Land and Food Systems, Faculty of
DSpace
UBCV
dos Santos Marques, Jessica Cristina
Master of Science - MSc
University of British Columbia
Greater progesterone (P4) levels prior to and post estrus in lactating dairy cows has been associated with improved fertility and greater intensity of estrous expression measured by automated activity monitors (AAM). Recently, greater intensity of estrous expression has also been associated with greater embryo production and quality in superovulated dairy heifers. However, it is unclear whether P4 causes greater expression of estrus and increased early embryo development in superovulated heifers. Therefore, the aim of this thesis was to evaluate the effect of different P4 concentrations in plasma during the follicular growth on the intensity of estrous expression, ovarian response to the superovulatory treatment, and embryo production and quality in superovulated Holstein heifers. In Chapter 1, scientific studies are reviewed to highlight gaps in the literature associated with P4 regulation, intensity of estrous expression and reproductive outcomes in dairy cattle. In Chapter 2, I found that different P4 levels during the follicular growth did not affect the maximum intensity of estrous expression or the duration of estrus in superovulated Holstein heifers. However, high P4 concentrations in plasma during the development of follicles increased embryo quality and reduced the number of embryos produced when compared to low P4 concentrations. Although estrous expression did not relate to the quality of embryos, the duration of estrus and the maximum intensity of estrous expression increased the number of embryos recovered 7 d post artificial insemination. In Chapter 3, study limitations and future directions are summarized. In conclusion, my results suggest that P4 concentrations during the follicular growth, and intensity of estrous expression, are important in regulating the quality of embryos and the number of embryos produced by superovulated heifers.
THE IMPACT OF PLASMA CONCENTRATIONS OF PROGESTERONE DURING SUPEROVULATION ON ESTROUS BEHAVIOUR, AND OVARIAN AND EARLY EMBRYONIC DEVELOPMENT IN HOLSTEIN HEIFERS by Jessica Cristina dos Santos Marques Techn., Agricultural Technical College "Jose Bonifacio", 2013 DVM., Federal University of Uberlandia, 2020 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Applied Animal Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) September 2022 © Jessica Cristina dos Santos Marques, 2022 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled: The impact of plasma concentrations of progesterone during superovulation on estrous behaviour, and ovarian and early embryonic development in Holstein heifers. submitted by Jessica Cristina dos Santos Marques in partial fulfillment of the requirements for the degree of Master of Science in Applied Animal Biology Examining Committee: Dr. Ronaldo L.A. Cerri, Associate Professor, Applied Animal Biology, UBC Supervisor Dr. Alexandra Protopopova, Assistant Professor, Applied Animal Biology, UBC Supervisory Committee Member Dr. Alexander G. Beristain, Associate Professor, Obstetrics and Gynaecology, UBC Supervisory Committee Member Dr. Barbara Stefanska, Assistant Professor, Food, Nutrition and Health, UBC Additional Examiner Additional Supervisory Committee Members: Dr. Daniel Weary, Professor, Applied Animal Biology, UBC Supervisory Committee Member iii Abstract Greater progesterone (P4) levels prior to and post estrus in lactating dairy cows has been associated with improved fertility and greater intensity of estrous expression measured by automated activity monitors (AAM). Recently, greater intensity of estrous expression has also been associated with greater embryo production and quality in superovulated dairy heifers. However, it is unclear whether P4 causes greater expression of estrus and increased early embryo development in superovulated heifers. Therefore, the aim of this thesis was to evaluate the effect of different P4 concentrations in plasma during the follicular growth on the intensity of estrous expression, ovarian response to the superovulatory treatment, and embryo production and quality in superovulated Holstein heifers. In Chapter 1, scientific studies are reviewed to highlight gaps in the literature associated with P4 regulation, intensity of estrous expression and reproductive outcomes in dairy cattle. In Chapter 2, I found that different P4 levels during the follicular growth did not affect the maximum intensity of estrous expression or the duration of estrus in superovulated Holstein heifers. However, high P4 concentrations in plasma during the development of follicles increased embryo quality and reduced the number of embryos produced when compared to low P4 concentrations. Although estrous expression did not relate to the quality of embryos, the duration of estrus and the maximum intensity of estrous expression increased the number of embryos recovered 7 d post artificial insemination. In Chapter 3, study limitations and future directions are summarized. In conclusion, my results suggest that P4 concentrations during the follicular growth, and intensity of estrous expression, are important in regulating the quality of embryos and the number of embryos produced by superovulated heifers. iv Lay Summary The use of sensor technologies to detect estrus has increased throughout the years in dairy farms. Data generated by these wearable sensors has helped researchers to study the link between intensity of reproductive behaviour and fertility in dairy cows. Greater intensity of reproductive behaviour has been associated with greater fertility and greater concentrations of the hormone progesterone before estrus in cows. This thesis investigated if different concentrations of progesterone before estrus impact the reproductive behaviour, ovarian response to superovulatory treatment, and the quality and number of embryos produced in Holstein heifers. This thesis demonstrated that different concentrations of progesterone before estrus did not affect the reproductive behaviour, however, greater progesterone levels were associated with greater quality of embryos and lower number of embryos produced. In addition, greater expression of reproductive behaviour was associated with greater number of embryos produced. Progesterone and reproductive behaviour are important markers of fertility in heifers. v Preface The work presented in this thesis was based on the work conducted at the University of British Columbia’s Dairy Education and Research Centre in Agassiz, Canada. All experimental procedures were approved by the Animal Care Committee of the University of British Columbia following the protocol # A20-0182. A version of the material in Chapter 2 will be submitted for publication with the present author as the lead author, Dr. Jose Denis-Robichaud as the second author and Dr. Ronaldo Cerri as last author. For all chapters, J.C.S Marques was the lead investigator responsible for all major areas of concept formulation, experimental design, material interpretation, data collection and processing, statistical analysis, and manuscript composition. Dr. Ronaldo Cerri and Dr. Jose Denis-Robichaud acted by helping in the idea formulation, data collection, statistical analysis and providing input, and editing all chapters of this thesis. vi Table of Contents Abstract .......................................................................................................................................... iii Lay Summary ................................................................................................................................. iv Preface............................................................................................................................................. v Table of Contents ........................................................................................................................... vi List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix List of Abbreviations .................................................................................................................... xii Acknowledgements ...................................................................................................................... xiv Dedication .................................................................................................................................... xvi Chapter 1: Introduction ................................................................................................................... 1 1.1 Follicular dynamics and endocrine regulation ................................................................ 4 1.1.1 Common growth phase of ovarian follicles ................................................................... 5 1.1.2 Dominance deviation process (selection) of ovarian follicles ....................................... 5 1.1.3 Dominance phase of ovarian follicles at initial phase of estrous cycle ......................... 6 1.1.4 Dominance phase of ovarian follicles at final phase of estrous cycle ........................... 7 1.1.5 Ovulation of ovarian dominant follicle .......................................................................... 8 1.2 Hormonal control of follicular dynamics and ovulation timing ..................................... 9 1.2.1 Single ovulation ............................................................................................................. 9 1.2.2 Superovulation ............................................................................................................. 10 1.3 Expression of estrus ...................................................................................................... 15 1.3.1 Automated monitoring of estrous behaviour ............................................................ 21 1.4 Fertilization and early embryonic development ........................................................... 23 1.5 Progesterone and early bovine embryo ......................................................................... 28 1.5.1 Progesterone levels prior to fertilization ................................................................... 29 1.5.2 Progesterone levels near fertilization ........................................................................ 31 1.5.3 Progesterone levels post-fertilization. ....................................................................... 32 1.6 Thesis objectives ........................................................................................................... 35 Chapter 2: The impact of plasma concentrations of progesterone during superovulation on estrous behaviour, and ovarian and early embryonic development in Holstein heifers. .............. 36 2.1 Introduction ......................................................................................................................... 36 2.2 Materials and methods ........................................................................................................ 39 2.2.1 Automated Activity Monitor........................................................................................ 40 2.2.2 Treatments.................................................................................................................... 40 2.2.3 Ultrasonography and blood samples ............................................................................ 41 2.2.4 Embryo collection ........................................................................................................ 42 2.2.5 Statistical analysis ........................................................................................................ 43 vii 2.3 Results ................................................................................................................................. 47 2.3.1 Ovarian structures and ovulatory response to superovulation ..................................... 48 2.3.2 Progesterone treatment and estrous expression ........................................................... 51 2.3.3 Number of embryos recovered 7 d post-AI ................................................................. 52 2.3.4 Progesterone treatment and embryo development ....................................................... 54 2.3.5 Estrous expression and embryo development .............................................................. 54 2.4 Discussion ........................................................................................................................... 56 2.5 Conclusions ......................................................................................................................... 66 Chapter 3: General Discussion...................................................................................................... 67 3.1 Thesis conclusions .............................................................................................................. 67 3.2 Limitations and future directions ........................................................................................ 68 Bibliography ................................................................................................................................. 74 viii List of Tables Table 1: Predicted means (± SEM) of the different ovarian structures of superovulated Holstein heifers by progesterone treatment. Holstein heifers received a pre-synch protocol, starting on d-26, followed by P4 treatment (High P4 or Low P4) with a protocol of superovulation, starting on d -11. Day 0 was defined as the day of timed AI. DF – Dominant follicle; CL – Corpus Luteum, FSH – Follicle-stimulating hormone, P4 – Progesterone, AI – Artificial insemination. ....................................................................................................................... 49 Table 2: Proportion of embryos recovered 7 d post-AI (following superovulation) for each stage of embryonic development and grade of quality by treatment group (High P4 and Low P4 during the follicular growth). Embryos were evaluated for grade of quality (IETS, 2013) and stage of embryonic development (Bó and Mapletoft, 2013). .............................................. 53 ix List of Figures Figure 1: Schematic depiction of estrous cycle follicular dynamics and endocrine regulation adapted from Forde et al., (2011b). Abbreviations represents: LH - luteinizing hormone and P4 – progesterone. Shaped circles icons represent growing ovarian follicles and icons black filled represent follicles in atresia. © 2011 N. Forde, adapted with permission #5275541368823. .................................................................................................................. 8 Figure 2: Illustration from the Magdalenian engraving in the “Grotte the la Mairie”, Teyjat, Dordogne, France. Circa 10,000 B.C. Reprinted from Roelofs et al. (2010). © 2010 J. Roelofs, by permission #5275550431357.. ......................................................................... 15 Figure 3: Temporal early embryo development in cattle according to number of cells and days after fertilization (© 2013 Bó and Mapletoft1 & © 2017 Q. Wei2 adapted with permission #5275550759609).. .............................................................................................................. 28 Figure 4: Schedule for the presynchronization and superovulation protocols of heifers exposed to Low or High progesterone (P4) during the follicular growth phase, in parallel with the schedule for reproductive tract ultrasound (US) exams, and blood samples (BS) performed during the study. Holstein heifers, between 10.5 and 12-months old, were randomly assigned into two treatment groups (High P4 and Low P4) to evaluate the effect of P4 concentration during the follicular growth on estrous intensity, superovulatory response, and embryo quality. AI - artificial insemination; EC - Embryo collection, FSH - Follicle-stimulating hormone, PG - Prostaglandin F2α, GnRH - Gonadotropin-releasing hormone.................... 43 Figure 5: Causal diagram used to inform the construction of the statistical models performed to evaluate the effect of different concentrations of progesterone (P4) during the follicular growth on the intensity of estrous expression and embryo development in superovulated x Holstein heifers. Possible confounders were kept in the models if P ≤ 0.2 (Dohoo et al., 2009). ................................................................................................................................... 47 Figure 6: Mean (± SEM) progesterone (P4) concentrations (ng/mL) during the study (where d0 was defined as the day of timed AI) by treatment group. Holstein heifers were randomly assigned into two treatment groups (High P4: n = 32 and Low P4: n = 31) to evaluate the effect of P4 concentrations during the follicular growth on estrous intensity, superovulatory response and embryo quality. .............................................................................................. 48 Figure 7: Effect plots from Poisson regression models summarizing the association between the duration of estrus (A) and maximum intensity of estrus (B) and the total corpus luteum (CL) number 7 d post-artificial insemination (AI), by treatment groups (High P4 and Low P4; heifers = 46). The maximum intensity of estrous expression was defined as the maximum relative increase (RI) during an estrous event. The duration of estrus was calculated using the number of hours which heifers exceeded 180% RI during an estrous event. ................ 51 Figure 8: Survival curve for time from CIDR removal (d -2) to estrous event by treatment groups (High P4 and Low P4), using a Cox proportional-hazards model (HR = 0.65; 95% CI = 0.38 – 1.09; P = 0.09). Observations were right censored at d 0 if estrus was not confirmed (n = 59 confirmed estrus vs. n = 4 not confirmed). Day 0 was defined as the day which timed artificial insemination (AI) was performed. ........................................................................ 52 Figure 9: Effect plots from Poisson regression models summarizing the effect of duration of estrus (A) and maximum intensity of estrus (B) on number of embryos recovered 7 d post-AI (n = 42 heifers), by treatment groups. Total follicle number at AI was considered a statistically significant confounder (P ≤ 0.2) and kept in the model (the effect plot fit were computed using the mean total of follicles). Relative increase (RI) in walking activity was calculated xi by determining changes in activity from d -2 to 0 in comparison to the baseline of each heifer. The maximum intensity of estrous expression was defined as the maximum RI during an estrous episode. Duration of estrus was calculated using the number of hours heifers exceeded 180% RI during an estrus event. .......................................................................... 55 xii List of Abbreviations AAM – Automated activity monitor AI – Artificial insemination AUC – Area under the curve BCS – Body condition score BS – Blood sample CL – Corpus luteum DF – Dominant follicle EC – Embryo collection eCG – Equine chorionic gonadotropin ERα – Estrogen receptors α-subtype ETP – Embryo transfer program FDF – Future dominant follicle FSH – Follicle-stimulating hormone GnRH – Gonadotropin-releasing hormone hCG – Human chorionic gonadotropin hmG – Human menopausal gonadotropin IETS – International embryo technology society IGF1 – Insulin-like growth factor 1 LH – Luteinizing hormone P4 – Progesterone PGF2α – Prostaglandin F2α RI – Relative increase xiii TMR – Total mixed ration US – Ultrasonography ZP3 – Zona pellucida glycoprotein 3 xiv Acknowledgements First, I would like to thank my advisor, Ronaldo Cerri for taking me under his wing first as an international visiting student and later as a master student. I cannot express in words how grateful I am for having the opportunity to learn about research and dairy cattle under his supervision. Thank you for your support and patience in the last two years, and for becoming not only my supervisor but my friend throughout this process. I also would like to thank my supervisory committee members – Dr. Daniel Weary, Dr. Alexander Beristain and Dr. Alexandra Protopopova for their guidance and support during my master program. I feel extremely blessed to be surrounded by such great professionals. To the Elizabeth R Howland Fellowship, James A. Shelford Memorial Scholarship and Pacific Dairy Centre Scholarship that were generously awarded to me by the faculty of Land and Food Systems and have been incredibly helpful for funding my education. To the repro team students, thank you so much to all current and past members of the laboratory for the support (I could never have done it without their help). Particularly, I would like to thank Joyce, Rodrigo, Amanda and Sydney for not given up on me in those long days of embryo collection and to Jose for having so much patience teaching me statistical analysis. Thanks to all students living at the UBC dairy centre residence for becoming my family in Canada and an enormous thanks to Nelson Dinn and all the farmers at UBC dairy for all the support during these years. Special thanks to the little heifers at UBC dairy that made my research possible. To my family, thank you all for your love and support during this time abroad. To my partner, Lyndon Striker, thank you for your love, for being such incredible support, and for sharing your amazing family with me and making me feel at home. And last but not least, a super thanks to my grandparents, the most beautiful stars in the sky, for always believing in me (even when I xv didn’t believe in myself) and for being my everything. Ataide and Maria, I know you are always with me, and you are the reason I must continue, I will love you forever and ever. xvi Dedication In memory of my grandparents, Ataide and Maria dos Santos. 1 Chapter 1: Introduction The dairy industry plays an important role in providing products to meet the nutritional needs of the global population (Špička, 2013). The consumption of milk and its derivates, beyond its nutritional effects, have been associated with reduced risk of diseases and improved weight management in humans (Amiri et al., 2021). In addition to its nutritional and health effects, milk production has an important contribution to the local economy (MNP, 2020). A recent economic study promoted by the Agriculture and Agri-Food Canada and the Government of British Columbia have shown that the local dairy industry contributed CAD$ 1.225 billion to the province’s gross domestic product and supported almost 12,500 jobs in 2019 (MNP, 2020). One of the major factors affecting profitability of dairy herds is reproductive performance (Sewalem et al., 2008). Although dairy cattle research and breeding programs were mainly focused on yield traits a few decades ago (Leitch, 1994), reproduction as well as longevity and health are currently traits of increased interest to dairy producers (Miglior et al., 2005). Suboptimal reproductive performance may lead to reduced milk yield, increased culling (for reproductive reasons), fewer available replacement heifers as well as increased veterinary costs and semen usage (Meadows et al., 2005). Thus, marginal production cost increase as reproduction performance decreases (Meadows et al., 2005). Studies have shown negative economic impacts of poor reproduction performance worldwide. In the Netherlands, Inchaisri et al. (2010) compared good and poor reproductive performance scenario (using variables such as ovulation and conception probability) and demonstrated a mean net economic loss of € 231/cow/year for poor reproduction performance. In Ohio, United States, Meadows et al. (2005) estimated a loss of USD$ 1.37/cow/year for each 1-d 2 increase in days open (calving to conception interval). De Vries (2006) have shown that average cost of pregnancy loss (abortion) was USD$ 555, by modeling a general Holstein herd in the United States. In Canada, Bauman et al. (2016) reported that farmers ranked reproductive performance as the most important priority factor to be researched. Likewise, recently Denis-Robichaud et al. (2018) found that estrus detection and conception risk were highly prioritized by respondents of their Canadian study survey. In this context, failure in estrus detection and early embryonic loss are substantial factors leading to poor reproductive performance in dairy cattle (Rutten et al., 2014). Improvements in estrus detection was reported to increase the likelihood of pregnancy (Rutten et al., 2014). However, rates of visual estrus detection were reported to vary from 40% (Roelofs et al., 2010) to 55% (Firk et al., 2002) across dairy farms. In addition to the suboptimal detection rates, decreases in the number of animals displaying estrous behaviour has been noted (Washburn et al., 2002) and associated with increasing milk production (Butler, 1998; Lopez et al., 2004). Therefore, several studies have evaluated estrus synchronization protocols (Inskeep, 1973; Pursley et al., 1995; Islam, 2011) as well as automated estrus detection technologies (Silper et al., 2015b; Burnett et al., 2017), to improve estrus detection and increase reproductive outcomes in the dairy industry. Given what has been said, scientific interest in automated activity monitors (AAM) data has increased recently (Gengler, 2019). Intensity of estrous expression, measured using physical activity detected by AAMs, has been associated with increased fertility of dairy cows (Madureira et al., 2015; Burnett et al., 2018). Several studies have demonstrated that greater increases in activity during estrus were correlated with greater pregnancy per artificial insemination (AI; Madureira et al., 2015; Burnett et al., 2018), greater pregnancy per embryo transfer (Madureira et al., 2022), as well as lesser ovulation failure (Burnett et al., 2018) and greater quality of embryos 3 (Madureira et al., 2020). However, the physiological mechanisms underlying the association between estrous expression and fertility is still unclear and further research is needed to fully understand the physiological processes and improve reproductive management of dairy cows. Furthermore, given that fertilization rate is relatively high (around 85%) in cattle (Santos et al., 2004), early embryonic loss is likely the main cause of inadequate pregnancy results following AI in cows (Ribeiro et al., 2016). Bovine embryos were reported to be more susceptible to death when transitioning from the morula to the blastocyst stage at 5 to 8 d of age (Maurer and Chenault, 1983). Several studies have evaluated predisposing factors affecting initial embryonic development in dairy cows (Sartori et al., 2002b; Santos et al., 2009; Ribeiro et al., 2013). These factors include inflammatory diseases prior to AI (Ribeiro et al., 2013), poor nutrition and body condition score (Santos et al., 2009), heat stress (Sartori et al., 2002b) as well as progesterone (P4) levels prior and post fertilization (reviewed in Lonergan and Sánchez, 2020). Although the causes of poor early embryo development are complex and multifactorial, circulating P4 levels seems to be an important component in the regulation of fertility of dairy cows (Wiltbank et al., 2012). Progesterone plays a key role in reproductive events related to establishment and maintenance of pregnancy through its effects on oocyte and embryo quality (Rivera et al., 2011) as well as its action on the uterine endometrium (Beltman et al., 2009; Forde et al., 2011a, 2012). Lower P4 levels have been associated with extended length of the follicular dominance phase (Inskeep and Dailey, 2005), lower quality of embryos (Rivera et al., 2011) as well as temporal changes in oviductal (Bylander et al., 2015) and endometrial (Beltman et al., 2009; Forde et al., 2011a, 2012) transcriptome. In addition, recently greater P4 levels (prior to and post AI) were associated with greater intensity of estrous expression in lactating dairy cows 4 (Madureira et al., 2021). Nevertheless, the association between P4, estrous intensity, and early embryo development in dairy cows is still only partially understood. Therefore, this chapter will focus on reproductive physiology, estrous behaviour, and early embryo development in cattle. To support the understanding of the bovine reproductive cycle, follicular dynamics and endocrine regulation throughout the estrous cycle will be described and associated with a schematic figure in the first section. The second section of this chapter will review the current management practices used to control follicular development and predict ovulation timing, as well as superovulation programs used for embryo collection. The third section will summarize the endocrine regulation of estrous behaviour as well as recent technologies developed to aid estrus detection in the dairy industry. The complexity of fertilization and early embryo development processes will be reviewed and linked by schematic depiction of early bovine embryos in the fourth section. Lastly, P4 and its association with early embryonic development will be reviewed. 1.1 Follicular dynamics and endocrine regulation Studies evaluating the morphologic changes in the bovine reproductive tract began with data obtained from slaughterhouse specimens, excised ovaries, and external inspection of the vestibule and vagina (Rajakoski, 1960). A breakthrough in follicle dynamics research occurred when ultrasound technology was implemented as a non-invasive method for precise in vivo monitoring of the bovine ovary morphology (Pierson and Ginther, 1984). Over the decades, the endocrine regulation and follicular dynamics of the estrous cycle have been published in hundreds of research reports (Ginther, 2016). The dynamics of the follicle population present in the bovine ovaries occurs in waves (Ginther, 2016). Follicular waves consist in the development of antral follicles clusters during an 5 inter-ovulatory interval (Ginther, 2016). In Bos taurus cattle, one estrous cycle may encompass 2 to 3 waves (Ginther, 2016), has a mean length of 21 days, and a starting point (day 0) defined as the day of ovulation (Rajakoski, 1960). Each follicular wave is composed by 3 phases: common growth, dominance deviation (also named as selection), and dominance (Ginther, 2016) and these phases will be fully described throughout this section. 1.1.1 Common growth phase of ovarian follicles The common growth phase consists in continuous increase in the diameter of the ovarian follicles and extends from the emergence of the antral follicle clusters (2 to 4 mm of diameter) on day 0 until the beginning of the deviation process (Ginther, 2016). In this stage, follicle-stimulating hormone (FSH) surge leads to increase in aromatase enzyme activity (in the granulosa cells of ovarian follicles) promoting the conversion of an androgen to estrogen (Hillier, 1994). In addition, luteinizing hormone (LH) binding to its receptors (in the theca cells) promote the conversion of cholesterol to testosterone, which is also converted to estrogen by the aromatase enzyme (Dorrington et al., 1975). Along with continuous follicular growth rates and estrogen production, the growing follicles develop FSH suppressors (Gibbons et al., 1997). Inhibin (Guilbault et al., 1993; Wood et al., 1993; Kaneko et al., 1995), proteinaceous fraction of follicular fluid (Kastelic et al., 1990) as well as estrogen (Ginther, 2016) have an inhibitory effect on FSH. The FSH suppression signals the beginning of the diameter deviation phase (Ginther, 2016). 1.1.2 Dominance deviation process (selection) of ovarian follicles The process of dominance deviation begins approximately on day 2.5 of the Bos taurus estrous cycle (Ginther, 2016) and is marked by changes in growth rates between the 2 largest follicles of the wave (Ginther, 2016). In this stage, the future dominant follicle (FDF) remains with 6 continued growth rate despite FSH surge decline, whereas future subordinate follicles growth rate decreases (Ginther, 2016). Intrafollicular factors such as LH receptors, free insulin-like growth factor 1 (IGF1), and estrogen are essential throughout this process (Ginther, 2016). Expression of LH receptors, concentration of estrogen, and free IGF1 were reported to increase in the FDF at the beginning of the deviation process whereas it decreases in the future subordinate follicles (Ginther, 2016). These changes in the FDF increases LH responsivity and consequently promote continuous growth rate, despite decreasing FSH concentrations (Forde et al., 2011b). The deviation process occurs 12 h before the visual diameter deviation (around 6 to 8.5 mm of diameter) and is followed by the dominance phase (Ginther, 2016). 1.1.3 Dominance phase of ovarian follicles at initial phase of estrous cycle The dominance phase is characterized by rapid continuous growth of the selected dominant follicle (DF) in conjunction with increases in estrogen production (Ginther, 2016). Sartori et al. (2001) reported that ovulation capacity was acquired when the follicle reached 10 mm of diameter in Holstein cattle. However, in the initial phase of the estrous cycle the growing DF does not ovulate (Ginther, 2016). Following ovulation in the previous cycle, marked as day 0 of the new cycle, P4 concentrations begin to increase due to the formation of the corpus luteum (CL). The CL is a transient gland formatted from luteinised granulosa and theca cells of the ovulated DF from the previous cycle (Ginther, 2016). The CL produces P4 for the establishment and maintenance of pregnancy and/or resumption of the estrous cycle (Forde et al., 2011b). Through negative feedback, P4 promotes insufficient amplitude and frequency of LH pulses necessary for final maturation of the DF and ovulation (Rahe et al., 1980), thus, the growing DF undergoes to atresia under higher P4 levels. Therefore, decreases in FSH suppressors occur due to follicular atresia 7 triggering the beginning of a new follicular wave and reoccurrence of the follicular phases (Forde et al., 2011b). 1.1.4 Dominance phase of ovarian follicles at final phase of estrous cycle During the estrous cycle mid-final phase, the P4 produced by the CL downregulate its own P4 nuclear receptor in the luminal epithelium of the endometrium (Kimmins and Maclaren, 2001). This downregulation allows an increase in expression of estradiol receptors which leads to expression of oxytocin receptors that upon binding with the ligand triggers prostaglandin F2α (PGF2α) synthesis cascade (Lamothe et al., 1977) around day 16 of the estrous cycle (Forde et al., 2011b). PGF2α is the major luteolytic hormone in ruminants (Baird et al., 1976). It reaches the ovary, via countercurrent exchange between the uterine vein and the ovarian artery, inducing CL regression and decreased P4 production (Forde et al., 2011b). Thus, decreasingly P4 and increasing estrogen levels (produced by the growing DF) induces a surge in gonadotropin-releasing hormone (GnRH) and allows display of estrous behaviour (Forde et al., 2011b). The GnRH surge leads to a LH surge (Sunderland et al., 1994) which promotes ovulation of the DF (Forde et al., 2011b). Figure 1 summarizes the follicular dynamics and endocrine regulation throughout the estrous cycle in ruminants adapted from Forde et al. (2011b). 8 Figure 1: Schematic depiction of estrous cycle follicular dynamics and endocrine regulation adapted from Forde et al., (2011b). Abbreviations represents: LH - luteinizing hormone and P4 – progesterone. Shaped circles icons represent growing ovarian follicles and icons black filled represent follicles in atresia. © 2011 N. Forde, adapted with permission #5275541368823. 1.1.5 Ovulation of ovarian dominant follicle The ovulation of the DF requires a series of synchronized biochemical events. Preovulatory LH surge promotes increased angiogenic factors and blood flow to the ovary as well as to the DF elevating the hydrostatic pressure around the follicle which may facilitate its eventual rupture (Senger, 2003). These factors combined also ensure that the DF is provided with necessary hormonal and metabolic nutrients for its final maturation (Senger, 2003). In addition to this, the LH surge shifts the hormonal production from estradiol to P4 which stimulates synthesis of collagenase in the ovary and consequently leads to weaken of the ovarian connective tissue facilitating the rupture of follicle and consequent ovulation (Senger, 2003). Lastly, after the LH surge, PGF2α causes contraction of the ovary smooth muscle (inducing increased intrafollicular pressure) and release of lysosomal enzymes (leading to connective tissue deterioration). It ultimately leads to release of the oocyte from the ovary to the oviduct (Senger, 2003). 9 1.2 Hormonal control of follicular dynamics and ovulation timing 1.2.1 Single ovulation The first attempts to control the bovine reproductive cycle occurred between 1948 and 1960 (Hansel, 1967). During this time, the application of hormonal manipulation was focused on dairy heifers that were bred naturally while at pasture with bulls of questionable genetic merit (Hansel, 1967). Thus, synchronization of estrus allowed convenient AI time using bulls of known genetic merit and consequently genetic progress of dairy herds (Hansel, 1967). At first, the technique included CL enucleation by ovary manipulation through rectal examination (Hansel, 1967). It resulted in estrus and ovulation within 3 to 5 d in high percentage of treated animals, however, this technique was criticized due to the development of adhesions in and around the oviducts (Hansel, 1967). The discovery processes of estrus synchronization techniques can be reviewed historically in four phases (Robinson, 1975; Patterson et al., 1989). The first phase is described by the development of methods to prolong or establish the P4 phase (luteal phase) administering exogenous P4 (Hansel and Malven, 1960; Zimbelman, 1963). It was followed by the second phase where progestins (synthetic P4) were combined with estrogens to induce estrous behaviour (Wiltbank et al., 1961; Whitman et al., 1972; Spitzer, 1982). The third phase included the luteolytic agents (promote lyse of CL) to decline P4 circulating levels (Inskeep, 1973; Lauderdale, 1975) and it was followed by the fourth phase where progestins and luteolytic agents were combined (Patterson et al., 1989). Over the decades, several protocols of estrus and ovulation synchronization (using hormonal treatment combinations) have been tested in dairy cattle reproductive programs (Pursley et al., 1995; Hiers et al., 2003; Sales et al., 2011). 10 Currently, one of the objectives of synchronization programs is to eliminate the need for estrus detection optimizing dairy farm management (Rabiee et al., 2005). In this context, the Ovsynch program (Pursley et al., 1995) is one of the most used synchronization protocols in dairy cattle (Rabiee et al., 2005). Over 50 research articles have evaluated the efficacy of Ovsynch program, and its variations, aiming to improve conception rates of bovine herds (reviwed by Rabiee et al., 2005). Based on GnRH and PGF2α, Ovsynch allows synchrony for successful AI without the need of estrus detection (Pursley et al., 1995). It includes administration of GnRH at random stage of the estrous cycle, which relies in the induction of atresia or ovulation of the DF leading to initiation of a new follicular wave (Pursley et al., 1995). It is followed by a PGF2α injection 7 d later to decline P4 circulating levels and a second dose of GnRH 48 h after PGF2α is administrated to induce ovulation (Pursley et al., 1995). In this protocol, AI is performed 16 to 24 h after second GnRH injection (Pursley et al., 1995). 1.2.2 Superovulation Conversely, in bovine embryo transfer programs (ETP) the majority of synchronization protocols include superovulation treatments to maximize the number of transferable embryos with high probability of producing pregnancies (Bó and Mapletoft, 2014). In cattle, these treatments rely on hormonal interventions applied before the dominance deviation process which leads to increased follicular growth and ovulation of several follicles (Hopper, 2021). Differently from synchronization protocols for single ovulating cattle, ETPs were commercially introduced in North America in the 1970s with the introduction of continental breeds (Bó and Mapletoft, 2014). At that time, continental breeds of cattle were in short supply and the use of ETP techniques offered the means by which their numbers could increase at a faster speed (Bó and Mapletoft, 2014). 11 Currently, superovulation and embryo transfer techniques are implemented in dairy herds to reproduce animals of high genetic merit (Bó et al., 2019). As a consequence of the genomic selection around 2008, most of the ETPs shifted the production of embryos primarily from cows to young, nulliparous heifers (Mikkola et al., 2020). In addition to its genetic contribution, ETPs have shown to improve the reproductive outcomes of commercial herds (Bó et al., 2019). Studies demonstrated improvement in fertility after embryo transfer in cows experiencing heat stress (Baruselli et al., 2010; Rodrigues et al., 2011) and in repeat breeder cows (Dochi et al., 2008; Ferreira et al., 2011). Rodrigues et al. (2007) reported that the transfer of embryos to repeat breeder cows, defined as cows that do not become pregnant after 3 or 4 breeding, resulted in increased pregnancy rates when compared to cows submitted to AI. Perhaps, these studies (Dochi et al., 2008; Ferreira et al., 2011) support the idea that the fertility problem in some repeat breeders may be associated with oocyte quality and/or failure in early embryo development (Mikkola et al., 2020). Although the commercial ETPs were introduced in the 1970s, one of the earliest scientific reports on superovulation in cattle was published in 1953 from the University of Wisconsin (Willett et al., 1953). In their study (Willett et al., 1953), cows were superstimulated using five daily injections of sheep or hog pituitary FSH. In this protocol, FSH was used to stimulate growth of several follicles and it was followed by an injection of human chorionic gonadotrophin (hCG) hormone 5 d later to induce ovulation (Willett et al., 1953). Besides FSH, equine chorionic gonadotropin (eCG) hormone (which has mostly FSH function) and human menopausal gonadotropin (hmG) hormone (which has FSH function) were also reported to induce superovulation in cattle (Bó and Mapletoft, 2020; Hafez et al., 1963). Whereas hmG was not used extensively by practitioners due to its high cost (Bó and Mapletoft, 2020), the use of eCG in 12 superovulation protocols became the standard approach for commercial ETPs due to its practicality (Hafez et al., 1963). A protocol developed by Hafez et al. (1963) was widely used in cows and included a single administration of eCG on day 16 of the cycle (to take advantage of the second follicular wave emergence), followed by administration of estradiol on day 19 and an injection of hCG on day 21 (Hafez et al., 1963). Nevertheless, subsequently results from scientific research promoted changes in practitioner’s approaches to superovulation (Bó and Mapletoft, 2014). Several studies demonstrated that cows submitted to eCG treatment had lower superovulatory response (Elsden et al., 1978; Monniaux et al., 1983), as well as abnormal P4 and LH levels (Greve et al., 1983; Dieleman et al., 1993) when compared to FSH-treated cows. In addition, when reviewing the historical perspective of superovulation in cattle, Bó and Mapletoft, (2014) stated that the main reason that most practitioners reduced the eCG use replacing it by FSH was due to eCG’s half-life. The long half-life of eCG, reported to be around 40 h (Murphy and Martinuk, 1991), was described to cause prolonged stimulation of the ovaries leading not only to abnormal endocrine profiles and non-ovulated follicles as also to reduced embryo quality (Mikel-Jensen et al., 1982; Callesen et al., 1986; Murphy and Martinuk, 1991) and increased risk of inducing an immune response, especially after repeated treatments (Mikkola et al., 2020). Nowadays, cows in most of the ETPs are superstimulated using pituitary extracts containing FSH (Bó and Mapletoft, 2014). Due to FSH low biological half-life, estimated to be 5 h or less (Laster, 1972), the usual regimen of FSH treatment has been twice daily for 4 or 5 d (Mikkola et al., 2020). Many practitioners prefer decreasing FSH dose schedules whereas others use constant-dose schedules (Bó and Mapletoft, 2014). Although, it seems that there is no experimental data supporting the use of constant or decreasing dose schedules (Bó and Mapletoft, 13 2014), the frequency of FSH administration has been the focus of several research studies (Hill et al., 1985; Lovie et al., 1994; Biancucci et al., 2016). It occurred based on the idea that reduced frequency of FSH administration could potentially improve protocol accuracy, safety and convenience from both humans and animal perspectives (Mikkola et al., 2020). In this context, several studies have been evaluating the superovulatory response of cows treated with FSH injections in reduced administration schedules such as full FSH dose given in a single injection (Hill et al., 1985) or full FSH doses given in different sites of injection (Hockley et al., 1992; Lovie et al., 1994). In addition, dilutions of full FSH dose in hyaluronic acid (Biancucci et al., 2016), aluminium hydroxide gel (Kimura et al., 2007; Tríbulo et al., 2011) or poly-vinyl-pyrrolidone (Chasombat et al., 2013) have been tested to decelerate FSH release and promote satisfactory superovulation using a single injection of FSH. However, some of these techniques were reported to have viscous solutions which were difficult to mix in the field (Tríbulo et al., 2011), lead to lower superovulatory response in comparison to the usual 4 d treatment schedule (Hill et al., 1985; Lovie et al., 1994; Tríbulo et al., 2011) or in others, the outcome appeared to depend on the animal’s body condition (Bo et al., 1994). In contrast with decreasing the number of FSH administrations, some authors (Bó et al., 2008; García Guerra et al., 2012) believe that the standard 4 to 5 d FSH treatment approach is insufficient for the development of small subordinate follicles (>1 mm) into ovulatory follicles. Using beef cows, García Guerra et al. (2012) evaluated the administration of FSH treatment for 7 d and demonstrated more follicles acquiring the capacity to ovulate with an increased number of ovulations when compared to 4 d FSH treatment. However, in their study (García Guerra et al., 2012), the mean number of total embryos recovered did not differ between treatment groups. In addition to the variations in the FSH schedule, most of the current superovulation protocols include 14 administration of PGF2α (to induce luteolysis) 48 or 72 h after initiation of FSH treatment with the objective to lead to occurrence of estrus in 36 to 48 h and ovulation 24 to 36 h later (Bó and Mapletoft, 2014). Although, superovulation protocols have evolved over the past 50 years, the unpredictable animals’ response to these protocols is still the major problem affecting the success of the embryo transfer industry (Bó and Mapletoft, 2014). A large number of factors can affect the superovulatory response in bovine herds, and some of these factors are inherent to the animal such as age (Kasimanickam et al., 2020), breed (Kasimanickam et al., 2020), nutrition (Sartori et al., 2017) as well as the environment in which the animal is housed including variations in temperature (Vieira et al., 2014). For example, Kasimanickam et al., (2020) demonstrated that lactating Holstein cows with body condition scores (BCS) lower than 2.5 or greater than 4 (in a 5-point scale) had lower number of total embryos recovered in comparison to cows with BCS between 2.5 and 4. In other study, Vieira et al. (2014) reported a significant difference in superovulatory outcomes between Holstein cows and heifers during the cooler and warmer periods of the year. In addition to factors inherent to the animal and its environment, one of the key components in the success of superovulation programs is the follicular status at initiation of ovarian superstimulation with FSH (Nasser et al., 1993; Adams et al., 1994). Several studies have shown that initiating FSH treatments as little as 1 d before or after the wave emergence significantly reduced the superovulatory response when compared with initiating treatments on the day of wave emergence (Nasser et al., 1993; Adams et al., 1994). However, given the duration of the follicular phases, only approximately 20% of the estrous cycle is available for initiating treatment at the time of follicular wave emergence (Bó and Mapletoft, 2014). Therefore, most of the superovulation programs include prior synchronization of wave emergence using either hormonal treatment 15 combinations with GnRH, P4, PGF2α or estradiol (Bó and Mapletoft, 2014) or using ultrasound-guided follicle ablation (Bergfelt et al., 1994). 1.3 Expression of estrus The first representation of a cow in estrus possibly occurred around 10,000 B.C. (Roelofs et al., 2010). Paleolithic cave paintings and engravings (Dordogne, France) may exhibit perhaps the first depiction of a reproductive event, a bull sniffing the vulva of a cow with representation of movement for mounting (Roelofs et al., 2010). Figure 2 shows an illustration from the Magdalenian engraving reprinted from Roelofs et al. (2010). Figure 2: Illustration from the Magdalenian engraving in the “Grotte the la Mairie”, Teyjat, Dordogne, France. Circa 10,000 B.C. Reprinted from Roelofs et al. (2010). © 2010 J. Roelofs, by permission #5275550431357. The expression of estrus is a behavioral sign that precedes ovulation, which occurs due to the effect of steroid hormones on specific centers of the hypothalamus in mammals (Roelofs et al., 2010). It is also a behavioral strategy to ensure that the female is mated close to ovulation (Roelofs et al., 2010). In cattle, the considered reference behaviour for estrus is standing immobile while being mounted (Roelofs et al., 2010), however, several behaviours were reported to be displayed more intensively during this phase. These behaviours are considered secondary signs of estrus and include restlessness, sniffing the vulva of another cow, flehmen, licking, rubbing, increase in 16 activity (number of steps taken), chin rest, mounting other cows and head butting (Roelofs et al., 2010). The hormonal profile of cows expressing sexual receptivity is characterized by low concentrations of P4, due to CL regression, and increasing concentrations of estradiol produced by the DF (Dieleman et al., 1986). Earlier studies have shown that P4 begins to decrease approximately 60 h prior to standing estrus whereas estradiol begins to increase about 40 h prior (Dieleman et al., 1986). Although the hormonal profile of cows during estrus is well known (Ginther, 2016), the endocrine mechanism regulating the intensity of estrous expression in cows is still only partially understood (Woelders et al., 2014). The hormonal control of estrus related behaviour have been the focus of several research studies over the past years (Fabre-Nys and Martin, 1991; Lyimo et al., 2000; Woelders et al., 2014). The role of P4 and estradiol in the control of estrous behaviour (Davidge et al., 1987; Lopez et al., 2004) as well as in the intensity of estrous expression (Aungier et al., 2015; Madureira et al., 2021) has been investigated. However, reports are not as consistent, and the physiological mechanism is still uncertain. Thus, research studies evaluating the role of P4 and estradiol on estrous behaviour will be the focus of discussion during this section. Increasing estradiol has been proposed as the trigger for estrous behaviour (reviewed in Roelofs et al., 2010). In dairy cows, positive correlations between estradiol and estrous expression were demonstrated in few studies (Lyimo et al., 2000; Lopez et al., 2004). Using continued sampling until 24 h after the last sign of estrus, Lyimo et al. (2000) showed a positive correlation between estradiol levels and total estrous score. Likewise, Lopez et al. (2004) correlated circulating estradiol at day of estrus with duration of estrous expression. In rats, estrogen receptors α-subtype (ERα) activated were reported to promote growth of hypothalamic neurons leading to 17 greater connections prior to sexual receptivity (Woelders et al., 2014). Interestingly in cattle, several genes associated with intensity of estrus were related to synaptic plasticity and remodeling of synaptic networks prior to estrus (Kommadath et al., 2011, 2010). Furthermore, estradiol levels around the time of estrus has been linked with influences on the genital tract such as changes in the cervical mucus (Rutllant et al., 2005), uterine tone (Bonafos et al., 1995), swelling and reddening of vulva (Diskin and Sreenan, 2000). However, reports evaluating the association between estradiol and estrous behaviour in cattle are not as consistent. Recently, Kumro et al. (2021) evaluated plasma estradiol levels every 2 h during estrous events in lactating and nonlactating dairy cows. In their study (Kumro et al., 2021), cows expressed signs of estrus as concentrations of circulating estradiol decrease. Although Lyimo et al. (2000) found a positive correlation between estradiol and total estrous score, in their study estradiol was shown to be sometimes elevated a day before onset of estrus and was not correlated with maximum score of estrous expression. In addition, Van Eerdenburg et al. (2000) demonstrated that no ERα were detected in the arcuate nucleus of the hypothalamus during estrous events of lactating dairy cows. In recent studies evaluating intensity of estrous expression using physical activity detected by AAMs, no correlation (Aungier et al., 2015) or weak correlation (Madureira et al., 2015; Silper et al., 2015b) between estradiol near the initiation of estrus and intensity of estrous expression was reported. Perhaps estradiol may not be the only steroid hormone playing a role in the regulation of intensity of estrous expression in cattle. Other studies have evaluated the role of P4 in the regulation of estrus related behaviour in cows (Simerly, 1998; Walker et al., 2008a; Madureira et al., 2021). Although earlier studies demonstrated that higher P4 levels during estrous phase had clear inhibitory effect on receptive behaviour (Asa et al., 1984; Ford, 1985; Davidge et al., 1987), circulating P4 prior estrus was 18 shown to be a key factor in the control of estrous expression (Simerly, 1998; Galvão et al., 2004; Madureira et al., 2021). In earlier studies, positive correlations between P4 priming and estrous expression (Bell et al., 1983; Stevenson et al., 1989) as well as intensity of estrous expression (Karsch et al., 1980; Fabre-Nys and Martin, 1991) were reported. Later studies investigating the use of estrus synchronization programs demonstrated that the addition of P4 during an Ovsynch-based (Kim et al., 2003) or estradiol-based (Galvão et al., 2004) synchronization protocol improved synchronization rates by preventing premature estrus. Recently, Madureira et al. (2021) reported that cows expressing greater intensity of estrous expression, measured by an AAM, had greater levels of circulating P4 4 d prior to estrus in comparison to cows expressing estrus of lower intensity. In addition, in the study of Madureira et al. (2021) a particular P4 profile (high concentration of P4 prior to AI, low concentration of P4 at AI and high concentration of P4 post-AI) was associated with increased expression of estrus and improved fertility of lactating dairy cows. Older reports evaluating the impact of P4 levels preceding estrus are not as consistent with more recent findings. Several earlier studies demonstrated no effect (Carrick and Shelton, 1969; Allrich et al., 1989) or negative effects (Davidge et al., 1987; Vailes et al., 1992) of previous P4 exposure in the estrous behaviour of dairy cows. Davidge et al. (1987) reported that higher P4 levels prior to estrus strongly inhibited chin rest and mounting behaviour in ovariectomized cows. Likewise, Vailes et al. (1992) demonstrated that higher circulating P4 prior to estrus was associated with reduced mounting activity in Holstein cows. Studies evaluating the association between P4 and estradiol at estrus demonstrated that prior exposure to P4 did not impact estradiol actions in dairy cows (Allrich, 1994) or in ovariectomized heifers (Carrick and Shelton, 1969; Allrich et al., 19 1989). In recent studies, negative associations between prior P4 exposure and estrous behaviour in dairy cows were not encountered. In the hypothalamus, neurons present in the medial preoptic nucleus are responsive to hormonal cues (such as P4 and estradiol) as well as olfactory signals (Saper and Lowell, 2014). The activation of these neurons leads to stimulation of the ventrolateral and ventromedial nucleus of the hypothalamus which in turn generates signals to the lateral hypothalamic area and midbrain periaqueductal gray matter, both responsible for motor and autonomic responses associated with sexual behaviour (Saper and Lowell, 2014). Although the presence of P4 and estradiol receptors in the ventromedial nucleus have been established many years ago (Sar and Stumpf, 1973; Rubin and Barfield, 1983), recent brain research conducted in rodents have shown that some hypothalamic neurons co-express P4 and estradiol receptors (Micevych and Sinchak, 2011; Sá and Fonseca, 2017). In addition, P4 might have the ability to downregulate or promote estradiol receptors in the hypothalamus depending on the phase of the estrous cycle (Sá and Fonseca, 2017). Although the definitive mechanism controlling the intensity of estrous behaviour have not been well established and the studies evaluating the associations between hormonal profile and sexual behaviour are inconsistent, it is plausible that P4 and estradiol, in conjunction, play a role in the control of estrous behaviour. The author of this thesis presumes an analogy of the actions of P4 and estradiol between the endometrium and the hypothalamus. In the endometrium, P4 controls the expression of its on receptor as well as estradiol receptor in a temporal matter, but also promotes accumulation of arachidonic acid and cycloxygenase, important for subsequent PGF2α production (Lamothe et al., 1977). Perhaps, the actions of P4 controlling estrous behaviour occurs in a similar process, where P4 binding to its receptor in the hypothalamus prior to estrus promotes accumulation of factors that facilitates posterior estradiol actions at the time of estrus increasing 20 the activation of motor and autonomic responses associated with sexual behaviour. This assumption could explain the inconsistence seen in scientific reports as the action of P4 on estrous behaviour depends on estradiol (and vice and versa), and the animal’s response to the hormonal profiles also depends on its receptor population in the hypothalamus. Despite the uncertainty in the mechanism of induction and control of estrus related behaviour in cattle, the behavioral changes seen in cows at estrus may possibly occur by triggering similar biochemical reactions as seen in rodents (Woelders et al., 2014). Prior to sexual receptiveness in rats, expression of opioid genes were reported to elicit effects of partial analgesia whereas oxytocin receptors activated in the presence of estrogen exerted effects of reduced anxiety and aggression (Woelders et al., 2014). The association of these factors in rats allows the female to engage in sexual behaviour easily despite the risk of pain or injury associated (Woelders et al., 2014). In cattle, expression of oxytocin genes in several brain areas were reported to be associated with estrous behaviour (Kommadath et al., 2011, 2010). In addition, the expression of genes known to modulate anxiety, depression, mood disorders, and stress responses, were found to be associated with the estrus score in cows (Kommadath et al., 2011). Beyond the endocrine control, many factors were reported to influence the expression of estrus in cattle (Roelofs et al., 2010). Reduction of estrous intensity have been associated with incidence of lameness (Walker et al., 2008b), higher milk production (López-Gatius et al., 2005; Rivera et al., 2010), poor nutrition (Wolfenson et al., 1997; Orihuela, 2000), lactation (López-Gatius et al., 2005), extended period with high environment temperatures (Gangwar et al., 1965) and genetic factors (Orihuela, 2000; Roxström et al., 2001). For example, intensity as well as duration of receptive behaviour were reported to vary between individuals with different genetic backgrounds (Roxström et al., 2001). In the study of López-Gatius et al. (2005), for each additional 21 lactation number there was a 20% decrease in walking activity at estrus. In addition, social interactions such as number of cows in estrus at any given time (Castellanos et al., 1997) as well as dominance rank (Orihuela et al., 1988) were reported to influence the behavioral manifestations of estrus. 1.3.1 Automated monitoring of estrous behaviour In the early 1970s, changes in housing systems and herd size of dairy farms increased the challenge of visual estrus detection (Stevenson and Britt, 2017). Cows were reported to be less likely to display estrous behaviour when housed on concrete flooring of free stall barns (Britt et al., 1986), and the number of cows managed per dairy worker has increased (Stevenson and Britt, 2017). Thus, throughout the decades several estrus detections tools have been developed and implemented to aid farmers reproductive management (Stevenson et al., 1996; Eradus et al., 1998; Roelofs et al., 2005). Insight about estrus detection aids is provided in details in several reviews (Foote, 1975; Senger, 1994; Diskin and Sreenan, 2000; Nebel et al., 2000; Firk et al., 2002). Technologies for automated monitoring of estrous behaviour, such as pedometers and accelerometers, will be the focus of this section. The first research report evaluating the use of pedometers for detection of estrus was published in 1977 (Kiddy, 1977). Accelerometers were introduced as physical activity monitors in North America in 2005 (reviewed in Stevenson and Britt, 2017). Whereas most pedometers measure activity using a horizontal spring-suspended lever arm which moves up and down when the cow moves, accelerometers assess the movement by changes in velocity (uniaxial, biaxial or triaxial depending on the technology) (Corder et al., 2007). Over the years, several studies have evaluated the efficiency of these technologies to detect incidence of estrus in cattle and have reported estrus detection rates ranging from 72 to 100% (Williams et al., 1981; Schofield et al., 1991; de Mol et al., 1997; Schweinzer et al., 2019). 22 Although the farmer decision to invest in these technologies depends on several factors such as farm size, financial status, profitability of the investment, as well as presence of a potential successor (Rutten et al., 2014), the adhesion of these systems have increased. Stevenson and Britt (2017) reported that perhaps 1 million individual sensors were sold in 2010, and at least a dozen companies were offering activity monitoring technologies. Unfortunately, to the best of my knowledge latest sales reports are not yet available. In addition to physical activity, these AAM are equipped to measure lying time, rumination or/and feeding time, ear temperature, and real-time positions in the barn (Stevenson and Britt, 2017). AAMs rely on increases in activity, a secondary sign of estrus, to detect the incidence of estrus in cattle (Firk et al., 2002). During estrus, cows increase activity by up to 9 fold compared to their own baseline activity (Silper et al., 2015a). Basically, pedometers and accelerometers systems are able to detect deviations in activity during estrus by comparing the activity value at estrus with activity values of a reference period (previous days or previous cycle depending on the technology) within the same cow (Stevenson and Britt, 2017). The threshold values which in turns trigger an estrous alert were reported to be calculated using mean value by one or two standard deviations (Williams et al., 1981; Schofield et al., 1991), confidence intervals (de Mol et al., 1997), or set relative increase (Eradus et al., 1992). However, nowadays the calculations and algorithms performed in most of the recent sensor technologies are protected by companies through intellectual property rights. The use of AAMs has helped researchers study the link between intensity of estrous expression and fertility of dairy cows. Greater intensity of estrus, measured by an AAM, was reported to be associated with greater pregnancy/AI (Madureira et al., 2015; Burnett et al., 2018) and lesser ovulation failure (Burnett et al., 2018) in spontaneous episodes of estrus in lactating 23 dairy cows. Expression of estrus was also reported to be compromised in anovular and older cows (Burnett et al., 2017). A study by Madureira et al. (2020) found that stronger estrous intensity was associated with greater number of total embryos collected and a greater percentage of viable embryos in superovulated heifers, and Madureira et al. (2022) reported that the occurrence and intensity of estrous expression improved pregnancy per embryo transfer in recipient lactating dairy cows. Although, utilization of AAM measurements to classify intensity of estrous expression in superovulated cows and heifers has been examined in few recent studies (Madureira et al., 2020, 2022), the association between behavioral estrous features and superovulatory response require more investigation. The intensity of estrous expression in single ovulating cows have been linked with endocrine profile of circulating P4 prior to and post-AI (Madureira et al., 2021), however, to date this association have not been examined in superovulated cows or heifers. Investigating this endocrine and behavioral relationship could provide evidence to use P4 profile and intensity of estrous expression as a tool to predict superovulatory response and assist in decision making of embryo transfer programs. 1.4 Fertilization and early embryonic development Successful fertilization requires prerequisites such as adequate oocyte maturation as well as adequate sperm transport and capacitation. In cattle, oogenesis (production of ovum) begins in the fetus pausing after birth with the nuclei of the oocytes in prophase I of their first meiotic division (Baker and Polge, 1976). The resumption of oocyte cell division is triggered during oocyte maturation by the preovulatory LH surge (Hyttel et al., 1989). In addition to resumption of the meiotic division, oocyte maturation includes several intracellular processes such as disruption of the gap junctions between cumulus-cell projections and oocyte, decreases in Golgi compartment 24 and transformation of the smooth endoplasmic reticulum (Hyttel et al., 1989). In cattle, release of the oocyte from the ovary to the oviduct occur between 22 and 29 h after preovulatory LH surge (Chenault et al., 1975; Bloch et al., 2006). The ampullary region of the oviduct hosts the fertilization process. However, prior to spermatozoa and oocyte binding, the sperm must be transported from the site of deposition in the female’s reproductive tract to the fertilization site (Baker and Polge, 1976). In cattle, spermatozoa flagellum motility as well as uterine elevated tone and contractility aid the spermatozoa transport to the oviduct (Baker and Polge, 1976). In addition, preceding fertilization the spermatozoa must undergo to a capacitation process in the female reproductive tract (Austin, 1951), which involves several biochemical processes for removal of seminal plasma coatings and surface molecules from the sperm (Baker and Polge, 1976). The capacitation will ensure that the spermatozoa have free binding molecular sites necessary for sperm-oocyte interaction (Baker and Polge, 1976). The interaction between oocyte and spermatozoon occurs due to specific binding proteins between the acrosome (membranous organelle of the sperm) and zona pellucida (membrane surrounding oocyte) (Senger, 2003). Although the zona pellucida was reported to contain 3 glycoproteins (Topper et al., 1997), the ZP3 protein is particularly responsible for the interaction between the zona pellucida and acrosome (Topper et al., 1997). In the ampullary region of the oviduct, ZP3-acrossome binding leads to initiation of acrosomal reaction (Topper et al., 1997) which is the fusion of spermatozoon membranes and the outer acrosomal membrane (Senger, 2003). This process leads to release of enzymes responsible for the digestion of a small portion of the zona pellucida and promotes spermatozoon penetration (Senger, 2003). In the interior of the zona pellucida, the contact between microvilli projections on the surface of the oocyte and the 25 posterior region of the spermatozoon head leads to oocyte-spermatozoon fusion (Szollosi and Ris, 1961). Following spermatozoon penetration, exocytosis of cortical granules occurs and induce biochemical changes in the zona pellucida, leading to activation of zona block mechanism which avoids polyspermy (Senger, 2003). It is followed by syngamy, the fusion of male and female pronuclei, and formation the single celled embryo named zygote (Senger, 2003). The zygote has a massive cytoplasmatic volume which is important for subsequent cell divisions confined within the zona pellucida (Senger, 2003). The large cytoplasmatic volume may be linked with large endogenous energy repository available to the developing embryo until implantation occurs (Sturmey et al., 2009). At this point, the zygote, undergoes to a series of mitotic divisions called cleavage division. A 2-celled embryo, product of the first cleavage division, has its cells named as blastomeres (Senger, 2003). Each blastomere will undergo to series divisions inside of the zona pellucida that maintains a fixed embryo volume throughout the sequential divisions (Senger, 2003). Generally, the diameter of the bovine embryo was reported to be approximately 150 to 190 μm including a zona pellucida thickness close to 12-15 μm (Lindner and Wright, 1983). The major source of endogenous energy reserve for the developing embryo is believed to be triglyceride, which in turn is the main component of the intracellular lipid in the oocyte (Homa et al., 1986). Additionally, exogenous nutrients such as pyruvate, lactate and amino acids may also be considered energy sources for the early embryo during the stages of pre-implantation development (Leese, 1995). Throughout development, active contractile patterns of the oviduct regulate slow movements of the embryo from the ampullary region of the oviduct to the uterus (Wijayagunawardane and Miyamoto, 2004). At 2, 4 and 8 celled developing embryo stage, the 26 blastomeres are totipotent, meaning that it has the ability to give rise to a complete individual organism (Senger, 2003). After the first divisions, a mass containing at least 16 cells is referred as morula (Bó and Mapletoft, 2013) and is described as a cellular mass which individual blastomeres are difficult to discern from one another (Bó and Mapletoft, 2013). The estimated age of the embryo at morula stage is 5 d (Lindner and Wright, 1983) and approximately at this stage the embryo reaches the uterus (Sponchiado et al., 2017). At the morula stage, embryo cells begin to distinguish into 2 populations: inner and outer cells (Senger, 2003). The inner cells are the population of cells that develop intercellular communication, whereas outer cells are the population that develop tight junctions (Senger, 2003). In the outer cells, the establishment of tight junctions alters the cell permeability promoting a buildup of ions and consequently fluid accumulation inside of the embryo (Senger, 2003). This process leads to the development of a fluid filled cavity within the embryo named as blastocele (Senger, 2003). At this phase, studies have shown a sharp rise in metabolic activity (measured by oxygen consumption) due to energy demands of sodium pumping for the formation of the blastocele and also increased protein synthesis (Brison and Leese, 1994; Donnay and Leese, 1999). Once a distinct cavity is recognizable, the embryo is called blastocyst (Bó and Mapletoft, 2013). A blastocyst has a highly prominent blastocele and is the stage where the differentiation between the 2 different cellular population is possible (Bó and Mapletoft, 2013). The outer cells, also named as trophoblastic cells, are the cells which will give rise to the fetal component of the placenta (extra embryonic membranes), whereas the inner cells will form the body of the embryo itself (Senger, 2003). The estimated age of the embryo at blastocyst stage is 7 d (Lindner and Wright, 1983). Throughout this stage, fluid continues to fill the blastocele, as the growth rate continues, which leads to increasingly internal pressure (Senger, 2003). At this point, thinning of 27 the zona pellucida also occurs due to the increasing diameter of the embryo (Bó and Mapletoft, 2013). With increasingly overall embryo diameter and thinning of the zona pellucida, the embryo is now named expanded blastocyst (Bó and Mapletoft, 2013). In addition to its continued fluid filled cavity and increasing diameter, the trophoblastic cells of the expanded blastocyst increase their production of proteolytic enzymes which further weakens the zona pellucida (Senger, 2003). At approximately day 9 or 10 (Sponchiado et al., 2017), the association of the factors mentioned above, combined with intermittent pressure pulses by the contractility of the embryo itself, leads to the rupture of the zona pellucida (Senger, 2003). Thus, the embryo becomes free from the zona pellucida and now is able to expand or elongate further within the lumen of the uterus (Senger, 2003). Embryos at this stage are named as hatched blastocyst (Bó and Mapletoft, 2013). The figure 3 demonstrate the temporal development of bovine embryos until the stage of hatched blastocyst. Structures are organized according to their number of cells (Mori et al., 2002) and days of development after fertilization process (Lindner and Wright, 1983). Microscopical embryo images are adapted from Bó and Mapletoft (2013) and Wei et al. (2017). 28 Figure 3: Temporal early embryo development in cattle according to number of cells and days after fertilization (© 2013 Bó and Mapletoft1 & © 2017 Q. Wei2 adapted with permission #5275550759609). 1.5 Progesterone and early bovine embryo In cattle, several studies have shown that the majority of the loss of potential offspring occurs during the early embryonic development stage (Santos et al., 2004; Inskeep and Dailey, 2005; Ribeiro et al., 2016). Reviewing several reports, Inskeep and Dailey (2005) summarized the distribution of failures of pregnancy in cows and demonstrated that perhaps 57% occurred due to the early embryo. Santos et al. (2004) shown that the viability of embryos collected from lactating, nonlactating and dairy heifers recovered 5 to 7d post-AI were in average 50, 57, and 71% respectively. Although the causes of poor early embryo development are complex and multifactorial, circulating P4 levels seems to be an important component in the regulation of Number of cellsDays after fertilization5d 8d2d1804Zygote12-cell em bryo14-cell em bryo18-cell em bryo1M orula2Blastocyst2Expanded Blastocyst2H atche d B lasto cyst2Hatching Blastocyst2110 29 fertility of dairy cows (Wiltbank et al., 2012). Recent studies have emphasized the role of P4 in reproductive processes at several points (reviewed in Lonergan, 2011; Wiltbank et al., 2014, 2012; Lonergan and Sánchez, 2020). Therefore, the focus of this section will be to review some of the studies evaluating the direct and indirect effects of circulating levels of P4 on bovine early embryonic development. 1.5.1 Progesterone levels prior to fertilization The effect of P4 on early embryo likely begins during the follicular development phase. In cattle, the number of follicular waves is determined by the length of the luteal phase and lower levels of P4 during the follicular growth was reported to lead, indirectly, to the persistence of the DF (Inskeep and Dailey, 2005). It is known that P4 regulates the pulsatile secretion frequency of GnRH which in turn regulates the frequency of LH (Kinder et al., 1996). In this context, low P4 levels during the follicular development was reported to promote increased frequency pulses of LH (indirectly) which in turn stimulate continued growth of the DF and thus, DF persistence (Inskeep and Dailey, 2005). In beef cows, Ahmad et al. (1995) have shown that oocytes from persistent DF are more likely to be in early stages of atresia and lead to embryonic death before the 16-cell stage. Likewise, in lactating dairy cows Cerri et al. (2009) demonstrated that extended length of the dominance phase led to fewer blastomeres and reduced embryo quality 6 d post-AI. Co-dominance and double ovulation, which is correlated with incidence of twins (Silva del Río et al., 2006), may also be affected by circulating P4 (Wiltbank et al., 2014). Several authors have shown a decrease in the number of double ovulations when circulating P4 levels are increased during the follicular development (Stevenson et al., 2007; Cerri et al., 2011b). Perhaps, low P4 levels during the follicular growth may underlie a delay in the FSH nadir and increase LH pulses surrounding dominance deviation resulting in dominance of two or more ovarian follicles 30 (Wiltbank et al., 2014). In addition, P4 may also have a direct role determining oocyte quality (Lonergan and Sánchez, 2020). Aparicio et al. (2011) reported the presence of P4 receptors in oocytes and cumulus cells and demonstrated that the inhibition of P4 synthesis (by the cumulus cells) during in vitro oocyte maturation led to significantly reduced blastocyst formation. However, the definitive mechanism by which P4 levels direct affect oocyte quality is still unclear (Lonergan and Sánchez, 2020). Although several studies have reported positive effects of P4 prior to estrus on pregnancy/AI (Fonseca et al., 1983; Diskin et al., 2006; Cunha et al., 2008; Bisinotto et al., 2010), recent research evaluating the effect of P4 levels prior to estrus on early embryo quality showed discrepant results. In the study of Rivera et al. (2011) cows in which the superovulatory treatment began with higher P4 levels (at the second follicular wave or first follicular wave supplemented with P4) had greater quality of embryos in comparison to cows where superovulation began with lower circulating P4 (at first follicular wave without P4 supplementation). Conversely, Cerri et al. (2011) reported no difference in embryo quality between cows when ovarian follicles did grow under low or high circulating P4 levels. However, whereas Rivera et al. (2011) investigated the effects of P4 using superovulated cows, Cerri et al. (2011) evaluated the effect of P4 using single ovulated lactating Holstein cows. The methodology by which these studies induced higher or lower concentrations of P4 during the follicular growth were also slightly different. Perhaps the inconsistency in the results demonstrated by these studies may be explained by the use of different study designs; or the impact of P4 levels on embryo quality differ between single ovulated and superovulated animals. In addition, the incidence or intensity of estrous expression, a possible intervening variable in the link between P4 concentrations prior to estrus and embryo quality, were not included into the analysis in these studies. In the study of Rivera et al. (2011), incidence or 31 intensity of estrus were not reported, whereas Cerri et al. (2011) demonstrated that only 5.3% of the lactating cows exposed to high concentrations of P4 were detected in estrus. When evaluating the effect of P4 levels prior to estrus on pregnancy/AI, Bisinotto et al. (2010) showed that lactating cows ovulating follicles that grew under low concentrations of P4 and showed estrus behaviour had similar pregnancy/AI than those ovulating follicles that grew under high P4 concentrations. Furthermore, in their study (Bisinotto et al., 2010) both groups of cows (high and low P4 showing estrus) had greater pregnancy/AI than cows not showing estrous behaviour regardless their P4 profile prior to estrus. 1.5.2 Progesterone levels near fertilization Beyond its effects during follicular growth, many studies have evaluated P4 levels surrounding the time of AI in cattle (De Silva et al., 1981; Waldmann et al., 2001; Ghanem et al., 2006). Several reports have shown that slight elevations of circulating P4 at the time of AI were associated with decreased fertility of dairy cows (Waldmann et al., 2001; Ghanem et al., 2006; Wiltbank et al., 2014). High concentrations of P4 near fertilization was found to impact indirectly oocyte fertilization by altering oviduct and uterine contractility affecting spermatozoon and oocyte transport (Hunter, 2005). In addition, a direct negative effect of higher P4 levels at fertilization possibly occurs; Silva and Knight (2000) demonstrated that the addition of P4 to an in vitro fertilization media reduced the rate of blastocyst formation. Although higher circulating P4 at the time of AI has been establish as a detrimental factor affecting cow fertility, the definitive physiological mechanism by which it occurs is still not well established (Wiltbank et al., 2014). 32 1.5.3 Progesterone levels post-fertilization. Expression of P4 receptors (nuclear and membrane) was reported to occur during all stages of bovine early embryo development (Clemente et al., 2009). Nevertheless, insufficient evidence exists for a direct effect of P4 on early embryos (Lonergan and Sánchez, 2020). Several studies have demonstrated no direct effect of P4 on embryo development by the addition of P4 during an in vitro culture media (Carter et al., 2008; Clemente et al., 2009; Larson et al., 2011). Conversely in vivo, Stronge et al. (2005) reported a positive linear correlation between P4 on days 5 to 6 post-AI and embryo survival; low P4 levels during these days were associated with low fertility of lactating dairy cows (Stronge et al., 2005). Although no direct effect on blastocyst formation has been found in vitro (Carter et al., 2008; Clemente et al., 2009; Larson et al., 2011), embryos developed under higher P4 levels have shown significant changes in gene expression (Carter et al., 2010) and post-hatching elongation (Carter et al., 2008). Carter et al. (2010) identified 194 differentially expressed genes between embryos collected from Simmental heifers non-supplemented and supplemented with P4 starting at 3 d post estrus. The pathways associated with the differentially expressed genes identified by Carter et al. (2010) were related to steroid hormone biosynthesis and cytokine-cytokine receptor interaction (Carter et al., 2010). Additionally, in beef-cross heifers, increased serum P4 post-AI was associated with increase in embryonic size on days 13 to 16 post-AI (Carter et al., 2008). However, considering the little evidence for a direct effect of P4 on early embryo development in vitro, many authors have suggested that the P4-induced changes in the oviductal and uterine environment are the major responsible for improved embryo development and increased fertility in cows (reviewed by Wiltbank et al., 2014 and Lonergan and Sánchez, 2020). 33 The P4 receptors (nuclear and membrane) was found in the oviduct of pregnant and cyclic cows during the post ovulatory period, suggesting that P4 may regulate functions in the oviduct through different pathways post-ovulation (Saint-Dizier et al., 2012). In addition, P4 supplementation was reported to increase amino acids composition of the oviduct fluid (Hugentobler et al., 2010) and to increase expression of endothelin 1 and its receptor in the oviductal cell membrane (Bylander et al., 2015). Endothelin was reported to have effects on oviductal muscular contractions and possibly be a P4 mediator of gametes and/or embryo transport (Bylander et al., 2015). On the other hand, increased amino acids in the oviduct fluid may benefit the development of the early embryo by acting as an exogenous energy source throughout the developing process (Leese, 1995). In the endometrium, P4 regulates P4 receptor in the luminal epithelium controlling PGF2α secretion (Kimmins and Maclaren, 2001). In addition, several studies have shown that reduced P4 production from the CL may lead to delay in temporal changes of the endometrial gene expression and reduced support for conceptus elongation (Beltman et al., 2009; Forde et al., 2012, 2011a). Conversely, P4 supplementation was reported to advance the temporal changes in the endometrial transcriptome (Forde et al., 2009) and contribute to conceptus elongation (Carter et al., 2008). Okumu et al. (2010) have shown earlier downregulation of P4 receptors in the endometrium of animals exposed to high P4 and suggested that uterine receptivity to implantation may be established earlier under increased P4 levels. In turn, low P4 has been linked with delay in downregulation of P4 receptors and associated with reduced capacity of the uterus to support conceptus development (Forde et al., 2011a). Although, some authors have stated that the changes in the endometrium gene expression occurs independently of pregnancy status, and that the major changes may be detected only on day 34 16 at maternal pregnancy recognition (Forde et al., 2011b; Bauersachs et al., 2012), a recent study (Sponchiado et al., 2017) demonstrated different perspectives. Sponchiado et al. (2017) evaluated the effect of early embryo development on endometrial function of bovines comparing pregnant and non-pregnant animals. In their study (Sponchiado et al., 2017), it was reported for the first time that the 7 d embryo was able to regulate interferon signaling and eicosanoid biosynthesis pathways in the endometrium. Perhaps, a viable 7 d embryo is able to regulate the endometrial function, by paracrine communication (Sponchiado et al., 2017), to favor its own development. This finding may raise perspectives that P4-induced changes in the oviductal and uterine environment may be mediated by the embryo itself. In summary, although the levels of circulating P4 throughout the bovine reproductive cycle have been the focus of several studies in the past decades, the definitive physiological mechanisms by which P4 regulates fertility in dairy cows is still partially understood. Higher levels of P4 during the follicular growth seems to be associated with reduced persistence of the DF (Inskeep and Dailey, 2005) and decreased number of double ovulations (Stevenson et al., 2007; Cerri et al., 2011b), however, the association of P4 levels during this phase and embryo viability is still uncertain (Rivera et al., 2011; Cerri et al., 2011). Low P4 levels near fertilization (time of AI) has been well establish as beneficial for fertility of dairy cows (Waldmann et al., 2001; Ghanem et al., 2006; Wiltbank et al., 2014) yet, the physiological mechanism by which it occurs is still partially understood. Lastly, higher P4 levels post fertilization has been reported as an important factor in the development of the early embryo by its effects on the oviduct (Hugentobler et al., 2010) and endometrium (Beltman et al., 2009; Forde et al., 2012, 2011a), however, a direct effect of P4 on early embryo is still uncertain. 35 1.6 Thesis objectives The objectives of this thesis were to evaluate if P4 concentration during the follicular growth is associated with (1) intensity of estrous expression, (2) superovulatory response, and (3) embryo production and quality in Holstein heifers submitted to a superovulation protocol and embryo collection at 7 d post AI. Investigating this endocrine, and behavioral (1), ovarian (2), and embryonic (3) relationship could provide evidence that the P4 profile and intensity of estrous expression could be used as a tool to predict and manipulate the superovulatory response in Holstein heifers, as well as, to maximize the number of transferable structures in embryo transfer programs. I hypothesized that heifers exposed to greater concentrations of P4 during follicular growth will (1) display greater intensity of estrous expression, (2) have growth of more DFs and ovulations, and (3) generate more embryos with greater quality when compared to heifers exposed to lower concentrations of P4. 36 Chapter 2: The impact of plasma concentrations of progesterone during superovulation on estrous behaviour, and ovarian and early embryonic development in Holstein heifers. 2.1 Introduction The hypothalamic mechanism by which the regulation of intensity of estrous behaviour occurs have not been fully elucidated (Woelders et al., 2014). In the literature reports evaluating the role of estradiol (Lyimo et al., 2000; Lopez et al., 2004; Kumro et al., 2021) as well as P4 levels prior to estrus (Vailes et al., 1992; Kim et al., 2003; Walker et al., 2008a) in the control of estrous expression in dairy cows are not as consistent. Recent studies evaluating intensity of estrous expression using physical activity detected by AAM reported no correlation (Aungier et al., 2015), or only a weak correlation (Madureira et al., 2015), between estradiol levels and intensity of estrous expression in lactating Holstein cows. However, greater levels of circulating P4 prior to estrus (diestrus phase) were reported to be associated with intensity of estrus detected by AAM (Madureira et al., 2021). Indeed, cows with greater levels of circulating P4 4 d prior to estrus expressed greater intensity of estrus in comparison to cows with lower levels of P4 (Madureira et al., 2021). In addition, a particular P4 profile (high concentration of P4 prior to AI, low concentration of P4 at AI and high concentration of P4 post-AI) was associated with increased expression of estrus and improved fertility in lactating dairy cows (Madureira et al., 2021). Studies have emphasized the effects of P4 levels prior to estrus on follicular development, embryo viability as well as temporal changes in PGF2α synthesis signals (reviewed in Lonergan, 2011; Wiltbank et al., 2014, 2012; Lonergan and Sánchez, 2020). Lower concentrations of P4 prior to estrus have been linked with higher pulsatile secretion frequency of 37 GnRH and thus frequency of LH, leading to continued growth and persistence of the DF (Inskeep and Dailey, 2005). In the study by Rivera et al. (2011), cows that began their superovulatory treatment with higher P4 levels had greater quality of embryos in comparison with cows that began with lower circulating P4. In addition, Cerri et al. (2011a) reported that reduced concentrations of P4 during the development of the ovulatory follicle led to premature luteolytic signals which might overwhelm the ability of the conceptus to block the PGF2α synthesis cascade. Greater increase in physical activity during estrus, measured by AAM, has been associated with improved fertility of lactating dairy cows (Madureira et al., 2015; Burnett et al., 2018). Studies have shown that greater intensity of estrous expression were correlated with greater pregnancy per AI (Madureira et al., 2015; Burnett et al., 2018), greater pregnancy per embryo transfer (Madureira et al., 2022), as well as lesser ovulation failure (Burnett et al., 2018) and lesser pregnancy losses (Madureira et al., 2019) when compared to lower intensity of estrous expression. Recently, Madureira et al. (2020) demonstrated that superovulated Holstein heifers expressing greater intensity of estrus had increased embryo viability and a greater number of embryos per flushing in comparison to heifers expressing moderate intensity of estrus. The intensity of estrous behaviour in heifers, as in lactating dairy cows, was shown not to be correlated with estradiol levels at time of estrous events (Silper et al., 2015b). Although, greater estrous expression in single ovulating cows have been linked with endocrine profile of circulating P4 prior to and post-AI (Madureira et al., 2021), to date this association in superovulated heifers remains to be researched. Differences between lactating cows and heifers in follicular dynamics and steroid secretion have been reported (Sartori et al., 2002a; Wolfenson et al., 2004). Characteristics of lactating animals, such as high feed intake and milk production, were reported to be related to 38 increased liver blood flow and steroid metabolism resulting in low P4 and estradiol concentrations in the circulation (Sangsritavong et al., 2002; Vasconcelos et al., 2003). In the study of Wolfenson et al. (2004), longer duration of the dominance phase, larger preovulatory follicle, lower preovulatory LH surge, as well as lower estradiol circulating, and lower circulating P4 concentrations were reported in cows when compared to heifers. Therefore, the association between the intensity of estrus expression and P4 regulation may be different between cows and heifers as the latter would not be confounded by milk production and all the related changes. With this perspective, exploring the effect of P4 concentrations prior to estrus, on the intensity of estrous expression, ovarian superovulatory responses, and embryo production and quality using the post-puberty heifer model would yield additional information to understand the role of these different components in the modulation of ETPs used in modern dairy cattle operations. Furthermore, to date a high degree of unpredictability in superovulatory response still exists affecting the efficiency and profitability of commercial ETPs (Bó and Mapletoft, 2014). Several reports have evaluated factors influencing multiple ovulations in dairy cows and heifers (reviewed in Bó and Mapletoft, 2014; Mikkola et al., 2020), including factors inherent to the animal such as age and breed (Kasimanickam et al., 2020), as well as related to the management such as nutrition (Sartori et al., 2017), climate (Vieira et al., 2014) and protocol of superovulation (García Guerra et al., 2012). However, the variability in superovulatory response still remains partially understood (Bó and Mapletoft, 2014). Although the effect of estrous expression (Madureira et al., 2020) and P4 levels prior to estrus (Rivera et al., 2011) have been investigated separately in superovulated animals, the association between these factors and successful superovulation require more investigation using randomized trials instead of only observational studies. Evaluating this endocrine and behavioral relationship could provide supplementary 39 information to predict and improve superovulatory responses in dairy heifers assisting in the decision making of ETPs. The aim of this study was to evaluate the effect of P4 concentrations in plasma during a superovulatory treatment on the intensity of estrous expression, ovarian responses, and embryo production and quality in Holstein heifers. We hypothesize that high concentrations of P4 during a superovulatory treatment will induce the display of greater intensity of estrous expression, induce the growth of a greater number of DFs and ovulation success, as well as yield a greater number of embryos with superior quality when compared to low concentrations of P4. 2.2 Materials and methods This study was conducted between November 2020 and July 2021 at the University of British Columbia’s Dairy Education and Research Centre in Agassiz, Canada. All experimental procedures were approved by the Animal Care Committee of the University of British Columbia following the protocol # A20-0182. Holstein heifers between 10.5 and 12.0 months had their ovaries scanned twice, four and two weeks prior to the expected start date, using a portable ultrasound scanner with 7.5-MHz linear-array transducer (Ibex Pro; E.I. Medical Imaging, Loveland, CO). During the ultrasound exams, follicles and CL were identified and their diameters measured. Heifers with a CL in at least one of the evaluation exams were enrolled into this experiment. Heifers were housed in sand-bedded free stalls with concrete flooring, headlock system, fans, and manure scraper. Animals were fed once daily with TMR formulated to exceed the requirements of a 350 kg Holstein heifer (NRC, 2001). Water and TMR were available for ad libitum intake. 40 2.2.1 Automated Activity Monitor Heifers were monitored continuously by an AAM (AfiPedometer Plus Tag, AfiMilk, Kibbutz Afikim, Israel) placed on the back leg above to the distal expansion of the metatarsal bone of each heifer at enrollment. Activity data was transmitted and recorded by the system hourly throughout the experiment period. Unprocessed step count data was retrieved from the AfiMilk system in time blocks of approximately 15 minutes. 2.2.2 Treatments Heifers were randomly assigned into two treatment groups: Low P4 and High P4. Randomization was performed by alternating treatments groups in chronological order by which heifers presented CL in their weekly exams. All heifers received a pre-synchronization protocol followed by the treatment, with a protocol of superovulation as described in Figure 4 (day 0 was defined as the day which AI was performed). Protocols schedule were as follows. Pre-synchronization protocol: on d -27, heifers received an injection of 500 mcg of cloprostenol (PGF2α; 2.0 mL i.m.; Estrumate, Merck Animal Health, Kirkland, QC, Canada), and on d -24, an injection of 100 µg of gonadorelin (GnRH; 2.0 mL i.m.; Fertiline, Vetoquinol, Lavaltrie, QC, Canada). On d -18, heifers received an intravaginal implant of progesterone (CIDR; 1,38 g progesterone, Zoetis, Kirkland, QC, Canada) and an injection of GnRH. Treatment and protocol of superovulation: on d -11 CIDR withdrawal occurred for all heifers and heifers in the Low P4 group received two doses of PGF2α 12 h apart on the same day. On d -9, all heifers received an injection of GnRH. On d -8, High P4 heifers received a new CIDR whereas Low P4 heifers received a second use CIDR. Starting on d -6, all heifers received decreasing doses of follicle stimulating hormone (FSH, i.m.; Folltropin; Bioniche, Inverin, Galway, Ireland) every 12 h for four days with total of 200 mg per heifer (starting at 40 mg/dose 41 and finishing at 10 mg/dose). Heifers in the Low P4 group received injections of PGF2α on d -5 (p.m.) and d -4 (a.m. and p.m.). On d -2 (60 h before AI), heifers in the High P4 group received an injection of PGF2α. Heifers from Low P4 and High P4 groups received an injection of PGF2α at the same time of CIDR withdrawal, 48 h before AI (d -2). Heifers from both groups also received two injections of GnRH on d -1, 12 h apart. Timed AI was performed twice (a.m. and p.m.) by a trained technician on day 0 with the same frozen-thawed semen. 2.2.3 Ultrasonography and blood samples Blood samples (10 mL) were taken on d -18, -11, -9, -6, -4, -2, 0 and 7 from the median coccygeal vein or artery into a sterile tube with EDTA (Vacutainer, Becton Dickinson, Franklin Lakes, NJ, United States). The samples were centrifuged at 2,000 x g for 15 min and stored at -80°C for future analysis. Plasma P4 concentrations were analyzed using a chemiluminescent enzyme immunoassay, as previously described by Nadalin et al. (2021). Briefly, P4 was extracted from plasma samples using petroleum ether and subsequently diluted in working conjugate solution (matching enzyme conjugate (3-CMO-Progesterone-HRP, East Coast Bio) in buffer). Samples were pipetted into coated and blocked plates, then incubation occurred for a minimum of 2 h and the plate was subsequently washed. After washing, the addition of substrate solution occurred, and the plate was incubated up to 1 h in the dark. Lastly, a stop solution was added. Optical density was measured on a microplate reader (450 nm) and the percentage binding was calculated by a visible spectrum absorbance reader. A log-logit regression line was used to calculate the concentration of P4. Concentration of P4 (ng/mL) were adjusted using correction factor for extraction inefficiency obtained from quality control samples. Real-time per rectum ultrasonography, using a portable ultrasound scanner with 7.5-MHz linear-array transducer (Ibex Pro; E.I. Medical Imaging, Loveland, CO), was used to monitor 42 ovarian structures and determine the presence and diameter of the follicles and CLs, as well as the ovulation of follicles. In addition to the evaluation exams (4 and 2 weeks prior to enrollment), ultrasonography exams were carried on days -26, -24, -18, -11, -9, -8, -7, -6, -4, -2, and 0, 24 h, 48 h, and 7 d post-AI. 2.2.4 Embryo collection Heifers were flushed 7 d post-AI for embryo collection. On the day of flushing, heifers received an injection of epidural anesthesia using 3 mL of lidocaine (Lidocaine HCL 2%, Bimeda Animal Health, Ontario, Canada). Flushing was performed by a transcervical procedure using Foley catheter (16 FR; 30 c.c.; Bioniche Animal Health., USA, Inc.) and the balloon of the catheter was placed approximately 3 cm past the external intercornual ligament of a uterine horn (ipsilateral to the CLs) and inflated. Approximately 400 mL of a flushing solution (ABT complete flush, ABT 360., Washington, USA) was used in the uterine horn in cycles of infusion/recovery to retrieve structures into a filter. For heifers with CLs on both ovaries, flushing was repeated in both horns. The content of the filter was transferred into a square petri dish with grid and embryo searching and evaluation was performed under microscope. Embryos were evaluated for fertilization and grades of quality (1 = excellent and good, 2 = fair, 3 = poor, and 4 = degenerated; IETS, 2013). Stage of embryonic development was also classified as follows: morula, compact morula, early blastocyst, blastocyst, or expanded blastocyst (Bó and Mapletoft, 2013). 43 Figure 4: Schedule for the presynchronization and superovulation protocols of heifers exposed to Low or High progesterone (P4) during the follicular growth phase, in parallel with the schedule for reproductive tract ultrasound (US) exams, and blood samples (BS) performed during the study. Holstein heifers, between 10.5 and 12-months old, were randomly assigned into two treatment groups (High P4 and Low P4) to evaluate the effect of P4 concentration during the follicular growth on estrous intensity, superovulatory response, and embryo quality. AI - artificial insemination; EC - Embryo collection, FSH - Follicle-stimulating hormone, PG - Prostaglandin F2α, GnRH - Gonadotropin-releasing hormone. 2.2.5 Statistical analysis Statistical analysis was carried out using SAS Studio (version 3.8, SAS Institute Inc., Cary, NC). Exclusively for the unprocessed physical activity data (steps count), data cleaning and preparation was performed using RStudio (version 1.3.1073, RStudio, PBC). A sample size of 26 heifers per group was calculated to identify a difference of 30% in the proportion of embryos of 44 excellent and good quality between heifers expressing greater and moderate intensity of estrous expression (53.1% vs 23.4%; Madureira et al., 2020) with a 1.14 adjustment factor for confounders, 95% confidence and 80% power. This sample size was also sufficient (sample size = 18 per group) to identify a difference of 35% in the proportion of embryos of excellent and good quality between heifers exposed to low and high concentrations of P4 (85.5% vs 50.0%; Rivera et al., 2011). An average of 3 embryos/heifer was considered in the sample size calculation. This sample size calculation also accounted for a 35% loss to follow up. Descriptive analysis was performed, and continuous variables were verified for normality and presence of outliers using graphical assessment (PROC UNIVARIATE). Unprocessed physical activity data (steps count) for 7 d before expected estrus (from d -9 to d -2) was used to calculate the baseline activity per hour, specifically for each heifer, using rolling mean procedure. Relative increase (RI) in walking activity was calculated by determining changes in activity from d -2 to 0 in comparison to the baseline of each heifer and the percentage of relative increase was calculated as follows: RI = [100 × (steps per hour/steps per hour at baseline)]. Heifers were considered to be in estrus when RI exceeded 180% for at least 2 hours. The end of an estrous episode was defined as RI < 180%. Heifers with 2 episodes of estrus within a 4-hour interval had their activity data combined into 1 event of estrus. The duration of estrus was calculated using the number of hours from the beginning to the end of an estrus event. The maximum intensity of estrous expression was defined as the maximum RI during an estrous episode. Mixed multinomial logistic regression models were used to analyze the effect of P4 treatment and estrous expression characteristics (maximum intensity and duration of estrus) on the stage of embryonic development and grades of quality (PROC GLIMMIX), considering embryo as experimental unit and including heifer as random effect. Binary logistic regression models were used to analyze the effect of treatment and estrous 45 expression characteristics on ovulation (defined by the presence of at least 1 new CL 7 d post-AI), considering heifer as experimental unit (PROC GLIMMIX). For logistic regression models, the linear relationship between the numeric variables and the log odds of the outcome were assessed graphically (PROC SGPLOT). The number of follicles at start of the FSH treatment, the total number of follicles at the time of AI, the total number of CL 7 d post-AI, and the total number of embryos recovered per flushing were used as count outcome variables. The effect of treatment and estrous expression characteristics (maximum intensity and duration of estrus) on count variables was tested using Poisson regression models (PROC GENMOD). In Poisson regression models, residuals and outliers were assessed graphically whereas overdispersion was analyzed by calculating the ratio between the raw variance and overall mean (a ratio greater than 2:1 was considered overdispersion; Dohoo et al., 2009). The effect plots of Poisson regression models were generated using PROC PLM procedure. Multivariable linear regression models were performed to analyze the effect of P4 treatment on estrous expression characteristics (maximum intensity and duration of estrus; PROC GLM). Plasma P4 levels during treatment days (d -9, -6, -4, -2) were used to calculate the area under the curve (AUC) of P4 during treatment for each heifer. The effect of P4 treatment on the AUC, the diameter of the DF and CL at beginning of presynchronization protocol and at the onset of P4 treatment was analyzed using multivariable linear regression models (PROC GLM). Plot of average plasma P4 levels during treatment days (d -9, -6, -4, -2) were generated using PROC SGRENDER procedure. The effect of estrous expression characteristics (maximum intensity and duration of estrus) on the plasma P4 levels (ng/mL) 7 d post-AI were analyzed using multivariable linear regression models (PROC GLM). The effect of P4 treatment on the diameter of the follicles at the time of AI was tested using mixed linear regression model (PROC MIXED), considering follicles as experimental unit, and including heifer 46 as random effect. For all linear regression models, homoscedasticity and normality of the residuals were assessed graphically using residual plots. A Cox proportional-hazards model was used to compare the time from CIDR removal (d -2) to estrus (RI exceeded 180% for at least 2 hours) between treatment groups (PROC PHREG). Observations were right censored at d 0 if estrus was not observed. Survival curves were generated from the proportion of heifers not in estrus at each hour. The assumption of proportional hazard was evaluated graphically and using Kolmogorov-type supremum test. For all models, heifers age and weight, as well as month of enrolment were tested (fixed effects) as possible confounders and kept in the models if P ≤ 0.2 (Dohoo et al., 2009). In models for which estrous expression variables were considered as an exposure, the total follicle number at AI was tested as possible confounder and kept in the models if P ≤ 0.2 (Dohoo et al., 2009). Confounders were identified using previously developed causal diagram (Figure 5). Results from logistic regression models are presented as odds ratios (OR), those from Poisson regression models are presented as log odds, and those from the Cox proportional-hazards model as hazard ratios (HR), all with 95% confidence interval (CI). Results were considered statistically significant when P ≤ 0.05, and results with biologically relevant difference but and a P ≤ 0.10 were presented as tendencies. 47 Figure 5: Causal diagram used to inform the construction of the statistical models performed to evaluate the effect of different concentrations of progesterone (P4) during the follicular growth on the intensity of estrous expression and embryo development in superovulated Holstein heifers. Possible confounders were kept in the models if P ≤ 0.2 (Dohoo et al., 2009). 2.3 Results A total of 63 Holstein heifers (High P4 = 32 and Low P4 = 31) were enrolled into this study. Heifers were on average (± SD) 10.9 ± 0.3 months and weighted 352 ± 20 kg, which did not differ between treatment groups (P = 0.9). Ninety-three percent of the heifers (n = 59) were considered to be in estrus between d -2 and 0. The overall average (± SD) of maximum intensity of estrous expression and duration of estrus were 520.4 ± 129.7 % RI and 13.9 ± 7.9 h, respectively. The AUC of plasma P4 concentrations during treatment (d -9, -6, -4, -2) were different between High P4 and Low P4 groups (Mean ± SE; High P4 = 11.9 ± 0.8 vs. Low P4 = 6.2 ± 0.8; P <.0001). Descriptive statistics of plasma P4 concentrations by treatment groups are summarized in Figure 6. 48 Figure 6: Mean (± SEM) progesterone (P4) concentrations (ng/mL) during the study (where d0 was defined as the day of timed AI) by treatment group. Holstein heifers were randomly assigned into two treatment groups (High P4: n = 32 and Low P4: n = 31) to evaluate the effect of P4 concentrations during the follicular growth on estrous intensity, superovulatory response and embryo quality. 2.3.1 Ovarian structures and ovulatory response to superovulation The diameters of the DF and CL were similar between High P4 and Low P4 groups at the beginning of presynchronization protocol (d -26), as well as at the onset of the P4 treatment (d -11; Table 1). The number of follicles at the start of the FSH treatment averaged (± SD) 3.8 ± 1.6 follicles and were not different between treatment groups (Mean ± SEM; High P4 = 3.5 ± 0.2 vs. Low P4 = 4.0 ± 0.3 follicles; P = 0.43). The total number of follicles at the time of AI was found to be associated with P4 treatment. Indeed, heifers exposed to High P4 had a greater number of Study days Progesterone levels (ng/mL) 49 follicles at time of AI in comparison to heifers exposed to Low P4 treatment (Mean ± SEM; High P4 = 16.6 ± 1.6 vs Low P4 = 13.9 ± 1.2 follicles; P = 0.005). However, the diameter of the follicles at the time of AI tended to be greater for Low P4 heifers in comparison to High P4 heifers (Mean ± SEM; High P4 = 11.3 ± 0.1 vs Low P4 = 12.0 ± 0.1 mm; P = 0.09). Table 1 summarizes the effect of P4 treatment on ovarian structures. Treatment Item High P4 Low P4 P-value Heifers, n 32 31 - Presynchronization (d - 26) DF, mm 13.4 ± 0.5 13.4 ± 0.4 0.98 CL, mm 19.9 ± 1.3 21.2 ± 1.3 0.46 Onset of P4 treatment (d - 11) DF, mm 12.3 ± 0.6 13.2 ± 0.6 0.21 CL, mm 22.5 ± 0.6 23.2 ± 0.6 0.48 Onset of FSH treatment (d -6) Number of follicles, n 3.5 ± 0.2 4.0 ± 0.3 0.43 Timed AI (d 0) Number of follicles, n 16.6 ± 1.6 13.9 ± 1.2 0.005 Size of follicles, mm 11.3 ± 0.1 12.0 ± 0.1 0.09 Table 1: Predicted means (± SEM) of the different ovarian structures of superovulated Holstein heifers by progesterone treatment. Holstein heifers received a pre-synch protocol, starting on d-26, followed by P4 treatment (High P4 or Low P4) with a protocol of superovulation, starting on d -11. Day 0 was defined as the day of timed AI. DF – Dominant follicle; CL – Corpus Luteum, FSH – Follicle-stimulating hormone, P4 – Progesterone, AI – Artificial insemination. 50 Seventy-three percent of the heifers (n = 46) ovulated following superovulation. Ovulation was not affected by P4 treatment (High P4 = 71.9% vs. Low P4 = 74.2%; OR = 0.8; 95% CI = 0.3 – 2.8; P = 0.81). However, estrous expression characteristics were associated with the odds of ovulating. The odds of ovulation increased by 1.84 for each 100% RI change in the maximum intensity of estrous expression (95% CI = 1.1 – 3.1; P = 0.01), and by 1.13 for each hour increase in the duration of estrus (95% CI = 1.0 – 1.2; P = 0.01). Although P4 treatment was not associated with the number of CLs 7 d post-AI (P = 0.20), the maximum intensity of estrous expression and the duration of estrus were associated with the total CL number 7 d post-AI. For each hour increase in the duration of estrus, the number of CLs 7 d post-AI increased (Figure 7A; log odds = 0.05; 95% CI = 0.03 – 0.06; P <.0001). Similarly, the number of CL 7 d post-AI increased for each RI change in the maximum intensity of estrus (Figure 7B; log odds = 0.003; 95% CI = 0.001 – 0.003; P <.0001). A) Total CL number 7 d post-AI Duration of estrus (h) 51 B) Figure 7: Effect plots from Poisson regression models summarizing the association between the duration of estrus (A) and maximum intensity of estrus (B) and the total corpus luteum (CL) number 7 d post- artificial insemination (AI), by treatment groups (High P4 and Low P4; heifers = 46). The maximum intensity of estrous expression was defined as the maximum relative increase (RI) during an estrous event. The duration of estrus was calculated using the number of hours which heifers exceeded 180% RI during an estrous event. 2.3.2 Progesterone treatment and estrous expression Heifers exposed to High P4 treatment tended to have longer time from CIDR removal (d -2) to estrous event in comparison to heifers exposed to Low P4 treatment (Mean ± SE; High P4 = 26.2 ± 2.0 h vs. Low P4 = 20.8 ± 1.8 h; HR = 0.65; 95% CI = 0.38 – 1.09; P = 0.09). Figure 8 illustrates the survival curve analysis for time to estrous event between experimental groups. Treatment did not the affect the maximum intensity of estrous expression (Mean ± SE; High P4 = 497.8 ± 23.9 % RI vs. Low P4 = 542.2 ± 23.5 % RI; P = 0.19) or the duration of estrus (Mean ± SE; High P4 = 13.5 ± 1.5 h vs. Low P4 = 14.3 ± 1.4 h; P = 0.70). However, estrous expression measurements were associated with P4 plasma levels 7 d post-AI in heifers that ovulated (n = 46). Total CL number 7 d post-AI Maximum intensity (Relative increase) 52 For each 100% RI increase in the maximum intensity of estrous expression, there was an increase of 1.31 ng/mL of plasma P4 levels 7 d post-AI (SE = 0.09; P = 0.04). Likewise, for each hour increase in the duration of estrus an increase of 0.51 ng/mL of P4 was found 7 d post-AI (SE = 0.09; P = 0.008). Figure 8: Survival curve for time from CIDR removal (d -2) to estrous event by treatment groups (High P4 and Low P4), using a Cox proportional-hazards model (HR = 0.65; 95% CI = 0.38 – 1.09; P = 0.09). Observations were right censored at d 0 if estrus was not confirmed (n = 59 confirmed estrus vs. n = 4 not confirmed). Day 0 was defined as the day which timed artificial insemination (AI) was performed. 2.3.3 Number of embryos recovered 7 d post-AI The flushing procedure was not performed for 21 heifers due to either ovulation failure (n = 17) or the impossibility to perform the transcervical technique (n = 4). A total of 42 animals were flushed 7 d post-AI, however, no structures were recovered after flushing procedure in 12 heifers. A total of 124 structures (High P4 = 53 and Low P4 = 71) were recovered from 30 heifers and evaluated for fertilization and stage of development. Due to technical difficulties, 105 embryos Time (h) Proportion of heifers not in estrus High P4 Low P4 53 (High P4: n = 42 and Low P4: n = 63) from 26 heifers were evaluated for quality. The overall average (± SD) of structures per heifer was 4.1 ± 3.8 (median = 2; range = 1 to 12). Per treatment group, the average (± SD) of number of embryos recovered per heifer was 3.3 ± 3.0 (median = 2; range = 1 to 9) for the High P4 group and 5.0 ± 4.5 (median = 3.5; range = 1 to 12) for the Low P4 group. Embryo/ova recovery (per ovulated follicle) was 51% (n = 124/243). Descriptive statistics of stage of embryonic development and grades of quality are shown in Table 2. Treatment Item Total High P4 Low P4 Embryos, n 124 53 71 Embryo stage, % Morula 34.9 41.8 30.9 Compact morula 4.9 1.9 7.0 Early blastocyst 13.3 19.6 8.5 Blastocyst 14.8 19.4 11.4 Expanded blastocyst 25.5 9.6 36.6 Unfertilized structure 6.6 7.7 5.6 Embryos, n 105 42 63 Embryo grade, % Excellent and good 53.3 64.2 46.0 Fair 27.6 21.5 31.8 Poor 14.4 9.6 17.5 Degenerated 4.7 4.7 4.7 Table 2: Proportion of embryos recovered 7 d post-AI (following superovulation) for each stage of embryonic development and grade of quality by treatment group (High P4 and Low P4 during the 54 follicular growth). Embryos were evaluated for grade of quality (IETS, 2013) and stage of embryonic development (Bó and Mapletoft, 2013). 2.3.4 Progesterone treatment and embryo development Progesterone treatment did not affect embryo stage (OR = 2.21; 95% CI = 0.69 – 7.06; P = 0.17). However, the odds of being of a greater quality grade were 1.98 times higher for heifers in the High P4 group than heifers in the Low P4 group (95% CI = 0.90 – 4.35; P = 0.08). In addition, heifers exposed to high concentrations of P4 had lower number of embryos per flushing in comparison to heifers exposed to low P4 concentrations (High P4 = 0.53 ± 0.13 log odds and Low P4 = 0.91 ± 0.11 log odds; P = 0.03). 2.3.5 Estrous expression and embryo development Embryonic developmental stage was not affected by the duration of estrus (OR = 1.00, 95% CI = 0.90 – 1.12, P = 0.90) or the maximum intensity of estrous expression (OR = 0.99, 95% CI = 0.99 – 1.00, P = 0.32). Likewise, embryonic quality grade was not affected by the duration of estrus (OR = 1.03, 95% CI = 0.95 – 1.11, P = 0.45) or the maximum intensity of estrous behaviour (OR = 1.00, 95% CI = 0.99 – 1.00, P = 0.49). However, the total number of embryos recovered 7 d post-AI was affected by estrous expression measurements (P <.0001). For each hour increase in the duration of estrus the embryo counts increased (Figure 9A; log odds = 0.04; P >.0001). Similarly, the embryo count increased for each 100% RI change in the maximum intensity of estrus (Figure 9B; log odds = 0.41; P >.0001). Both Poisson regression models included the total follicle number at AI as confounder. 55 A) B) Figure 9: Effect plots from Poisson regression models summarizing the effect of duration of estrus (A) and maximum intensity of estrus (B) on number of embryos recovered 7 d post-AI (n = 42 heifers), by treatment groups. Total follicle number at AI was considered a statistically significant confounder (P ≤ 0.2) and kept in the model (the Duration of estrus (h) Number of embryos recovered 7 d post-AI Number of embryos recovered 7 d post-AI Maximum intensity (Relative increase) 56 effect plot fit were computed using the mean total of follicles). Relative increase (RI) in walking activity was calculated by determining changes in activity from d -2 to 0 in comparison to the baseline of each heifer. The maximum intensity of estrous expression was defined as the maximum RI during an estrous episode. Duration of estrus was calculated using the number of hours heifers exceeded 180% RI during an estrus event. 2.4 Discussion The main objective of this study was to evaluate the effect of P4 concentrations in plasma during a superovulatory treatment on the intensity of estrous expression, ovarian responses, and embryo production and quality in Holstein heifers. This study demonstrated that different concentrations of P4 during the follicular growth had an effect on the number and the diameter of follicles following superovulation, the embryo quality, and the number of embryos recovered 7 d post-AI. Ovulation, embryonic stage, and estrous expression characteristics, however, were not affected by P4 treatment. Furthermore, this study demonstrated that the duration and the intensity of estrous expression were associated with the odds of ovulation as well as the number of follicles that ovulated, the number of embryos recovered, and the P4 concentrations 7 d post-AI. However, embryonic stage and quality grades were not associated with estrous expression characteristics. The P4 AUC during the follicular phase showed that the protocol used in this study for High P4 and Low P4 treatments successfully induced high and low circulating concentrations of P4, respectively. Administration of PGF2α injections to heifers assigned to Low P4 treatment were intended to induce complete luteolysis or to compromise the synthesis of P4 during initial CL development (Inskeep, 1973). Studies have shown that treatment with PGF2α successfully induces reduced concentrations of circulating P4 during the early luteal phase in crossbred heifers (Beltman et al., 2009) and during the development of the ovulatory follicle in lactating Holstein cows (Cerri et al., 2011b). The utilization of a second use progesterone-releasing intravaginal device in heifers assigned to Low P4 treatment probably contributed to the differences observed in circulating 57 concentrations of P4 during follicular growth, but another reason to use the intravaginal device in the Low P4 group was to avoid premature ovulations. Studies have shown that cows receiving a second use intravaginal implant containing 1.9 g (van Cleeff et al., 1992) or 1.38 g (Herrmann and Wallace, 2007) of P4 had lower serum concentrations of P4 in comparison to cows receiving new intravaginal devices. In this study, the diameter of the DF and CL was similar between High P4 and Low P4 groups, both at the beginning of presynchronization protocol and at the onset of P4 treatment. The ovarian structures at the onset of the synchronization protocols were shown to affect the synchronization response (Moreira et al., 2000). Indeed, it was demonstrated that dairy heifers starting synchronization on day 2 of the estrous cycle, with lower diameter of DF (4.6 ± 7 mm) and CL (3.6 ± 1 mm), had a decreased synchronization response compared with heifers that began with established DF and CL (DF greater than 10 mm and CL greater than 19 mm). The similarity of ovarian structures between treatment groups found in this study indicates that heifers from both experimental groups possibly had similar ovarian capacity to respond successfully to presynchronization and treatment protocols. The number of follicles at the start of the FSH treatment were not different between the High P4 and Low P4 groups. It is well known that the emergence of follicular waves is stimulated by an increase in FSH (Adams et al., 1992). However, the factors controlling the number of follicles at the follicular wave emergence are not yet fully understood (Burns et al., 2005). When evaluating 138 different follicular waves in dairy cows, Burns et al. (2005) reported that the variability in the number of follicles (3 mm or greater in diameter) was inversely correlated with the concentrations of FSH and was not associated with estradiol, inhibin, or insulin-like growth factor 1 (IGF1) levels. High and low concentrations of P4 were reported to have no effect on FSH 58 secretion in Holstein heifers in an prior study (Ireland and Roche, 1982), however, whether different circulating P4 levels have an impact in the mechanisms regulating the recruitment of a new cohort of antral follicles for the next growth wave is still uncertain. The similarity between treatment groups found in this study suggests that different circulating levels of P4 during follicular wave emergence did not affect the recruitment of follicles, yet the physiological mechanisms underlying this association needs to be further investigated. In addition, the similarity in number of follicles at the onset of the FSH treatment between the experimental groups indicates that heifers exposed to High P4 or Low P4 treatment likely had similar gonadal competence to respond to superstimulation. One of the major sources of variability in superovulatory protocols was reported to be the number of follicles at onset of the FSH treatment (Mikkola et al., 2020). In Holstein cows, Kawamata (1994) reported that the number of small follicles (3 to 6 mm of diameter) 1 d before the initiation of a superovulatory treatment with FSH was significantly correlated with the number of CLs after superovulation and the number of transferable embryos recovered 7 d post estrus. Likewise, Ireland et al. (2007) reported that beef heifers with consistently high follicle numbers during their follicular waves had greater response to superovulation in comparison to heifers with low follicle numbers. In the present study, the High P4 heifers had a greater number of follicles at the time of AI in comparison to Low P4 heifers. However, the diameter of follicles tended to be greater for Low P4 group in comparison to High P4 group. Cerri et al. (2011a) investigated the effects of P4 concentrations during the development of the ovulatory follicle and demonstrated that cows exposed to high concentrations of P4 had greater concentration of IGF1 in the follicular fluid in comparison to cows exposed to low P4 concentrations. In the same study (Cerri et al., 2011a), low circulating P4 was associated with increased fluid concentrations of estradiol, which has been 59 negatively correlated to IGF binding proteins and free IGF1 (Echternkamp et al., 1994). Insulin like growth factor 1 acts stimulating the proliferation and differentiation of granulosa cells in synergy with gonadotropins (Velazquez et al., 2009). In the study from Gong et al. (1996), heifers treated with recombinant bovine somatotropin, which has been associated with elevations in IGF1 (Gong et al., 1991), had increased superovulatory response to FSH treatment in comparison to control heifers. Perhaps high P4 levels during the follicular development in superovulation treatments is also associated with greater IGF1 and greater stimulation of ovarian follicles growth. However, our study did not evaluate IGF1 levels, and this association needs to be further investigated in future research. In fact, Low P4 heifers had greater diameter of follicles at the time of AI in comparison to High P4 heifers. In single ovulating cows, studies have shown this association (Cerri et al., 2011b) or no association (Bisinotto et al., 2015) between lower circulating P4 (during follicular growth) and larger diameter of the DF at final GnRH injection of synchronization protocols. Lower P4 concentrations during the follicular development has been associated with increased frequency of the pulses of GnRH and thus LH, resulting in increased LH stimulatory effect on follicle development (Inskeep, 2004; Wiltbank et al., 2006). In the study by Cerri et al. (2011a), the basal concentration of LH tended to be greater for cows exposed to lower concentrations of P4 during development of the DF. Conversely in superovulated cows, Rivera et al. (2011) reported that the concentrations of LH on day 4 of a superstimulation protocol with FSH were not different between cows that began the superstimulation during the first or second follicular wave, i.e., with different P4 levels. Unfortunately, the diameter of the follicles at the time of AI was not reported in their study (Rivera et al., 2011). The measurement of follicle diameter in superstimulated ovaries via ultrasonography may be challenging, due to the large number of follicles, and requires diligent attention. Our results suggest that lower levels of P4 have impacts 60 on follicular diameter in superovulation treatments, however, the physiological mechanism underlying this association was not assessed and future research is needed to further investigate the relationship between P4, LH secretion and diameter of follicles in superovulatory treatments. Although the number of follicles at time of AI was shown to be affected by P4 treatment in this study, ovulation and number of follicles that ovulated (total CL number 7 d post-AI) were not different between treatment groups. Earlier studies evaluating the effect of P4 levels at initiation of superstimulation with eCG (Saumande, 1980) or FSH (Tamboura et al., 1985) reported no correlation between P4 concentrations at the start of treatment and subsequent number of follicles that ovulated in dairy cattle. However, using Simmental and Charolais cross heifers, Goulding et al. (1990) reported that animals starting superstimulation at day 10 of the estrous cycle (i.e., with higher P4 levels) had greater proportion of follicles that ovulated compared with heifers which stimulation with FSH started on day 2 (i.e., with lower P4 levels). In more recent studies evaluating the effect of P4 concentrations, information on number of follicles that ovulated were not reported in superovulated animals (Rivera et al., 2011), whereas Cerri et al. (2011b) reported no difference in ovulation rates between single ovulating cows with high and low concentrations of P4 during the development of the DF. As a consequence of a greater number of follicles at the time of AI in heifers exposed to high P4 concentrations shown in our study, we expected that High P4 heifers would have greater number of ovulations (or total CL number 7 d post-AI) when compared to Low P4 heifers. It is not clear why there was no differences in number of CLs 7 d post-AI between treatment groups, however, it is possible that the smaller diameter of follicles from heifers exposed to High P4 treatment, when compared to heifers from Low P4 treatment, affected the ovulatory capacity of High P4 heifers. In the study of Sartori et al. (2001), although acquisition of ovulatory capacity occurred when the DF was 10 to 12 mm of diameter, cows 61 required a greater dose of LH to induce ovulation, whereas when the DF reached ≥ 12 mm ovulation was induced in all cows using standard LH doses. The odds of ovulation and the number of follicles that ovulated (total CL number 7 d post-AI) were shown to increase as the duration of estrus and the maximum intensity of estrous expression increased. The association between greater intensity of estrous expression and ovulation has been reported in several studies using single ovulating dairy cows (Burnett et al., 2018; Madureira et al., 2019, 2015). Although, a recent study from our laboratory have reported that greater intensity of estrous expression was associated with greater embryo production in superovulated dairy heifers (Madureira et al., 2020), information on the probability of ovulation and the number of follicles that ovulated were not assessed on that study. To the best of our knowledge, this is the first study reporting the association between intensity of estrous expression measured by an AAM and number of ovulated follicles in superovulated Holstein heifers. Ovulation failure has been reported as a consequence from the lack of responsiveness of the hypothalamus to increased estradiol, resulting in failure to stimulate the GnRH surge and thus the LH surge (Wiltbank et al., 2002). In addition to this, a several factors were reported to impact the ovulatory process in superovulated cows (Mikkola et al., 2020). For example, ovulation rates have been reported to be affected by the follicular status at the initiation of the ovarian superstimulation with FSH (Nasser et al., 1993; Adams et al., 1994), as well as FSH dose and FSH/LH ratio (Kanitz et al., 2002). However, considering the fact that the heifers in this study experienced similar superovulatory protocol, and that the P4 treatment imposed did not affect the odds of ovulation, it is possible that the expression of estrus of greater intensity and duration is, to some extent, linked with the responsiveness of the hypothalamus to the preovulatory stimulus and the ability of the ovarian follicles to ovulate after superstimulation treatment. However, specific hypothalamic 62 mechanisms potentially associated with the relationship between expression of estrus, preovulatory stimulus and ovulatory capacity of follicles, such as GnRH, LH profiles as well as components of the follicular fluid, needs to be further investigated. In this study, High P4 heifers tended to have longer time from CIDR removal (d -2) to estrous event in comparison to Low P4 heifers. Overall, the interval from CIDR removal to estrous event in the current study was shorter than in other studies on dairy cows (Dalton et al., 2000; Hittinger et al., 2004). In single ovulating cows, the time from CIDR removal to standing estrus was reported to average 49 h (Hittinger et al., 2004), whereas it was described to be on average 39 h in superovulated cows (Dalton et al., 2000), possibly due to increased circulating estradiol produced by several growing follicles. Perhaps the use of different behaviours to detect estrus (standing estrus vs. physical activity detected by AAM) may explain some of the variation in time to estrus event between studies. Using visual observation of standing estrus behaviour, it is feasible to determine if a heifer is in estrus or not, however, little can be said whether that is the onset or the end of the episode of estrus. In addition, even using AAMs, Silper et al. (2015a) reported differences in onset and end of estrous episodes when comparing two different technologies to monitor estrous behaviour in Holstein heifers. Due to lower circulating concentrations of P4 and greater diameter of follicles in Low P4 heifers in the present study, we expected shorter time from CIDR removal to estrous event in those animals when compared to heifers exposed to High P4 treatment. In the study of Savio et al. (1993), the circulating concentrations of P4 was shown to decrease faster in heifers receiving a used CIDR (lower circulating P4) when compared to heifers receiving a new CIDR (higher circulating P4). In the same study (Savio et al., 1993), the diameter of follicles was inversely proportional to time to estrous event. Although we expected such differences, the fact that High P4 heifers had greater number of follicles at time of AI may raise 63 questions regarding the differences in estradiol profile between experimental groups and its relationship with the interval from CIDR removal to estrus. Because our study did not evaluate estradiol profiles, it is not possible to rule out the possibility that the shorter time to estrus event in Low P4 heifers is due to increased circulating estradiol. However, it is possible to point out that the differences in P4 profiles prior to estrus are linked with time to estrous interval. In a study conducted by our laboratory using a similar protocol to induce different P4 levels in single ovulating heifers (Moore and Cerri, 2022), lower P4 levels during the development of the DF was shown to be correlated with shorter interval to estrous event when compared to higher P4 levels. Progesterone treatment did not affect estrous expression characteristics in this study. Given the fact that greater P4 levels 4 d prior to estrus was reported to be associated with increased intensity and duration of estrus in Holstein dairy cows (Madureira et al., 2021), we expected that High P4 heifers would have increased maximum intensity of estrous expression and duration of estrus when compared to Low P4 heifers. Due to differences in follicular dynamics and steroid secretion between lactating cows and heifers, such as lower estradiol and P4 circulating in lactating cows (Sartori et al., 2002a; Wolfenson et al., 2004), it is possible that different P4 levels prior to estrus in heifers do not impact the mechanism regulating the intensity of estrus expression. However, in the study from our laboratory using single ovulating heifers (Moore and Cerri, 2022), greater intensity and duration of estrous expression were positively associated with higher circulating P4 levels during the development of the DF in a crossover experimental design. In addition, high P4 levels 7 d post-AI was associated with estrous expression characteristics in this study. It is not clear why P4 levels prior to estrus and post estrus have different associations with estrous expression characteristics in the current study. Considering the fact that the profile of steroid hormones in superovulated and unstimulated Holstein heifers has been reported to differ 64 during the follicular growth phase (Roberge et al., 1995), we speculate that the effect of P4 concentrations and intensity of estrus in superovulated Holstein heifers may be, to some extent, different than in single ovulating heifers. In addition, the number of animals used in the current study may be insufficient to detect the effect differences of P4 levels on estrous expression characteristics. The embryonic developmental stage was not affected by P4 treatment, however heifers in the High P4 group tended to have embryos of greater quality and a lower number of embryos recovered 7 d post-AI in comparison to heifers in the Low P4 group. Likewise in superovulated dairy cows, Rivera et al. (2011) demonstrated lower embryo quality from superovulated animals induced to ovulate follicles from the first follicular wave (i.e., with reduced P4 levels during follicle growth) than from the second follicular phase of their estrous cycle. Antral follicles exposed to low concentrations of P4 were reported to have hastened growth, increased estradiol production, and premature oocyte maturation (Inskeep, 2004). Concentrations of estradiol were reported to not affect fertilization in lactating dairy cows (Cerri et al., 2011b), however premature oocyte maturation was associated with early stages of follicular atresia (Inskeep, 2004). In this context, it is plausible that growing follicles from heifers exposed to Low P4 levels could be undergoing to gradual atresia and oocytes were able to be fertilized, however, their developmental competence was, to some extent, compromised resulting in lower quality of embryos. The probability of producing pregnancies in ETPs was reported to increase as the quality of the transferred embryos increase (reviewed in Bó and Mapletoft, 2013). Bovine embryos graded as excellent and good quality were reported to result from 9% to 20% greater pregnancy per embryo transfer when compared to embryos graded as poor quality (Chebel et al., 2008; Madureira et al., 2022). Thus, supplementation with P4 prior to estrus might have the potential to increase the 65 probability of pregnancy in ETPs by increasing the quality of transferable embryos. In fact, Low P4 heifers had a greater number of embryos recovered 7 d post-AI in comparison to High P4 heifers. Considering that heifers from both experimental groups had similar number of follicles ovulated, it is not clear why the number of embryos recovered 7 d post-AI was different between High P4 and Low P4 treatments. In the study of Rivera et al. (2011), no difference in the number of embryos recovered 6.5 d post-AI was found between cows superovulated during different periods of the follicular wave (different P4 profiles). Because High P4 heifers tended to have longer time from CIDR removal to estrous event and had numerically lower percentage of expanded blastocyst when compared to Low P4 heifers, we speculate that the time for ovulation of follicles was longer in High P4 heifers. This factor could be influencing the transport of embryos from the oviduct to the uterus and affecting the number of recovered embryos in this study. However, time for ovulation was not assessed in this study and the association between P4 concentrations, ovulation process and embryo transport in superovulated heifers should be further investigated in future research. Estrous expression characteristics did not affect the embryo stage or quality grades, however, the total number of embryos recovered 7 d post-AI was found to be affected by estrous expression measurements. Greater intensity of estrous expression, measured by AAM, has been previously correlated with increased embryo viability and production in Holstein heifers (Madureira et al., 2020) as well as increased P4 levels 7 d post-AI in lactating dairy cows (Madureira et al., 2021, 2019). Progesterone levels post estrus were reported to impact embryonic gene expression (Carter et al., 2010), post-hatching elongation (Carter et al., 2008) and fertility outcomes of dairy cows (Stronge et al., 2005). Considering the association between intensity of estrus expression, P4 and fertility in dairy cows (Madureira et al., 2021, 2019), we expected a 66 positive effect of intensity of estrous expression on quality of embryos and number of embryos recovered 7 d post-AI in dairy heifers. The results demonstrated in the current study may suggest that, although intensity of estrous expression impacted embryo production, it did not affect embryo quality in superovulated Holstein heifers exposed to different concentrations of P4 during follicular growth. Perhaps the effect of estrous expression on quality of embryos is, somehow, minimized when different levels of P4 are induced prior to estrus in Holstein heifers. Nevertheless, the expression of estrus of greater intensity and duration was shown, once more, to be an important marker of improved embryo production in superovulated dairy heifers. 2.5 Conclusions In conclusion, this study demonstrated that high P4 levels during the follicular phase had a positive impact on the number of follicles at time of AI and quality of embryos following superovulation in Holstein heifers. However, decreases in the diameter of follicles at time of AI and number of embryos recovered 7 d post-AI were found when heifers were exposed to high P4 levels. In addition, estrous expression characteristics were not affected by different concentrations of P4 during the follicular growth, yet intensity of estrous expression was shown to affect the odds of ovulation as well as the absolute number of follicles that ovulated following superovulation, the number of embryos recovered, and the plasma P4 levels 7 d post-AI. 67 Chapter 3: General Discussion 3.1 Thesis conclusions In dairy cows, intensity of estrous expression have been associated with impacts on fertility over the past 5 years (Burnett et al., 2018; Madureira et al., 2022, 2019). However, the definitive physiological mechanism regulating the intensity of estrous behaviour in cows is not yet fully understood (Woelders et al., 2014). The overall objective of my thesis was to contribute to the current knowledge regarding the association between intensity of estrous expression and fertility in dairy cattle by demonstrating the effect of different P4 concentrations during the follicular growth on the intensity of estrous expression, superovulatory response, and embryo production and quality in superovulated Holstein heifers. In Chapter 1, the physiological mechanism of the estrous cycle, fertilization process and early embryonic development was described along with biotechnologies to manipulate follicular growth, behavioural estrus, and automated monitoring of estrous expression. Chapter 1 was concluded with a review concerning recent findings on P4 levels at different phases of the reproductive cycle and its associations with fertility outcomes in dairy cattle. Throughout the first chapter, gaps in the scientific literature were highlighted in order to connect the role of the scientific study (described in Chapter 2) in filling the gaps related to the association between P4, estrous expression and early embryo development in superovulated dairy heifers. In Chapter 2, it was demonstrated that heifers exposed to high P4 levels during the follicular growth had greater number of follicles at the time of AI, tended to have greater quality of embryos, but had lower number of embryos recovered 7 d post-AI when compared to heifers exposed to low P4 levels. Findings described in Chapter 2 also demonstrated that greater intensity and longer duration of estrous expression, measured by AAM, resulted in increased odds of ovulation and 68 number of follicles ovulated, as well as greater number of embryos recovered 7 d post-AI. With this, once more intensity of estrous expression was shown to be an important marker of improved fertility in superovulated dairy heifers. Furthermore, different concentrations of P4 during the follicular growth did not affect the intensity of estrous expression in superovulated Holstein heifers in the study described in Chapter 2. However, P4 plasma levels 7 d post-AI was shown to be associated with the maximum intensity of estrous expression and the duration of estrus. Perhaps P4 may be associated with the intensity of estrous expression in superovulated dairy heifers, however, the results presented in Chapter 2 suggest that it may occur differently than hypothesized by the author of this thesis. It is possible that the link between P4 and the intensity of estrous expression is not exclusive to this hormone profile prior to estrus, but it is also dependent on the decline of P4 levels at time of estrus and estradiol levels. 3.2 Limitations and future directions In Chapter 2, twenty-seven percent of the animals submitted to the superovulatory treatment failed to ovulate at least one follicle. Although it is well known that the variability in superovulatory response have been among the primary limiting factor affecting the success of the embryo transfer industry (Lonergan and Boland, 2011), superovulation programs have reported that in average 80% of the donors have three or more ovulations following superstimulation (Lonergan and Boland, 2011). Given the scenario of the scientific study described in Chapter 2, it is possible that the increased anovulatory rates have impacted the expected number of embryos recovered in the study. However, currently in most of the embryo transfer programs, the selection of embryo donors is based on their genetic lines and their efficiency in superovulatory response (based on previous stimulations) and not in a randomized experimental condition as described in 69 Chapter 2. In fact, the absence of prior selection of animals, based on their previous response to superstimulation, may explain the increased anovulatory rates reported in the study. As mentioned throughout Chapters 1 and 2, several studies have focused on investigating different factors affecting the superovulatory response in dairy cattle aiming to improve the response to superstimulation and thus, the success of the embryo transfer industry. For example, studies have evaluated the frequency of FSH administration (Hill et al., 1985; Lovie et al., 1994; García Guerra et al., 2012; Biancucci et al., 2016), different FSH dilutions (Kimura et al., 2007; Tríbulo et al., 2011; Chasombat et al., 2013), as well as the effects of age (Kasimanickam et al., 2020), breed (Kasimanickam et al., 2020), nutrition (Sartori et al., 2017), environment (Vieira et al., 2014) and follicular status (Nasser et al., 1993; Adams et al., 1994). However, to date the variability in superovulatory response is still a challenge (Bó and Mapletoft, 2014). Future research is needed to continue investigating the mechanisms affecting the efficiency of the superovulatory response in dairy cattle. Perhaps greater understanding of this variability can assist in the development of an improved superstimulation method and promote a more repeatable superovulatory response in dairy heifers reducing the need of prior selection of animals. In Chapter 2, I hypothesized that High P4 heifers had compromised ovulatory capacity in comparison to Low P4 heifers. This hypothesis was brought out due to the fact that High P4 heifers had lower diameter and greater number of follicles at the time of AI in comparison to Low P4 heifers, however, the number of follicles ovulated (total CL number 7 d post-AI) was shown to be similar between experimental groups. Studying the acquisition of the ovulatory capacity, Sartori et al. (2001) demonstrated that ovulation was induced in all cows using a standard LH dose when the DF reached ≥ 12 mm. Considering the fact that the diameter of follicles for High P4 heifers averaged 11 mm at the time of AI, extending the follicular phase during the superovulatory 70 protocol could be a potential strategy to improve the ovulatory capacity of High P4 heifers. Perhaps maintaining the P4 intravaginal device or extending the FSH treatment by 1 d, using similar superovulatory treatment, could lead to increase in the diameter of follicles and consequently increase in the number of follicles able to ovulate in heifers exposed to high concentrations of P4. This strategy can potentially lead to not only greater quality of embryos as well as greater number of embryos produced 7 d post-AI when high concentration of P4 is induced during the follicular growth. Future research is needed to investigate the association between the length of the follicular phase, P4 levels and ovulatory capacity in superovulated Holtein heifers. In the study described in Chapter 2, no embryos were recovered after flushing procedure in 19% of the heifers. In addition, the embryo recovery rate (per ovulated follicle) was 51%. To date, most of the basic methodologies in uterine flushing are based on the experience of veterinary practitioners and yet little information is provided by research investigation (Sartori et al., 2003). When using nonsurgical embryo recovery methods, studies have shown rates of embryos recovery (per ovulated follicle) varying widely from as high as 60–80% (Kelly et al., 1997; Kim et al., 2001) to as low as 20–25% (Fricke et al., 1994; Mitchell, 1998). The effect of different uterine flushing techniques on embryo recovery rates have been evaluated in few studies (Sartori et al., 2003; Castro Neto et al., 2005). Sartori et al. (2003) compared recovery of embryos from superovulated Holstein heifers by flushing the uterine horns through insertion of the catheter very close to the tip of the horn (deep) or just after the uterine bifurcation (shallow). In their study (Sartori et al., 2003), the shallow flushing technique was superior to the deep technique for number of embryos recovered per horn (5.4 ± 1.1 vs 3.9 ± 0.8) as well as percentage of embryos recovered per CL (63.9 ± 8.6% vs. 37.4 ± 6:5%). Castro Neto et al. (2005) demonstrated that double uterine flushing increased the number of embryos recovered in superovulated Limousin (Bos taurus) and Guzera 71 (Bos indicus) cows (Double = 6.7 ± 0.4 vs. Single = 4.6 ± 0.2). In their study (Castro Neto et al., 2005), the flushing technique was performed placing the catheter in the uterine body and after the flushing the catheter was either removed (single flushing) or left in the same position and an additional 150 ml of flushing solution was infused and kept in the uterus for 30 minutes (double flushing). Although in most of the studies (including the study described in Chapter 2) the flushing technique and trained personal is kept constant to reduce the variability in the embryo recovery rate, it is evident that the embryo transfer industry requires a potential gold standard, scientifically tested, method for uterine flushing. Limited literature exists evaluating flushing procedures and future research is needed to investigate different flushing techniques, its variations and pros and cons aiming to promote consistent rates of embryo recovery in ETPs. The area under the curve of plasma P4 levels during the follicular growth were different between experimental groups in the study described in Chapter 2. However, there was a variability in P4 profiles across individuals within the same treatment group (data not shown). This variability could have impacts in the results obtained during this study. Several studies have evaluated P4 profiles (using milk and/or blood samples) in lactating dairy cows during successive estrous cycles (Martin et al., 2013; Blavy et al., 2016; Antanaitis et al., 2020). Nevertheless, to the best of my knowledge similar evaluation has not been conducted in dairy heifers. Throughout the estrous cycle, P4 seems to have a typical profile including continuous increase post estrus, maintaining at luteal phase and decreasing close to the estrous event; however, there is no clear understanding of what is deviant and what is a normal pattern (Blavy et al., 2016). In addition, optimum concentrations of P4 at each stage of this typical profile have not been set (Blavy et al., 2016). For example, it is well know that circulating concentrations of P4 greater than 1 ng/mL at the time of AI impact fertility (Waldmann et al., 2001; Ghanem et al., 2006; Wiltbank et al., 2014). However, 72 at what speed P4 levels needs to increase after estrus or what is the range concentration of circulating P4 during the follicular growth that is beneficial for the growing follicle is still uncertain. Thus, in the context of the study described in Chapter 2, it is complex to define normal or abnormal variability of plasma concentrations of P4 within the treatment groups and select a subset of animals that responded to the treatment according to expected. More research is needed to create knowledge on optimal range of P4 profile associated with fertility, as well as how and when P4 profiles are different, comparing different farms, different management systems, different breeds, stage of lactation, among others. These comparisons could be a first step toward the development of a point of reference to identify deviations in P4 profiles throughout each phase of the estrous cycle and to support researchers working in this field. In conclusion, this thesis contributed to the knowledge concerning hormonal regulation of the intensity of estrous expression and its association with embryo production and quality in superovulated dairy heifers. It is evident that greater understanding of the intensity of estrous expression and its hormonal regulation have the potential to increase the profitability of dairy herds by improving reproductive performance of dairy cows. In addition, given the importance of superovulation and embryo transfer programs to reproduce animals of high genetic merit (Bó et al., 2019), it is clear that greater understanding of P4 regulation, behavioral estrus, and embryo production have positive impacts in the genetic selection programs. In the past 2 decades, genetic selection programs of dairy cattle have shown increased attention to fertility traits (Chebel and Veronese, 2020). 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Thesis/Dissertation
eng
Applied Animal Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Cerri, Ronaldo
The impact of plasma concentrations of progesterone during superovulation on estrous behaviour, and ovarian and early embryonic development in Holstein heifers
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