partly because
ER are both upregulated and downregulated by E2 itself. Generally, the expression of ER is upregulated by oestrogens and down-regulated by progesterone (Batra and Iosif, 1989;
Ciesiółka et al., 2016). This is reflected in cycling sows as the expression of the predominant
ER in the uterus, ER-α, peaks at a time when E2 concentrations peak in the oestrus cycle and then declines with the subsequent progesterone increase (Sukjumlong et al., 2003). However, oestradiol reduces expression of ER-α mRNA in the endometrium of ovariectomised gilts
(Sahlin et al., 1990). This may also be the case in prepubertal gilts with underdeveloped ovaries and HPG axes, as it is in prepubertal lambs (Meikle et al., 2000), giving theory to why gilts with greater D80 E2 tend to have reduced uterine capacity later on.
It was previously proposed that gilts with lower D80 E2 concentration experience less inhibition from E2 on gonadotropin release, promoting earlier establishment of the HPG axis
Page 150 of 213 and follicular growth and sexual maturation. However, the findings failed to show a relationship between juvenile serum E2 concentration and age at first heat in the gilts observed at Farm B. The lack of relationship could be due to discrepancies between the event of ovulation and displaying behavioural signs of oestrus. Put simply, a threshold peak of E2 is needed to induce overt oestrous behaviour, but sub-threshold concentrations of E2 may still be enough to trigger an LH-surge that induces ovulation. So-called “silent” heats are known to occur when females experience their first oestrous cycles (Cronin, 1982). Additionally, gilts were not checked for heat until 150 days of age at Farm B, thus, any gilts that had their first oestrus prior to this age would not have been detected until their next heat. Repeat studies accounting for such sources of variability are necessary to determine whether E2 could be a useful marker of gilts that obtain puberty early.
In the present study, basal serum AMH concentrations in juvenile gilts were not associated with mating and litter qualities, similarly to Experiment One. There was also no relationship between AMH and early puberty attainment in this study, nor between AMH and antral follicle counts (a marker of ovarian function) in Experiment Two. Most previous literature investigating AMH as a marker of ovarian properties in other species have been carried out in mature females. There have been very few studies that have examined the association between circulating AMH concentration and reproductive measures in juvenile females, though, Torres-Rovira et al. (2014) found that prepubertal lambs with high AFC had
13-fold higher circulating AMH concentrations. Also, serum AMH concentrations were positively correlated to the size of the ovarian reserve in lambs (Torres-Rovira et al., 2014). In
8-year-old girls, AMH concentrations appear to be linked with precocial puberty (Lashen et al., 2013). Positive relationships between AMH and ovarian reserve and AFC have also been shown in sexually mature humans, rodents (reviewed by Broer et al. (2014)), cows (Ireland et al., 2008; Hirayama et al., 2012), ewes (Lahoz et al., 2014) and mares (Claes et al., 2014). A
Page 151 of 213 more detailed, longitudinal assessment is required to determine whether AMH can be a useful marker of ovarian properties and therefore fertility in female pigs.
In conclusion, a negative relationship between D80 E2 concentration and the total number of piglets born was found, but only at parity three and there was no association between D80
AMH concentration and litter size. The relationship observed between E2 concentration and litter size at parity three would not likely be a useful marker for increasing gilt retention rates, as gilts are at highest risk of culling prior to this point (Stalder et al., 2004; Hughes et al., 2010;
Plush et al., 2016). However, it may be a useful marker for choosing sows that have greater reproductive output later, which may ultimately lead to greater productivity after parity three.
Further studies, in which all sows are kept for at least three parities, are needed to determine whether D80 E2 concentration could be used to select gilts that have superior lifetime performance. Finally, a more detailed assessment of AMH concentration is needed to tease apart the complex relationships of this hormone with E2 concentration, ovarian properties and uterine capacity.
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5.5 Acknowledgements
This project was funded by Australian Pork Limited (Grant number: 2014/217). The authors would like to thank Assoc. Prof. P. Thomson for his guidance in statistical analysis and the staff at both farms for their assistance.
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CHAPTER SIX: EXPERIMENT FOUR
Serum Concentrations of AMH and E2 and Ovarian and Uterine
Traits in Gilts
Abstract
Poor sow retention due to reproductive failure is a common reproductive inefficiency amongst piggeries. This shows that traditional methods of gilt selection are inadequate and a marker of reproductive success is needed. The aim of this study was to determine whether circulating levels of AMH and E2 at D80 and D160 are associated with uterine and ovarian traits at D160.
Uterine weight, horn length and horn diameter were measured, and ovarian follicle counts were determined histologically. There was a negative relationship between both D80 and D160
AMH levels and D160 ovarian follicle populations. There was also a positive relationship between D80 E2 levels and uterine capacity in gilts that were pubertal at D160. The findings indicate that D80 and D160 AMH could be used to predict ovarian reserve and that D80 E2 levels may be indicative of uterine capacity in precocial gilts.
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6.1 Introduction
Globally, poor sow retention is a common reproductive inefficiency amongst piggeries.
In Australia, it is estimated that around 40% of sows are culled prior to parity three (Hughes et al., 2010; Serenius et al., 2006; Plush et al., 2016). This premature culling has resulted in an average herd parity of just 2.7 (Plush et al., 2016). This is concerning considering gilts only become profitable between parities three to six (Stalder et al., 2004). This is attributed to younger sows having lower pregnancy rates, smaller litter sizes, higher chances of savaging and greater non-productive days (D’Allaire et al., 1999), as well as to the higher costs required to read piglets bred from young females (Kroes et al,. 1979). Increasing the average herd parity by a single unit has been shown to be equivalent to a 0.5% increase in lean pork percentage at slaughter (Stalder et al., 2004). In other words, increasing sow retention rates would decrease the input required per kilogram of lean pork. Not only would this be in the best interest of pork producers from an economic standpoint, but also from an efficiency, sustainability and welfare perspective.
It has been known for some time that, in Australian piggeries, the most significant factor contributing to the premature culling of replacement breeding females is reproductive failure
(Hughes et al., 2010). However, the reproductive traits that are considered at gilt selection typically remain limited to teat number, body conformation and dam performance. Selection normally occurs around 160 days of age and it is not until well after entry into the breeding herd that a gilt’s reproductive potential becomes more evident. Maintaining unproductive individuals up to this point is costly. Hence, an early-age predictive marker for reproductive success is required to aid with the gilt selection process.
Mammals are born with a limited number of ovarian follicles. The number of dormant primordial follicles, which constitutes ovarian reserve, is an important determinant of
Page 155 of 213 reproductive potential in females (Monniaux et al., 2014). Obtaining counts of these microscopic follicles is relatively difficult. However, counts of more mature, growing follicle populations can reflect ovarian reserve. This is due to the ovary recruiting primordial follicles to grow in cohorts. The size of these cohorts, at particular stages of development, is proportionate to the pool of primordial follicles the ovary has available to recruit from. The fleets of follicles may not remain proportionate to ovarian reserve at all stages of development due to the differing rates of apoptosis of follicles along the way. Warren et al. (2015) have shown that in pigs, the number of growing follicles at intermediate and primary stages are correlated with ovarian reserve, but populations of follicles at later stages are not.
Reproductive potential is also associated with antral follicle populations within ovaries.
In young cycling cattle, antral follicle counts (AFC) have been directly linked to the number of morphologically healthy oocytes (Ireland et al., 2008). Further, AFC is a good marker of ovarian response to exogenous gonadotrophins in assisted reproductive technology cycles (10).
In species other than the pig, the circulating level of anti-Mullerian hormone (AMH) has been shown to be a good indicator of ovarian reserve and AFC in cattle (Hirayama et al.,
2012; Batista et al., 2014); sheep (Torres-Rovira et al., 2014); mares (Claes et al., 2014); goats
(Monniaux et al., 2011); humans (La Marca et al., 2010); mice (Kevenaar, et al., 2006)). This may be attributed to antral follicles being the site of maximal AMH production and AFC being proportionate to primordial follicle populations in these species. The relationship between circulating AMH and follicle populations in pigs is yet to be distinguished. There is some evidence that indicates AMH may have a unique role in pigs. Expression commences in recruited primordial follicles and, in most species, is maximal at the antral stage before diminishing. However, in pigs, AMH continues to be expressed at similar levels to the antral stages and intensifies in preovulatory follicles (Almeida et al., 2018).
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Circulating oestradiol (E2) is another candidate hormone that could be indicative of gilts with high reproductive potential. We previously demonstrated that circulating E2 levels in juvenile gilts aged 60, 80, or 100 days of age were positively associated with the probability of stillbirth and negatively associated with the number of piglets born alive in parities one to three (Steel et al., 2018). This period was chosen because this is a critical window for ovarian development in pigs (Schwarz et al., 2008) and the levels at 80 days of age were found to be the most useful to measure. In humans, girls who experience sexual maturity early appear to have lower serum E2 levels than normal-maturing girls when compared at the same sexual developmental stage (Bidlingmaier et al., 1977). Considering the findings of Bidlingmaier et al. (1977) and given that E2 exerts a negative-feedback effect on the hypothalamic-pituitary- gonadal (HPG) axis prior to sexual maturity, it may be that the lower E2 levels in juveniles promote earlier establishment of the HPG axis. In gilts, this early development results in regular cycling patterns being established earlier, leading to an increased number of piglets born alive, and higher retention rates to parity three (Nelson et al., 1990; Patterson et al., 2010).
Furthermore, uterine traits such as weight and horn length in gilts aged 160 days of age have been shown to be linked with uterine capacity (Young et al., 1996, Lents et al., 2014) and there is evidence that increasing uterine capacity alleviates issues associated with overcrowding, including early foetal losses, low birth weights and reduced litter size (Foxcroft et al., 2009).
Thus, we hypothesized that the levels of ovarian hormones in juvenile gilts are related to their ovarian and uterine properties.
The aim of this study was to measure circulating levels of AMH and E2 in young gilts at 80 and 160 days of age an examine their relationship with ovarian and uterine traits at the age that selection normally occurs.
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6.2 Methods
Animals and ethics
All animal procedures were conducted with prior institutional ethical approval under the requirements of the NSW Prevention of Cruelty to Animals Act 1985, in accordance with the National Health and Medical Research Council /Commonwealth Scientific and Industrial
Organisation / Australian Animal Commission’s Code of Practice for the Care and Use for
Animals for Scientific Purposes.
Fifty-four multiplier gilts (F1: Large White x Landrace, PrimeGrowTM Genetics,
Corowa, NSW) aged 80 days of age were used in this study. There were five pairs, two trios and one quartet that originated from the same litter.
From weaning at 28 days of age up until 70 days of age gilts were housed in conventional weaner pens in groups fo 45. Gilts were moved to large commercial grower pens and housed on concrete slatted floors in groups of 200 until they were around 130 days old.
Gilts were then re-grouped into pens of 50 and housed in conventional finisher pens on concrete slatted and solid floors (50:50) until around 160 days when gilts were slaughtered.
The feeding system varied throughout the gilts’ lifetime. From weaning until about 125 days of age, gilts were given ad libitum access to a number of commercial weaner and grower diets. Gilts were then fed a specific gilt developer diet ad libitum until slaughter.
Blood samples and assays
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Blood samples were collected over consecutive weeks from two groups of gilts (Group
1: N = 26, Group 2: N = 28) at 80 days of age and again at 160 days of age (±4 days). Blood was collected into serum separator tubes using 18 g x 25 mm vacutainer needles and left to clot for 2 h at room temperature. The tubes were then centrifuged at 1000 x g for twenty minutes and sera separated and stored at 80ºC for less than two months. Five serum samples at D160 were excluded.
After thawing, serum samples were diluted 1:2 in PBS, and AMH was quantified using a competitive inhibition ELISA kit (CEA228Po: Cloud-Clone Corp, TX,USA) using monoclonal antibodies specific for porcine AMH. In-house validation of this kit was performed as described previously (Steel et al., 2018). The minimum detectable dose for the assay kit was
135.8 pg/mL. The intra- and inter-assay precision was <11.4% and <12.9%, respectively.
Oestradiol was measured using a competitive inhibition ELISA kit (CEA461Ge: Cloud-
Clone Corp, TX, USA) using a monoclonal antibody specific to E2. The minimum detectable dose for E2 was 46.2 pmol/L and the intra- and inter-assay precision was <2.9% and <14.5%, respectively.
Assessment of uterine and ovarian development
At 160 days of age, gilts were slaughtered at an on-site abattoir. Trimmed carcass weight (CW) and P2 back-fat scores were recorded and reproductive tracts were recovered.
The uteri and ovaries from each animal were weighed and the lengths and diameters of each uterine horn were recorded. Uterine diameter measurements were taken at the tubal, middle and cervical ends of each uterine horn. One ovary from each gilt was fixed in 10% neutral buffered formalin for 72 h in preparation for histological analysis. Two uterine measurements were excluded due to damage.
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Histological preparation and analysis
Ovaries were sliced in half before being embedded in paraffin wax. Starting from the cut in the midline, half of each embedded half-ovary was serially sliced in 5 μm sections using a rotating microtome (Leica®, Wetzlar, Germany). Every 40th section was placed in a water bath containing foetal calf serum to aid in fixing the section onto a glass slide. Slides were dried overnight prior to staining with Harris’ Haematoxylin and alcoholic Eosin Y (0.01%).
Slide sections were photographed via the Zeiss Axioscan.Z1 at 20x magnification.
Follicles that contained cross-sections of the oocyte were counted and classified by follicle type (primary, secondary, preantral, antral) as described previously (Warren et al.,
2015) with the exception that intermediate follicles were classified as primary follicles. Atretic follicles were not classified by follicle type. Gilts were classified as cycling or non-cycling by the presence of corpora lutea (CL). The total number of follicles was determined according to the methods described by Ireland et al. (Ireland et al., 2008). For each gilt, total follicle count was calculated by multiplying the total number of follicles for each quarter-ovary by a correction factor of 320 (40 x 4 x 2: 40 accounts for counting every 40th section; four accounts for only slicing one-quarter of the ovary; and two accounts for only testing one ovary per gilt).
Statistical analysis
Statistical analysis was conducted using R software version 3.3.3 (R Foundation for
Statistical Computing, Vienna, Austria). A significance determination threshold of α = 0.05 was used for all statistical analysis in this study. Replicate (Rep), dam and sire were considered as random factors and were nested to account for genetic and in utero effects. Paired t-tests were performed to assess differences between right and left ovarian and uterine traits. Principal
Page 160 of 213 component analysis (PCA) was performed using the stats::prcomp function in R to combine uterine weight, length and diameter parameters into principal components (PC) that could best summarise the variation between uteri, and to combine small, primary, secondary, preantral, antral and atretic follicle counts into PCs that best describe the variation in ovarian follicle populations between gilts. Data were scaled prior to PCA. The gamm::mgcv function was used to fit multi-dimensional spline models to assess interactions between continuous variables and non-linear relationships. Linear relationships were assessed using lme4::lmer and lme4::glmer for continuous and binary outcome variables. Carcass weight and P2 fat scores and, where applicable, pubertal status at D160 (as determined by the presence of CL) were considered as predictor variables. Interactions were assessed and removed if insignificant. Missing values were omitted from the analyses.
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6.3 Results
Serum hormone levels, carcass traits, ovarian properties
Table 16 shows a summary of the serum AMH and E2 levels at D80 and D160 as well as carcass, ovarian and uterine measurements at D160 for 54 gilts. Serum levels of AMH increased an average of 8.3% from D80 to D160 ( t(47) = -3.17, P = 0.003), while E2 levels increased around 11.9% ( t(48) = -3.74, P < 0.001). Ten gilts were pubertal by D160, as indicated by the presence of CL.
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Table 16: Descriptive statistics for serum concentrations of AMH and E2 in gilts at 80 and 160 days of age and carcass, uterine and ovarian properties at 160 days of age
Age (days) Median Mean SD Range
Serum hormone levels:
AMH (ng/mL) 80 11.1 10.9 1.3 (7.9-13.9)
AMH (ng/mL) 160 12.0 11.8 1.2 (8.9-14.0)
E2 (pmol/L) 80 94.7 94.7 15.7 (65.5-131.0)
E2 (pmol/L) 160 104.2 106.2 14.7 (72.6-143.1) Carcass traits: CW (kg) 160 78.0 78.1 10.0 (58.1-112.4) P2 Fat score (mm) 160 13.2 13.4 2.9 (8.4-24.0) Uterine traits:
Weight (g) 160 77.6 102.0 70.9 (26.7-370.9)
Diameter (mm) 160 12.3 12.3 2.3 (8.6-17.2)
Length (mm) 160 496.3 533.4 176.5 (252.5-1450.0) Ovarian follicle counts: Primary 160 49,440 51,543 24,376 (14,000 – 120,000) Secondary 160 31,360 37,374 21,636 (9,600 – 104,640) Preantral 160 3,840 5,502 5,479 (0 -29,964) Antral 160 5,600 7,004 5,430 (640 – 29,091) Atretic 160 22,880 28,756 21,970 (1,920 – 107,636) Total (Healthy) 160 92,840 101,423 44,126 (42,880-275,814) Total (Healthy + Atretic) 160 112,427 130,179 58,613 (52,160-383,451) AMH=Anti-Müllerian hormone; E2=Oestradiol, CW=Trimmed carcass weight;
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Correlations between ovarian hormones and uterine and ovarian traits
Levels of serum E2 were positively correlated with preantral follicle counts at D160
(Table 17).
Table 17 Pearson’s correlation coefficients between ovarian hormones at 80 and 160 days of age (D80 and D160, respectively) and uterine and ovarian traits at D160.
D80 D160
AMH E2 AMH E2
Uterine Weight −0.25^ 0.23 0.02 −0.08
Horn Diameter −0.23 0.04 0.10 0.02
Horn Length −0.25^ 0.21 0.17 −0.09
Primary 0.02 0.02 0.21 0.00
Secondary 0.04 −0.18 0.23 −0.03
Preantral 0.00 −0.24^ −0.07 0.37*
Antral 0.02 −0.13 0.08 0.26^
Atretic −0.10 −0.16 0.18 0.11
^p < 0.1 *p < 0.05; AMH: Anti-Müllerian hormone; E2: Oestradiol
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Intra- and inter-hormone correlations
The inter-age and inter-hormone correlations can be found in Table 18. There was a negative correlation between E2 levels taken at different ages, but not between AMH measurements. Further, AMH and E2 levels at D160 were negatively correlated, but this relationship was not observed at D80.
Table 18: Pearson’s correlation coefficients between ovarian hormones at 80 and 160 days of age (D80 and
D160, respective).
D80 D160
AMH E2 AMH E2
AMH
D80 E2 0.23^
AMH 0.13 -0.05
D160 E2 -0.07 -0.32* -0.29* ^P<0.1 *P<0.05; AMH=Anti-Müllerian hormone; E2=Oestradiol
Intra-uterus correlations
There were significant correlations between all uterine traits measured (Table 19).
Table 19: Pearson’s correlation coefficients between uterine traits at 160 days of age.
Uterine Horn Horn Weight diameter Length
Uterine Weight
Horn diameter 0.76 * Horn Length 0.84 * 0.52 * * P<0.001;
Uterine mass indices (UMIs)
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Due to the significant intra-uterine trait correlations, PCA was performed. The PCA revealed two PC values that accounted for the significant variation in uterine traits (80.7% and
16.3%, respectively). The first PC characterises the overall size of the uterus as it is proportionate to uterine weight (loading = 0.62), horn length (loading = 0.57) and horn diameter
(loading = 0.54) and is referred to as the UMIsize. The second PC (UMIshape) describes uterine shape as it is proportionate to horn length (loading = 0.64) and uterine weight (to a weaker extent; loading = 0.08) and strongly inversely proportionate to horn diameter (loading = -0.76).
A visual summary of UMI values for each uterus is shown in Figure 13.
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Figure 13: Principal component analysis for uterine traits. Two variables, or Uterine Mass Indices (UMIs),
UMIsize, and UMIshape, were created to summarise uterine weight, horn length and horn diameter measurements for each gilt. The two UMIs cumulatively account for 97.0% of the variation observed between uteri. Higher
UMIsize values correspond to uteri with greater uterine weight, horn length and horn diameter, whereas, higher
UMIshape values correspond to uteri with greater horn length and uterine weight and lesser horn diameter. Each point on this graph represents a uterus, plotted according to their two UMI values. Points distributed towards the arrowheads of the red vectors have greater measurements of the corresponding uterine trait (labelled in red) than points located towards the tail-end of the vectors. Positively correlated traits have vectors that point in the same direction, negatively correlated traits have vectors that point in opposite directions and traits that have no correlation have vectors that are perpendicular to each other.
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Intra-ovary correlations
There were significant correlations between follicle types including between primary and secondary, secondary and atretic, preantral and antral, preantral and atretic and antral and atretic (Table 20)
Table 20: Pearson’s correlation coefficients between ovarian follicle populations at 160 days of age.
Primary Secondary Preantral Antral Atretic Primary Secondary 0.52 * Preantral 0.17 0.28^ Antral 0.22 0.23 0.84 * Atretic 0.22 0.48 * 0.61 * 0.72 *
^P<0.1 *P<0.001;
Ovarian Follicle Indices (OFIs)
Due to the significant intra-ovary trait correlations, PCA was performed. The PCA revealed two PC values that accounted for the significant variation in ovarian follicle populations between gilts (55.8% and 24.4%). The first PC characterises the overall follicle populations as it is proportionate to all follicle types measured and is referred to as Ovarian
Follicle Index (OFI)tot. The second PC characterises the ratio of primary and secondary follicles to preantral, antral and atretic follicles and is referred to as OFIProp. A visual summary of OFI values for each ovary pair is shown in Figure 14 and the variable loadings for each OFI can be found in Table 21. There were no significant differences in either OFI values between cycling and non-cycling gilts (P>0.05).
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Figure 14: Principal component analysis for ovarian follicle counts. Two variables, or Ovarian Follicle Indices
(OFIs), were created to summarise the primary, secondary, preantral, antral and atretic follicle counts of each gilt.
This graph shows two OFIs that were created, OFItot and OFIprop, which cumulatively explained 80.2% of the variation in follicle populations observed between gilts. Each point on this graph represents an individual gilt, plotted according to her two OFI values. Gilts with higher OFItot values have greater follicle numbers of all types, whereas gilts with higher OFIprop values had greater primary and secondary follicle counts and lesser preantral, antral and atretic follicle counts. Points distributed towards the arrowheads of the red vectors have greater counts of the corresponding ovarian follicle type (labelled in red) than points located towards the tail-end of the vectors.
Positively correlated traits have vectors that point in the same direction, negatively correlated traits have vectors that point in opposite directions and traits that have no correlation have vectors that are perpendicular to each other.
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Table 21: Table of variable loadings from the principal component analysis
Loading Follicle Type OFItot OFIprop
Primary 0.28 0.66 Secondary 0.36 0.57 Preantral 0.50 -0.33 Antral 0.52 -0.34 Atretic 0.51 -0.10 OFI = Ovarian Follicle Index
Association between E2 levels and UMIsize
There was a two-way interaction effect between pubertal status and D80 E2 on D160
UMIsize. There was a positive, linear association between D80 E2 levels and UMIsize in cycling gilts at D160 (Figure 15; slope = 0.13, s.e = 0.03, χ2(1) = 13.7, P < 0.001) but no relationship between D80 E2 levels and UMIsize in gilts that were non-cycling at D160 (P>0.05).
Cycling Gilts
Figure 15: The relationship between serum oestradiol (E2) levels at 80 days of age (D80) and Uterine Mass Index
(UMI)size in gilts that were cycling at D160 (slope=0.13, SE = 0.03, χ2(1)=13.7, P<0.001)
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Association between E2 levels and OFIprop
The relationship between E2 at D80 and OFIprop at D160 varied with pubertal status
(Figure 16; P=0.027, Adj-R2 = 0.14). There was no association between AMH and OFIprop in gilts that had CL present, whereas, there was a negative relationship between E2 at D80 and
OFIprop in gilts that did not have CL apparent at D160. Such that, non-cycling gilts with elevated
D80 E2 (>110 pmol/L) had ovaries with lower primary and secondary follicle counts and greater preantral, antral and atretic follicle counts.
Non-cycling Gilts
Figure 16: Smoothing spline showing the relationship between serum oestradiol (E2) levels at 80 days of age
(D80) and Ovarian Follicle Index (OFI)prop values in non-cycling gilts at D160 with Bayesian confidence intervals.
Greater serum E2 levels corresponded to lower the OFIprop values (P=0.027, Adj-R2 = 0.14), which represents ovaries with a smaller ratio of primary and secondary follicles to preantral, antral and atretic follicles.
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Association between AMH levels and OFItot
There was a three-way interaction effect of D80 AMH, D160 AMH and pubertal status on D160 OFItot (P = 0.045, Adj-R2 = 0.12). In non-cycling gilts, there was an interacting association between D80 and D160 AMH levels and D160 OFItot (Figure 17), whereas there was no association in gilts that had CL present at D160. The relationship was such that, the lower the D80 and D160 AMH levels, the greater the overall follicle counts (primary, secondary, preantral, antral and atretic) in gilts that were non-cycling at D160.
(a) (b)
Figure 17: Three-dimensional smoothing spline model showing the (a) two-way interaction effect between serum anti-Mullerian hormone (AMH) levels at 80 (D80) and 160 days of age (D160) on OFItot in non-cycling gilts at
D160 (P=0.045, Adj-R2 = 0.12). The graph is also shown (b) with standard errors (in grey), rotated for perspective.
Pubertal status was determined by the presence of corpora lutea at D160. Non-cycling gilts with lower D80 and lower D160 AMH had greater OFItot values. Greater OFItot values correspond to gilts with greater overall follicle counts (primary, secondary, preantral, antral and atretic).
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Other body condition, uterine and ovarian parameters
When comparing gilts that were cycling with those that were non-cycling at D160, there were no significant differences in either D80 or D160 ovarian hormone levels, nor were there differences in carcass weight or P2 fat scores (p > 0.05). Measurements of serum AMH and E2 at D80 and D160 were not associated with either UMIshape, CW or P2 fat scores at D160 (p >
0.05). Values of UMIsize, OFItot or OFIprop were not associated with CW or P2 fat scores (p >
0.05). However, UMIshape varied significantly with P2 fat scores (slope = 0.08, SE = 0.04,
χ2(1)= 4.32, p = 0.038), such that gilts with greater P2 fat scores had heavier uteri with longer, thinner uterine horns.
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6.4 Discussion
This study examined the relationships between ovarian hormones at D80 and D160 and ovarian and uterine traits at D160. Examining this relationship is difficult due to complex ovarian follicle growth dynamics and the significant correlations amongst uterine traits. In order to achieve this, principal component analyses were performed, creating simple summary variables that combined the uterine traits and counts of different follicle types to explain the variation between uteri and ovaries of individual gilts. When considering ovarian follicle populations, most of the variation between gilt ovaries was due to overall follicle counts (as summarised by OFItot), and, to a lesser extent, the ratio of primary and secondary follicles to preantral, antral and atretic follicles (as summarised by OFIprop). The ovarian follicle populations measured were independent of carcass weight and fat content. This is consistent with the findings of Schwarz et al. (2013) that showed age- and weight-matched gilts have great variation in ovarian activity. There is some evidence that antral follicle populations can vary with growth (Knox, 2019). However, these measurements were not able to be obtained due to the study being conducted in a commercial production system in which growth rate is not typically measured. Moreover, there were positive correlations between primary and secondary follicle numbers as well as between secondary and atretic and preantral, antral and atretic follicle numbers. A previous study by Warren et al. (2015) was similar in that it found pre-antral and antral follicle counts to be positively correlated in porcine ovaries, but differed in that it found no correlation between primary and secondary follicles, and additional positive correlations between secondary and preantral, primary and preantral and primary and antral follicles.
Results revealed that juvenile (D80) levels of serum E2 were associated with future ovarian follicle populations at D160, the age of selection into the breeding herd. Serum E2
Page 174 of 213 levels at D80 were not linearly correlated with individual follicle counts at D160 in gilts grouped as a whole. However, when grouped according to their pubertal status, there was a non-linear relationship between D80 E2 levels and the ratio of different follicle types at D160
(OFIprop) in non-cycling gilts. More specifically, as D80 E2 levels increased over 100 pmol/L,
OFIprop values declined, equating to ovaries with smaller proportions of primary and secondary follicles compared to the other, larger (and high-E2 producing) follicle types. Serum concentrations of E2 at D160 were not associated with either OFI, regardless of cycling status.
There were no significant correlations between single measurements of serum AMH concentrations and uterine and ovarian traits when gilts were grouped as a whole, nor was there a correlation between D80 and D160 AMH levels. However, non-linear models showed a two- way interaction between D80 and D160 AMH levels and ovarian follicle populations (OFItot) in non-cycling gilts, such that gilts with lower levels of AMH at both D80 and D160 had greater ovarian follicle populations at D160. The findings suggest that using a combination of both measurements could predict ovarian follicle populations.
Primordial follicle count (ovarian reserve) was not determined in the present study.
Previously, Warren et al. (2015) found that the number of primary follicles in pig ovaries was positively correlated with the number of primordial follicles, suggesting that the gilts with lower AMH levels at D80 and D160 (and therefore OFItot) in the present study had a greater ovarian reserve. This negative association contrasts those in other species where AMH has been found to be positively associated with ovarian reserve and antral follicle counts. It should be noted that most livestock studies have examined this relationship in adult females undergoing superovulation cycles, whereas juvenile gilts were examined in the present study.
However, though AMH appears to oppose the effects of FSH within the ovary (Di Clemente et al., 1994), recent studies have found that it stimulates FSH production in the pituitary of
Page 175 of 213 prepubertal rats (Garrel et al., 2016). A negative relationship between AMH levels and HPG development and/or ovarian activity has also been demonstrated in humans as girls with lower
AMH at eight years of age are much more likely to have precocial puberty (Lashen et al., 2013).
Thus, the negative relationship between AMH and ovarian follicle populations in this study may be due to AMH having different actions depending on the target tissues and maturational age.
Both AMH and E2 were found to be associated with ovarian follicle populations but each was associated with a different OFI and, therefore, each represented a different ovarian follicle population dynamic; that is, E2 was associated with the ratio of different follicle types
(OFIprop) and AMH was associated with overall follicle counts (OFItot). Previously, the porcine ovary has been classified morphologically into two types: The “grape type” with a large number of large surface antral follicles (>5 mm) and atretic follicles and a low number of smaller antral follicles, and the “honeycomb type”, with a large number of small follicles and no large antral follicles (Schwarz et al., 2013; Dufour et al., 1988). When prepubertal ovaries of each type were observed by laparoscopy, after a few days these proportions would change synchronously, and once pubertal, the disparity in follicle numbers between ovary types evened out, showing that porcine ovaries undergo stages of follicular growth in waves (Schwarz et al.,
2013; Dufour et al., 1988). Due to the relationship between AMH levels at D80 and D160 and
OFItot, the two AMH concentrations determined at D80 and D160 are proposed to be more predictive of ovarian reserve in gilts than the single E2 concentration determined at D80. This could explain why ovarian hormones were only reflective of growing follicle populations in non-cycling gilts and not those that had already begun cycling at D160.
There was no association between D80 and D160 AMH levels, regardless of pubertal status at D160. A study in sheep also showed no intra-individual correlation of AMH levels
Page 176 of 213 between pubertal (3, 4.5 and 6 months) and adult ages (19 months) (Lahoz et al., 2014). The lack of intra-individual correlation of AMH levels may be due to prepubertal AMH peaks being experienced at different ages. Longitudinal studies are required to profile AMH in more detail and determine the maturational age at which AMH would most strongly predict ovarian potential.
When considering uterine traits, most of the variation in uterine measurements between gilts at D160 was due to differences in overall uterine size (as summarised by UMIsize) and a small amount of the variation was due to different uterine shape; that is, uteri tended to be either heavier with longer, thinner horns or lighter with shorter, thicker horns (as summarised by UMIshape). Though neither UMI values or ovarian hormone levels varied between cycling and non-cycling gilts, pubertal status appeared to affect the relationship between ovarian hormones and uterine traits. That is, there was a positive relationship between D80 E2 levels and UMIsize in gilts that were cycling at D160, independent of carcass weight and P2 fat scores.
This indicated that gilts with greater D80 E2 levels had greater uterine capacity (Young et al.,
1996; Lents et al., 2014). On the other hand, UMIshape values were independent of ovarian hormone measurements, but varied significantly with P2 fat scores, such that gilts with greater
P2 fat scores had heavier uteri with longer, thinner uterine horns. The lack of association between CW and UMI values, which are positively proportionate to uterine weight, does not support previous findings that uterine weight is positively correlated with gilt growth and body weight (Tummaruk, et al., 2014).
6.5 Conclusions
The results showed a relationship between D80 E2 levels and the proportion of primary and secondary follicles in ovaries at D160. There was also a two-way interaction between D80 Page 177 of 213 and D160 AMH levels on overall follicle populations in gilts that were non-cycling at D160, such that the lower the circulating AMH levels at both D80 and D160, the greater the follicle populations. It was deduced that AMH levels at D80 and D160 are predictive of ovarian reserve and that D80 E2 levels are reflective of the follicular waves that occur in gilts prior to puberty.
There was a positive association between D80 E2 levels and uterus size in gilts that were pubertal at D160, but the strength of this finding is limited due to the small sample size. Overall, the findings suggest that gilts with lower than average serum levels of AMH at D80 and D160 and E2 levels lower than 100 pmol/L at D80, have greater reproductive potential. Future longitudinal studies are needed to further characterise the relationships observed and to determine whether such hormonal measurements could be effectively applied on-farm to aid in the gilt selection process.
6.6 Acknowledgements
This project was funded by Australian Pork Limited (Grant number: 2014/217). The authors would like to thank Assoc. Prof. P. Thomson for his guidance in statistical analysis and the farm staff for their assistance.
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CHAPTER SEVEN: GENERAL DISCUSSION
7.1 Summary
This thesis presents results from four studies investigating the ovarian hormones E2 and
AMH as potential markers for future reproductive success in gilts at a young age. The objectives of this thesis were:
• To determine the serum concentrations of E2 and AMH in juvenile gilts prior to and
after treatment with gonadotrophins at 60, 80 and 100 days of age
• To determine whether the serum concentrations of E2 and AMH at either 80 or 160
days of age correlate with uterine and ovarian properties at slaughter
• To determine whether the serum concentrations of E2 and AMH at either 60, 80 or 100
days of age correlate with fertility and reproductive performance
The overarching hypothesis was that the production of the ovarian hormones E2 and AMH during a period of ovarian development (60 to 100 days) correlates with reproductive properties and fertility in adulthood.
Prior to experimentation, a commercially available ELISA kit for porcine AMH was verified for measuring serum AMH in juvenile gilts. The initial experiment was conducted to examine serum AMH and E2 levels in gilts aged 60, 80 and 100 days, at 0, 2 and 4 days after gonadotropin administration. Mating, litter and culling information subsequently collected from these animals over three parities were then analysed with the corresponding hormone data to identify any associations. The second experiment examined whether D80 E2 and AMH levels were associated with ovarian and uterine properties upon slaughter at D160 (the age that selection into the breeding herd commonly occurs). A larger-scale trial was then conducted across two geographically and genetically different farms to validate the results of Experiment Page 179 of 213
One, comparing serum AMH and E2 levels at D80 and mating, litter and culling information over three parities. The final experiment was longitudinal as the hormone measurements were conducted at D80 and again just prior to slaughter at D160. Similar to the second experiment, ovarian and uterine traits were measured, however, the assessment of the ovarian traits was more detailed.
The results of the four studies suggest that D80 serum E2 levels in juvenile gilts are associated with future fecundity. The relationship between AMH and E2 levels at a juvenile age and uterine traits was inconsistent and warrants further investigation. Moreover, the results showed there was an association between juvenile levels of AMH and ovarian follicle populations, but this did not translate to differences in reproductive output or premature culling.
The results presented highlight the uniqueness and complexities of endocrinology in the pig and emphasise the difficulty of determining reliable hormonal markers for future reproductive success in a production setting.
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7.2 Discussion
AMH ELISA Assay
Up until recently, ELISAs specific for porcine AMH were not readily available.
Previous studies using ELISAS with non-porcine-specific AMH antibodies reported drastically lower intrafollicular concentrations of the hormone in pigs compared to other species, suggesting porcine AMH had low affinity with antibodies in the assay (Monniaux et al., 2012).
In Experiment One, we aimed to use a porcine-specific AMH ELISA assay (CEA228Po:
Cloud-Clone Corp, TX, USA) to determine serum levels of AMH in prepubertal gilts for the first time.
In-house verification of the porcine-specific AMH ELISA assay was conducted.
Dilutional linearity was examined via serial dilutions of two standard-spiked serum samples and back-calculated concentrations were found to be within the acceptable range of 20% of real values. Parallelism was also confirmed by comparing the absorbance curves from serial dilutions of two serum samples and a standard. The minimum detectable limit of the kit was
354.9 pg/mL and the intra- and inter-assay variations were <11.4% and <12.9%, respectively.
The verification process that was conducted in-house was restricted due to financial, ethical and scientific limitations. It should be noted that the cross-reactivity between AMH and analogues was not examined and dilutional linearity and parallelism was determined using serum from in-tact females. Prior to the commencement of the studies in this thesis, the number of porcine-specific AMH ELISA kits available on the market was very limited and most were financially unobtainable. Therefore comparison to other porcine-specific AMH kits was not examined and the repeatability of the results using different assays is unknown. Further, this
Page 181 of 213 assay did not discriminate between the biologically active and inactive forms of AMH and future research should consider this.
Quantifying serum AMH in juvenile gilts
Once the porcine-specific AMH ELISA kit was verified, AMH levels in juvenile gilts
60, 80 and 100 days of age were quantified, which, to the best of our knowledge, had yet to be reported. Compared to other species including cattle, sheep and humans, juvenile gilts had greater circulating AMH levels (Kelsey et al., 2011; Lashen et al., 2013; Guerreiro et al., 2014;
Lahoz et al., 2014; Torres-Rovira et al., 2014; Baruselli et al., 2015) which may be due to pigs having greater ovarian follicle populations (Warren et al., 2015), and polyovular cycles. Pigs also have a unique intra-follicular expression pattern of AMH. Follicles begin expressing AMH in the primordial phase, similarly to other species (Almeida et al., 2018). However, in other species, AMH expression peaks in the early antral phase and diminishes in preovulatory follicles, whereas, in the pig, AMH continues to be expressed at similar levels throughout the antral stages and intensifies in preovulatory follicles and continues to be expressed in the CL
(Almeida et al., 2018). Further cross-species studies would be beneficial in supporting the proposal that circulating AMH levels reflect the dynamics of ovarian follicle development that are specific for each species.
Studies examining serum AMH and E2 levels and production
parameters
In species other than the pig, AMH has been shown to be a good marker of ovarian follicle count, which, in turn, is indicative of the level of HPG development and gonadotropin
Page 182 of 213 responsiveness. E2 hormonal profiles in response to gonadotropin administration have also been shown to indicate fertility. In order to examine this in pigs, we measured serum E2 and
AMH levels at 0, 2 and 4 days after PG600 injection in juvenile gilts aged 60, 80 or 100 days and related the hormone levels to mating outcome, gestation length, total number of piglets born, number of piglets born alive, mummified and stillborn, and weaning to oestrus intervals
(WOI) for parities one to three.
Results from Experiment One showed:
• Basal E2 levels were positively associated with the probability of stillbirth
• Serum E2 levels before and after gonadotropin stimulation were negatively
associated with the number of piglets born alive.
• Serum AMH levels after gonadotropin treatment were associated with gestation
length.
• There was no effect of age (60, 80 or 100 days) on any of these relationships
Of the hormonal markers assessed, the most economical to use in a production setting would be basal levels, eliminating the cost and labour associated with injecting pigs with
PG600 and collecting and assaying multiple blood samples. Though the best predictor for the number of piglets born alive were E2 levels taken four days after PG600 injection, basal E2 levels were also able to predict the number of piglets born alive. The only trait that was associated with hormone levels after gonadotropin stimulation and not associated with basal hormone levels was gestation length, the value of which in a production setting can be argued.
Thus, for subsequent experiments, gonadotropin injections were not performed. As age had no significant effects on any of the relationships observed between ovarian hormones and production characteristics, gilts that were 80 days of age were used in subsequent experiments.
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In Experiment Three, a larger number of animals was assessed at two geographically separated farms. Results showed:
• Basal levels of E2 were negatively associated with total born (parity three only)
The results from both experiments indicate that basal levels of serum AMH in juvenile gilts were not associated with mating and litter qualities and that that lower basal levels of E2 in juvenile gilts were associated with future fecundity. This finding suggests that gilts with lower E2 levels at D80 experience less negative feedback on gonadotropin release during this time of ovarian development, causing earlier establishment of the HPG axis and an increase in basal FSH levels, thereby resulting in earlier onset of puberty and improved reproductive capacity at the first mating.
Both experiments showed no association between the hormone levels and the chance of culling prior to parity three. It was interesting to see that even in parities where litter size varied greatly, culling due to litter size was rare. This raises the question of whether farms are culling gilts appropriately; however, the sample size was insufficient to strongly support this proposal. The results suggest that the selection of gilts on the basis of their juvenile E2 levels may result in a greater number of piglets/sow/year but that this may not necessarily lead to an increase in sow retention rates under current practice.
Studies examining serum AMH and E2 levels and ovarian and uterine
properties at slaughter
Evidence in the literature shows that AMH is positively associated with ovarian follicle populations in humans, cattle, sheep and rodents (refer to Table 1). Further, there is evidence that circulating AMH and E2 may be linked with precocial puberty in women and it is well
Page 184 of 213 known that E2 is responsible for uterine growth and development. Thus, it was hypothesised that the ovarian hormones AMH and E2 related to the age of puberty onset, AFC and uterine dimensions in gilts. Whether these two hormones in gilts aged 80 days of age were associated with ovarian and uterine properties upon slaughter at 160 days of age was examined in
Experiment Two. Uterine dimensions, ovarian weight and small (1-3mm), medium (3-6 mm) and large (>6mm) surface antral follicle counts were recorded. The results of Experiment Two showed:
• An interaction between AMH and E2 levels at 80 days of age and uterine shape at
160 days of age, such that, lower AMH and E2 levels at 80 days of age were
associated with heavier uteri and longer and thinner uterine horns
• No relationship between ovarian hormones and surface AFC
Though there was an association between D80 AMH levels and uterine mass at 160 days of age, the relationship between D80 E2 levels and uterine shape was much stronger.
The lack of relationship between ovarian hormones and surface antral follicle counts did not support the original hypothesis. However, Warren et al. (2015) found that in pigs, ovarian reserve was positively associated with the number of primary follicles, and not AFC, as it is in cattle and humans. To address this, Experiment Four was designed to include a histological assessment of ovarian follicle populations. It was also expected that the histological assessments may give a more accurate measure of antral follicle counts than the surface assessments. An additional blood sample was collected at the time of slaughter to determine whether there was an association between concurrent circulating ovarian hormone levels and ovarian and uterine traits. The presence or absence of CL was used to classify gilts as either pubertal (cycling) or immature (yet to cycle) at D160. Results from Experiment Four showed:
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• A positive relationship between D80 E2 and UMIsize in pubertal gilts at D160, such
that gilts with higher E2 levels had heavier, longer and thicker uterine horns
compared to gilts with lower E2.
• A two-way interaction between D80 AMH, D160 AMH and ovarian follicle
populations in immature gilts, such that lower D80 and D160 AMH levels were
associated with greater ovarian populations at D160.
• Elevated D80 E2 levels were associated with reduced ovarian follicle populations
in gilts that were immature at D160.
Though there was a negative association between D80 AMH levels and future ovarian follicle counts at D160, the negative relationship between serum AMH measurements and ovarian follicle counts taken on the same day was much stronger, making D160 AMH levels a more useful marker for ovarian follicle populations and ovarian reserve.
Regarding the relationship between D80 E2 levels and uterine capacity, the findings of
Experiments Two and Four were inconsistent. In Experiment Two, the D80 E2 levels were negatively associated with D160 uterine capacity, whereas in Experiment Four, this association was not evident. In fact, in the cohort of gilts that had their first ovulations by D160, the relationship was positive. This disparity could be attributed to the different methods used to assess the presence of CLs, classifying gilts as pubertal. When examining the ovaries in
Experiment Four, it was clear that some ovaries that contained CLs had CLs that were obviously located on their surface, while others had less obvious CLs that were predominantly internal. Thus, some of the gilts classified as immature in Experiment Two may have indeed ovulated, potentially affecting data analysis.
Regarding the relationships between the ovarian hormones at D80 and the ovarian traits at D160, the findings in Experiments Two and Four were, again, not consistent. The
Page 186 of 213 histological assessment of follicle numbers in Experiment Four provided a much more detailed and accurate measurement than the count of surface antral follicles in Experiment Two. This highlights the limitation of using the surface AFC method to investigate ovarian development.
The finding that serum AMH levels were associated with ovarian follicle populations was not surprising, as this has been found in various studies in other species (refer to Table 1).
However, previous literature shows the relationship between circulating AMH concentrations and ovarian follicle populations to be positive, rather than the negative association found here.
This variation may be due to AMH having a different role in the pig, as suggested by their unique AMH expression patterns within the ovary (Almeida et al., 2018), as well as AMH having a different action depending on maturational age, as indicated by Garrel et al. (2016).
Further, a more detailed assessment of the active and inactive forms of AMH is needed to better understand these relationships in pigs as well as in other species.
Reproductive properties at slaughter vs. production outcomes
Previous studies have linked uterine capacity in the pig with reduced foetal losses, greater birth weight and increased litter sizes (Foxcroft et al., 2009). Also, increased ovarian follicle numbers have been associated with the reproductive potential of an individual
(reviewed by Ireland et al. (2011). The findings of Experiments One and Three suggest that gilts with lower E2 levels at D80 experience less negative feedback on gonadotropin release during this time of ovarian development. Such reduced inhibition by E2 may allow earlier establishment of the HPG axis and an increase in basal FSH levels, potentially leading to earlier onset of puberty and improved reproductive performance. Consistent with this proposal, the results of Experiment Four showed that elevated E2 levels were associated with reduced ovarian follicle populations.
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The on-farm assessments were not able to establish a link between lower D80 E2 levels and the early onset of puberty. Age at first heat data was not obtained from gilts at Farm A as gilts were not checked for this until being moved into the mating shed at 190 days of age. Also, while age at first heat was recorded at Farm B, checking for heat did not commence until 150 days of age, when some gilts may have already commenced cycling. It should be noted that some variability in production parameters between farms may be attributed to the differences in mating selection criteria between farms; Farm A basing mating on gilt weight, and Farm B basing mating on gilt age. Furthermore, premature culling of non-productive gilts in the on- farm studies within this thesis limits the ability to predict the effect of using ovarian hormones were used to select non-productive gilts and productive gilts. Despite the shortcomings of the on-farm data collection, the results of this thesis show that properties such as uterine capacity and ovarian follicle number, as well as measures of fecundity, were associated with serum E2 levels at 80 days of age, thereby providing an opportunity to identify gilts with favourable reproductive characteristics prior to selection for the breeding herd.
The results also showed a relationship between serum AMH levels and ovarian follicle counts, with lower levels of AMH at D80 and D160 associated with greater ovarian follicle counts. In Experiments One and Three D80 AMH was not associated with mating, litter or culling data recorded for the first three parities. It should be noted that the relationship between
D160 AMH levels and D160 ovarian follicle counts was much stronger than that between D80
AMH levels and D160 ovarian follicle counts. Therefore, while the D80 AMH levels were not associated with mating or gestation outcomes, D160 AMH levels might be. Further research is required to confirm this. Regardless of the lack of fecundity improvement in the first three parities, selection of gilts with greater ovarian follicle counts seems inherently sensible, as females with a greater ovarian reserve would be expected to have a longer reproductive lifespan.
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Dam and sire were considered random factors and gilts of visually uniform weight were selected for the studies. Unfortunately, recording all piglet weights, growth rates and litter sex- ratios was not part of standard production practice at either farm. Such factors have previously been shown to affect reproductive potential in gilts (Elsaesser and Parvizi, 1979; Magnabosco et al., 2016). The fact that such previously identified indicators of reproductive qualities in gilts are not regularly measured on-farm is concerning and poses the questions: Are efforts into finding endocrine markers of reproductive success futile? Why are expensive new technologies and procedures being developed when previously proven, cheaper, less labour-intensive processes are not being implemented? While these questions are perhaps beyond the scope of this discussion, it highlights that for any effective gilt selection procedure to be adopted widely, it must be simple and convenient to use, as well as cheap enough that the immediate expense is justified by the long-term gain. While further studies are needed to assess the usefulness of
E2 and AMH as markers of reproductive performance, significant development of the assays would also be needed before routine implementation for production purposes is feasible.
7.3 Final Conclusions
The results of this project addressed all three objectives: the serum concentrations of
E2 and AMH before and after treatment with gonadotrophins at 60, 80 and 100 days of age were measured successfully in Experiment One; serum E2 and AMH levels at D80 and/or D160 were correlated with uterine and ovarian properties at slaughter in Experiments Two and Four; and serum E2 and AMH levels at 60, 80 or 100 days of age were correlated with fertility and reproductive performance in Experiment One and Three.
The results support the hypothesis that circulating levels of ovarian hormones E2 and
AMH during a period of ovarian development (60 to 100 days) correlate with reproductive Page 189 of 213 properties and fertility in adulthood. The findings indicate that circulating levels of AMH and
E2 could be used to select for traits such as ovarian follicle count and litter size in gilts. The traits that gilts are proposed to possess according to their AMH and E2 hormone profiles at 80 days of age are outlined in Table 22. It is therefore recommended that when selecting gilts into the breeding herd, gilts with low E2 and low AMH levels should be preferred, followed by gilts with low E2 and high AMH levels, gilts with high E2 and low AMH levels, and lastly high E2 and high AMH levels.
Table 22: Proposed traits according to AMH and E2 hormone profiles at 80 days of age and the recommended selection preference ranking.
D80 AMH Greater ovarian Greater Selection Preference D80 E2 levels levels follicle count litter size Ranking
Low Low 1st
Low High 2nd
High Low 3rd
High High 4th
For such a selection process to become a reality, a cheaper method for determining circulating levels of AMH and E2 in gilts must first be developed. Using the methods described here, the cost-to-benefit ratio would be too large to be deemed feasible in a production setting.
Future studies should also consider confounding factors such as litter sex-ratio, piglet weight and growth rate of the study animals. Recommended unit thresholds of hormone levels to be used for selecting gilts were not determined in this thesis as this is dependent on a multitude of factors including the outcome unit change desired and the size of the piggery operation. With this information, receiver operator curves could be used to determine the optimal thresholds at which to select gilts into the breeding herd.
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To our knowledge the relationship between circulating AMH levels and ovarian, uterine, and production traits in gilts have not previously been examined. One of the most notable findings from this thesis is the negative relationship observed between AMH levels and ovarian follicle populations. This contrasts studies in other species that have shown AMH and ovarian follicle populations to be positively associated (refer to Table 1). The present study suggests that AMH may have a different temporal role in the pig, which may explain the unique
AMH expression patterns observed previously within porcine ovaries (Almeida et al., 2018).
Perhaps the levels of AMH in porcine ovaries is proportionate to the inhibitory signal required to control the rate of recruitment, in order to ‘save’ reserves in gilts that were born with ovaries that contain fewer follicles.
The findings of this thesis indicate that improvements to production efficiency may be achieved through better prediction of uterine capacity and reproductive lifespan using AMH and/or E2 serum levels to inform replacement gilt selection. However, due to the considerable inter-individual variation in hormones and hormonal response to gonadotropin stimulation observed, further studies underpinning the role of AMH through more in-depth, longitudinal studies to examine the relationships between AMH and E2 levels and ovarian, uterine and production traits in female pigs are key for further determining the suitability of E2 and AMH as indicators of reproductive potential. If the selection of replacement gilts using measures of these hormones was feasible in a production setting, large-scale prospective studies would then need to be undertaken. Improving the current gilt selection process is essential in order to reduce the high culling rates of gilts in the initial parities and to increase the longevity and lifetime productivity of sows. Increasing the efficiencies of our food production systems is imperative to being able to meet the growing demands of our ever-increasing human population.
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