Chapter 33 Ram Kasimanickam Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, Washington, USA Reproductive competence is a major aspect affecting the production and economic success of dairy and beef cow herds. For herds using artificial insemination (AI), detection of estrus (submission rate) and calving rate are the two major determinants of inter-calving interval. The economic consequences of low efficiency and poor accuracy in the detection of estrus are the main reasons why cattle reproduction research programs focus on developing practical breeding protocols. The important requirements for any effective estrous synchronization protocol are predictable and high estrus and ovulation responses during a specified interval, followed by a higher pregnancy rate to a single insemination carried out in a predetermined time.1 To achieve better control of the estrous cycle in cattle it is necessary to synchronize emergence of new follicular waves, ensure a luteal phase, terminate the luteal phase, and synchronize ovulation. Growth of ovarian follicles in cattle occurs in distinct wave-like patterns during the estrous cycle, during pregnancy, and in certain anestrous conditions.2 Each follicle wave has a lifespan of 7–10 days (range 6–12 days, depending on two vs. three waves per cycle and 18 vs. 24 days length of cycle) as the follicle progresses through the different stages of development, namely emergence, selection or atresia, and dominance and atresia or dominance and ovulation. Emergence of a new follicular wave is supported by a transient (1–2 days) increase in follicle-stimulating hormone (FSH) coinciding with the emergence of a follicular wave. Peak FSH concentrations occur just before wave emergence, and then subsequently decline over several days; concurrently, follicles grow and reach approximately 8.0 mm in diameter.3,4 On average, these follicles grow at a constant and comparable rate and then this group of growing follicles is divided into a single dominant and several subordinate follicles. Typically, deviation begins when the diameter of the largest follicles reaches 8.0 mm.3,4 The time difference is approximately 8 hours in cattle.5 Once functional dominance is established, the selected dominant follicle continues to grow while the remaining follicles that emerged during the same follicular wave cease growth and become atretic. Selection of the dominant follicle occurs during declining FSH concentrations; however, the dominant follicle continues to grow (despite FSH at nadir concentrations) until it either ovulates or undergoes atresia.6 During the final stages of selection of the dominant follicle, there appears to be a transition from mainly FSH to luteinizing hormone (LH) dependency.6 The selected dominant follicle uniquely expresses mRNA for LH receptors in granulosa cells, which are likely the key to allowing its continued growth under LH stimulation.6,7 Estrogen-active dominant follicles are largely LH-dependent; positive feedback of estrogen releases a large amount of gonadotropin-releasing hormone (GnRH), resulting in an LH surge and ovulation of the dominant follicle.6,7 Conversely, if the dominant follicle fails to ovulate, the consequences are continued growth of the dominant follicle dependent on increased LH pulse frequency which, if prolonged, can lead to persistence of the dominant follicle.8 Therefore, hormonal treatments that modify both FSH and LH clearly affect the fate of a follicular wave. Manipulation of the follicle wave, in turn, may alter systemic hormonal concentrations, the intrafollicular environment, and the oocyte. Thus, a dominant follicle capable of ovulation is present only at specific times during each wave. Therefore the interval from exogenously induced luteolysis and/or withdrawal of a progestagen treatment to estrus and ovulation depends on the stage of the follicle wave at luteolysis and/or at progestagen withdrawal (whichever occurs last if this does not occur at the same time); cattle with a selected dominant follicle will be in estrus within 2–3 days, whereas those in which follicular growth is at the preselection stage, estrus will occur 3–7 days later.9–12 To achieve maximum synchrony of onset of estrus in a synchronization program, it is necessary to have a recently selected dominant follicle present at the end of treatment. Therefore, new wave emergence must be synchronized, because both the stage of the follicular wave and the duration of dominance affect the duration of the follicular phase and the interval from treatment to estrus. Synchrony of estrus is optimal when the duration of dominance is either less than 4 days (short) or more than 10–12 days (very long). Further, duration of dominance of the preovulatory follicle can also affect fertility. When the duration of dominance of the ovulatory follicle exceeds 10 days, there is a dramatic decline in pregnancy rate,10–12 primarily due to aged oocytes with premature activation. Therefore it is desirable to precisely control follicular dynamics (and in particular new wave emergence) to minimize both the variation in timing of onset of estrus and to ensure high pregnancy rates. The method for controlling follicular wave emergence is based on two principles (Figure 33.1): (i) emergence of a new follicular wave is primarily FSH-dependent; and (ii) an estrogen-active dominant follicle is largely LH-dependent. Thus, the negative feedback effects of progestagens and estrogens can be used to synchronize follicle wave emergence by suppression of FSH and/or LH. However, the extent to which follicles are dependent on gonadotropins during various stages of follicular wave development is poorly defined. Further, details of local mechanisms that play key roles in regulating the sequential progression of follicles through sequential physiological stages of the wave are not well defined. This makes it difficult to develop a simple exogenous hormonal treatment that gives predictable new wave emergence in all animals treated, irrespective of the stage of the follicle wave and stage of estrus at treatment.9,13 Figure 33.1 Phases of follicular wave. A follicular wave consists of growing, static and regression phases. Growing phase includes recruitment, and selection and dominance and atresia of subordinate follicles; static phase includes preparation of ovulation or regression; and regression phase includes regression of dominant follicle and preparation for subsequent wave. The method for controlling follicular wave emergence is based on two principles: (i) emergence of a new follicular wave is primarily FSH-dependent; and (ii) an estrogen-active dominant follicle is largely LH-dependent. The induction of new follicle wave emergence using exogenous hormonal treatments requires (i) consistent termination of an existing follicle wave, (ii) predictable induction of a transient increase in FSH to induce emergence of a new wave, and (iii) normal growth of the dominant follicle after selection. The primary dependence of the follicle wave progression on gonadotropin support has resulted in the use of steroids to suppress FSH and LH and thus terminate the existing wave. However, it should be realized that FSH and LH are differentially regulated.6 In the case of FSH, the dominant follicle is the key regulator of the recurrent increases or decreases that occur during the wave. In contrast, although progesterone concentrations are important in regulation of LH pulse frequency, there are changes in LH pulse frequency that occur during the luteal phase which are difficult to clarify by changes in peripheral progesterone or estradiol concentrations. An alternative nonsteroidal approach is the administration of GnRH to induce endogenous LH and FSH surges that cause luteinization or ovulation of an existing dominant follicle, thereby inducing emergence of a new wave.14 Functional removal of the dominant follicle or cohort follicles by inducing either ovulation or luteinization using exogenously induced gonadotropin release is a practical approach for induction of a new follicular wave. Administration of GnRH induced an immediate LH and FSH surge, the magnitude of which was independent of progesterone concentration or stage of the follicle wave.15 However, the effect of GnRH on the existing follicle wave is dependent on the presence or absence of a dominant follicle. GnRH administered after dominant follicle selection caused it to ovulate, with emergence of a new wave 1.5–2.0 days later.14 However, when GnRH was administered before selection, it had no effect on progression of the existing follicular wave. In all cows treated after dominant follicle selection, the induced gonadotropin surge was followed by a transient increase in FSH (but not LH) which was associated with new wave emergence.15 Therefore GnRH synchronizes new wave emergence only when administered in the presence of a functional dominant follicle, whereas if given before dominance it would appear not to affect the subsequent progress of that wave, presumably because of lack of LH receptors on the granulosa cells of such growing follicles. The dichotomy of GnRH effects on progression of the follicle wave is a constraint that needs to be considered when using it as a treatment to synchronize new wave emergence at the start of either progesterone or prostaglandin (PG)F2α synchronization regimens.16 The subsequent use of PGF2α to cause regression of the induced corpus luteum (CL) is mandatory when GnRH is used to synchronize follicle waves. In addition, not only the stage of development of the dominant follicle,17 but also stage of the estrous cycle18 at the time that GnRH is administered affects results. If GnRH is administered when the dominant follicle is pre- or post-dominance, ovulation may not occur and a new follicular wave will not emerge.17 An alternative is to ensure that a viable growing dominant follicle is present at the time of GnRH treatment. In that regard, cattle will respond most consistently when GnRH is administered between days 5 and 12 of the estrous cycle.18,19 Even though combined treatment with progestin and estradiol has been known for decades, knowledge of the physiological effects and mechanisms of action of exogenous estradiol and progesterone on follicles is a prerequisite to the development of improved methods of controlling the estrous cycle. Estradiol suppresses the growing phase of the dominant follicle; suppression is more profound when given in combination with progesterone.20 The mechanism responsible for estrogen-induced suppression of follicle growth appears to involve suppression of both FSH and LH.21 Further, it exerts its negative effect on both the hypothalamus and pituitary. Studies have focused on the effects of short-term estradiol and progesterone treatments, either alone or in combination at the time of pre-follicle wave emergence, on LH and FSH, progression of the follicle wave, and changes during follicle wave development.1 The period of elevated FSH associated with wave emergence was shortened by approximately 10 hours following estradiol, progesterone, or a combination of estradiol and progesterone treatments, compared with control. Frequency of LH pulses was reduced following progesterone treatment and was further reduced in response to a combination of estradiol and progesterone treatments. This steroid combination also significantly reduced pulse amplitude. The emergence of the follicle wave and selection of the first dominant follicle of the cycle were delayed approximately 1.5 and 2.5 days, respectively, by the combination of estradiol and progesterone treatment. Intrafollicular estradiol concentrations were suppressed in the largest follicle following combination estradiol and progesterone treatment, whereas under estradiol treatment they were unchanged. However, they were suppressed in smaller follicles following both estradiol and combination estradiol and progesterone treatments. Therefore, suppression of LH pulses by the combination estradiol and progesterone treatment is important for suppressing follicular estradiol in the largest follicle, whereas suppression of FSH alone may be sufficient to reduce follicular estradiol concentrations in smaller follicles. In estrous synchronization protocols, estradiol is usually injected at the initiation of the protocol, concurrent with insertion of a progestin-releasing device. The combination of estradiol and progestin was used to determine if suppression of follicle growth would induce new wave emergence at a consistent interval after treatment regardless of the phases (i.e., growing, static, or regressing phases) of follicle development at which treatment was initiated. The use of estradiol-17β in progestin-implanted cattle was followed consistently by the emergence of a new wave, on average, 4.5 days later.9,16 Once the estradiol was metabolized, there was an FSH surge and a new follicular wave emerged.22,23 The administration of 2.5 or 5 mg estradiol-17β (or 2 mg of estradiol benzoate or estradiol valerate) in progestin-implanted cattle at random stages of the cycle was followed by the emergence of a new follicular wave approximately 4 days later, with little variability.24–26 The mechanism of action of estradiol would appear to be through FSH and LH, causing follicle atresia followed by synchronous release of FSH and emergence of a new follicle wave. Hence, in general, estradiol has been the hormone treatment used most successfully to synchronize follicle wave emergence in cattle. However, the use of estradiol is being prohibited in an increasing number of countries, so alternatives must be sought. Similarly, the use of GnRH or LH to induce ovulation, and to remove the suppressive effects of a dominant follicle on FSH, result in a surge in FSH and a new follicle wave at a predictable time thereafter. Therefore, synchronization of follicle wave emergence is likely to be through FSH release, which may involve manipulation of ovarian (follicular) metabolic pathways or feedback mechanisms to the hypothalamic–pituitary axis. Luteolysis is initiated by estradiol from the developing preovulatory follicle, which triggers the release of hypophyseal oxytocin, which in turn stimulates release of a small quantity of uterine PGF2α. PGF2α then initiates a positive feedback loop involving release of additional luteal oxytocin and PGF2α of both luteal and uterine origin.27 Oxytocin stimulates synthesis and secretion of PGF2α from the uterus. It has recently been proposed that release of luteal PGF2α amplifies the luteolytic signal in an autocrine or paracrine manner.27,28 PGF2α causes decreased blood flow to luteal parenchyma and morphological changes that reduce both the number of small steroidogenic luteal cells and the size of large luteal cells. In addition, it is involved in intracellular signaling that facilitates apoptosis of large luteal cells and stimulates intraluteal production of PGF2α, thereby resulting in the demise of the CL.29,30 Since the discovery of PGF2α as the luteolytic agent in cattle,31,32 it has been the most commonly used treatment for elective induction of luteal regression and/or synchronization of estrus.33 The injection of PGF2α causes immediate regression of the CL after approximately day 5 of the estrous cycle: progesterone concentrations decline rapidly to basal levels within 24 hours, and LH pulse frequency increases, causing a significant increase in estradiol from the dominant follicle and the induction of estrus and ovulation. Despite rapid luteolysis, the interval from treatment to the onset of estrus is variable and dependent on the stage of the follicle wave at treatment. Animals with a functional dominant follicle are typically in estrus within 2–3 days, because the dominant follicle at the time of induced luteolysis ovulates shortly (Figure 33.2a); however, animals at the pre-dominance or regression phase of the wave will require 4–6 days to form a dominant follicle and hence have a longer and more variable interval to onset of estrus (Figure 33.2b). Figure 33.2 Interval from PGF2α administration to estrus. (a) Animals with a functional dominant follicle are in estrus within 2–3 days because the dominant follicle at the time of induced luteolysis ovulates soon thereafter. (b) Animals at the pre-dominance or regression phase of the follicular wave will require 4–6 days for a dominant follicle to reach preovulatory diameters and hence have a longer and more variable interval to onset of estrus. PGF2α failed to effectively induce luteolysis during the first 5 or 6 days following estrus. Lack of responsiveness was due to lack of PGF2α receptors in immature CL, but recent work has demonstrated the presence of PGF2α receptors in the CL as early as 2 days after ovulation.34 However, it appears that the mature CL may possess a positive feedback loop that results in intraluteal PGF2α production that may continue the process of luteolysis initiated by a single treatment of exogenous PGF.35 It should be noted that although luteolytic response was variable when a single dose of PGF2α was given on days 5 or 6 of the estrous cycle, two doses of prostaglandin analog (12 hours apart) consistently induced luteal regression (95–100%) at 2–5 days after estrus.36,37
Pharmacological Intervention of Estrous Cycles
Introduction
Ovarian follicular dynamics
Pharmacological control of new wave emergence
Use of GnRH
Use of estradiol
Controlling the lifespan of the corpus luteum
Shortening the luteal phase (premature luteolysis)
Extending/simulating a luteal phase with exogenous progesterone
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