2009: Synchronization of estrus and ovulation: a practitioner's perspective Synchronization of estrus and ovulation: a practitioner’s perspective 1 H. Maxwell 2 Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, 3 Auburn, AL, USA 4 Abstract 5 Artificial insemination as a management tool can be facilitated by synchronization 6 of estrus or ovulation. Although estrus detection will remain an important part of 7 successful breeding programs, timed artificial insemination is increasingly used in cattle 8 herds. The practicing theriogenologist must utilize his or her knowledge of the 9 physiology of the bovine estrous cycle and reproductive pharmacology, consider 10 constraints imposed by availability of labor and facilities, and choose among the 11 available protocols the one which is most likely to be successfully implemented. This 12 article reviews the physiology of the bovine estrous cycle, and some of the protocols 13 currently in use. 14 Keywords: Bovine estrous cycle, estrus synchronization, artificial insemination 15 Introduction 16 Artificial insemination as a management tool has been extensively utilized by 17 dairy producers for over 50 years, and use in beef cattle has become increasingly 18 common over the last 2 decades. Heat detection and insemination based on estrus signs 19 and behavior remain necessary components for most successful artificial insemination 20 programs, but the veterinary practitioner is increasingly called upon to suggest and 21 monitor protocols aimed at increasing efficiency or convenience of artificial insemination 22 programs by synchronizing estrus or ovulation. Knowledge of the physiology of the 23 195 estrous cycle, reproductive pharmacology, and management of cattle and people are all 24 necessary components of successful programs. 25 Estrous cycle control has developed in distinct phases, as the events of the estrous 26 cycle have been understood and pharmacologic agents have become available. Today, 27 the most successful protocols combine control of the length of the luteal phase with 28 control of the emergence of follicular waves, often with control of the time of ovulation 29 and timed artificial insemination. An understanding of the physiology of the estrous 30 cycle, including emergence of follicular waves, development of the dominant follicle, 31 follicular atresia, ovulation, development of the corpus luteum (CL), and luteolysis is a 32 necessary prerequisite to evaluate and implement synchronization strategies. 33 Integration of the physiology of the estrous cycle with reproductive 34 pharmacology, the age, breed and lactation status of the targeted group, the animal 35 handling skills and facilities present on the farm or ranch, the availability of sufficient 36 skilled personnel at critical times, effective communication and record keeping are all 37 important to success of protocols. The practicing theriogenologist must take all of these 38 into consideration as he or she consults with their clients. 39 A brief outline of critical points is presented below, and discussion of the some of 40 the more common protocols which have evolved follows. 41 The bovine estrous cycle 42 Cattle are reproductively non-seasonal, polyestrous, monovulatory, with an 43 interestrus interval ranging from 18-23 days. Observable estrus behavior is detectable 44 even in the absence of the male. 45 196 Heat, or estrus, is best demonstrated by standing to be mounted, whether by a bull 46 or by other females in the herd. Typical estrus behavior is triggered by rising estradiol in 47 an environment where progesterone concentrations are falling. 48 The rising concentrations of circulating estradiol accompanying estrus induce the 49 leutinizing hormone (LH) surge, and the LH surge induces ovulation. Ovulation follows 50 the onset standing heat by 24 to 32 hours. 51 Ovulation is followed by development of the CL, a transient endocrine organ that 52 arises on the ovary at the site of ovulation. The CL produces a number of endocrine 53 products, the most important for this discussion being progesterone, which is necessary 54 for maintenance of pregnancy following insemination. Should pregnancy not occur, the 55 CL will regress and another ovulatory follicle will develop. This follicle will produce 56 sufficient estradiol to again induce psychic estrus and the LH surge, and ovulation will 57 follow. The cycle will repeat indefinitely in the absence of pregnancy. This very basic 58 outline of the events of the estrous cycle has proven to be sufficient for insemination 59 programs based on heat detection. As we move to more precisely control the estrous 60 cycle, an understanding of other, less obvious events becomes necessary. 61 The cycle is sometimes conveniently divided into the luteal and follicular phases, 62 based on the dominant ovarian structure present. The follicular phase begins with the 63 regression of the CL and encompasses about 20% of the cycle, the period from the onset 64 of luteolysis until ovulation. Estradiol from the developing dominant or preovulatory 65 follicle is the predominant sex steroid during the follicular phase. The luteal phase 66 begins at ovulation, and continues until regression of the CL. During this time, the sex 67 steroid dominance shifts to progesterone produced by the CL. The luteal phase includes 68 197 both metestrus, the period of CL development following ovulation, and diestrus, the 69 period spanned by the life of the mature CL. Follicles continue to emerge and grow 70 during the luteal phase, and although they produce estradiol, luteal progesterone inhibits 71 LH sufficiently to prevent development to preovulatory size 72 The endocrine signal that results in regression of the CL in the absence of 73 pregnancy has been known to be prostaglandin F2alpha (PGF2ά) for several decades. 74 More recently, the signal responsible for maternal recognition of pregnancy, which 75 effectively blocks luteolysis, has been shown to be interferon tau produced by the fetal 76 trophoblast.1 The signals that result in emergence, growth, and regression of ovarian 77 follicles became evident following the advent of ultrasound as a research tool coupled 78 with endocrine assays in the early 1980’s.2 As mechanisms regulating the ovarian cycle 79 were discovered and pharmacologic agents became available, opportunities to develop 80 protocols to control the events of the estrous cycle developed. 81 Follicular dynamics 82 During the 1980’s, ultrasound technology allowed basic research that described 83 waves of follicular growth and regression. These revelations were coupled with endocrine 84 assays to explain the dynamic nature of ovarian follicles. Understanding of the processes 85 that initiated follicular emergence, selection, atresia, dominance and ultimately ovulation, 86 coupled with strategic administration of the limited number of available pharmacologic 87 agents have become the basis for programs to manipulate the estrous cycle and in many 88 instances precisely control the time of ovulation. The basic research on follicular 89 dynamics has been reviewed extensively in recent publications,3,4 and the reader is 90 198 referred to these resources for a more detailed description. A brief summary, applicable 91 to clinical practice follows. 92 Estrus is characterized by the presence of a large ovarian follicle, producing 93 sufficient estrogen to induce the psychic changes associated with estrus behavior and to 94 induce a surge of LH, which eventually results in ovulation of the follicle and release of 95 the oocyte. Following ovulation, the granulosa and thecal cells surrounding the follicular 96 antrum transform into luteal cells (luteinization) and the CL develops. Steroid production 97 shifts from estrogen to progesterone and this period of progesterone dominance initiates 98 the luteal phase. 99 At the time of the LH surge which initiates the events of ovulation, both LH and 100 follicle-stimulating hormone (FSH) are released from the pituitary. This release of FSH 101 is followed closely by a second surge of FSH, and these peri-ovulatory surges of FSH are 102 associated with the emergence of a cohort of small follicles from the ovarian follicular 103 pool shortly after ovulation. Emergence, defined as the last day the potential dominant 104 follicle was less than 4 mm, is perhaps more easily understood as the time that follicles 105 are easily detectable with commonly used ultrasound equipment. Emergence of follicles 106 is generally coincident with the peak of the FSH surge. As growth of these follicles is 107 stimulated by FSH, follicular hormonal activity increases and the antral follicles produce 108 a number of endocrine products, including the protein hormone inhibin, and later 109 estradiol.5 Estradiol and inhibin exert negative influence on the anterior pituitary to 110 decrease or inhibit release of FSH from the anterior pituitary, so that following follicular 111 emergence, FSH concentrations decline. FSH has a long half life relative to LH, but 112 concentrations of FSH remain sufficient to support follicular growth only to a diameter of 113 199 8-9 mm,6 although exogenous FSH administration will allow follicular development to 114 continue past this stage.7 115 Within the cohort of emerged follicles, a single follicle either has or gains a slight 116 developmental advantage as FSH concentrations approach their nadir. As this 117 developmentally advantaged follicle grows, it acquires additional LH receptors in the 118 granulosa cells surrounding the follicular antrum and oocyte.8 The acquisition of LH 119 receptors is critical for continued development of the follicle, and LH support allows this 120 follicle to continue progressive development as the growth rate of the other follicles in 121 the cohort declines. The time at which the growth rate of the future dominant follicle 122 exceeds the growth rate of the largest dominant follicle is referred to as follicular 123 deviation and the largest follicle is referred to as the dominant follicle. As this selected 124 follicle continues to grow in response to pulsatile release of LH it continues to produce 125 estradiol and inhibin, and FSH support for the remaining follicles is lost. These 126 subordinate follicles, lacking LH receptors in a low FSH environment, begin the process 127 of atresia as the dominant follicle continues to grow. As the dominant follicle grows and 128 produces estradiol, a positive feed back loop with the hypothalamus increases the 129 frequency of gonadotropin-releasing hormone (GnRH) pulses, which in the face of 130 continued inhibition of FSH release, results in increased pulsatile LH, but not FSH, 131 release from the pituitary. 132 Coincident with the development of this first post ovulatory follicular wave, and 133 eventual development of a single dominant follicle, is the development of the CL and the 134 associated increase in circulating progesterone concentrations. As noted above, the 135 dominant follicle grows and increasingly produces estrogen which feeds back on the 136 200 hypothalamus to increase GnRH and elicit LH pulses from the pituitary. However, in the 137 high progesterone environment of the luteal phase, GnRH release is limited, and the 138 resultant LH pulsatility is insufficient to support continued development of the dominant 139 follicle to pre-ovulatory size. The dominant follicle, deprived of sufficient LH for 140 continued development, eventually joins the other members of the cohort, and undergoes 141 atresia. Atresia of the dominant follicle removes the source of estradiol and inhibin, and 142 shortly after its demise, another surge of FSH from the anterior pituitary stimulates 143 emergence of a second follicular wave.9 The second follicular wave develops in a 144 manner similar to the first, and a new dominant follicle develops. The dominant follicle 145 of the second follicular wave may go on to become the ovulatory follicle, or may undergo 146 atresia and be replaced by a dominant follicle from a third follicular wave which will 147 become the ovulatory follicle. 148 In the non-pregnant animal, the endometrium releases PGF2ά by day 16 -17 post 149 ovulation, and destruction of the CL (luteolysis) follows. As circulating progesterone 150 concentrations decrease following luteolysis, the inhibitory effect of progesterone on 151 GnRH release is removed. LH pulse frequency increases, and the dominant follicle 152 responds with continued growth. This growth is accompanied by increasing estrogen 153 production, and feedback on the hypothalamus further increasing pulsatile release of 154 GnRH, which it turn acts on the pituitary to increase LH pulse frequency. Follicular 155 development continues in this low progesterone environment with the production of 156 estrogen eventually reaching the threshold necessary to trigger the LH surge. 157 Most cattle exhibit either 2 or 3 follicular waves during an estrous cycle. It is 158 necessary to recognize that follicles which achieve dominance can only reach pre-159 201 ovulatory status in the low progesterone environment that follows luteolysis, and that any 160 of the dominant follicles produced in either 2 or 3 wave cycles can grow to ovulatory size 161 if luteolysis is induced and the inhibitory effect of progesterone removed. Figure 1 is a 162 schematic representation of ovarian follicular development during a typical three wave 163 estrous cycle. 164 Pharmacologic control of the estrous cycle 165 Programs to control the time of estrus and ovulation developed as the events 166 controlling various portions of the estrous cycle were recognized, and as products for 167 pharmacologic manipulation of these events became available. Even though the number 168 of pharmacologic agents available is limited, producers and veterinarians are offered an 169 ever increasing number of protocols for control of the estrous cycle, with seemingly 170 endless variations and refinements. Veterinarians actively practicing in the field as 171 theriogenologists are asked and expected to evaluate estrus and ovulation synchronization 172 programs, and make recommendations regarding selection and implementation on 173 specific premises. Understanding the available hormones and their interactions with the 174 events of the estrous cycle are essential in this task. 175 Pharmacologic agents to control the bovine estrous cycle 176 Opportunities for manipulation of the estrous cycle in cattle are limited to control 177 the length of the luteal phase, initiation a new follicular wave, and control the time of 178 ovulation. Many of the currently available programs rely on control of all three. Precise 179 control of ovulation has increasingly led to adoption of timed artificial insemination 180 (TAI) protocols. 181 202 Drug availability is influenced by legal constraints which prohibit the use of some 182 drugs and classes of drugs. Products must be available from commercial sources, and the 183 use of compounded drugs for estrous cycle control is prohibited by the Animal Medical 184 Drug Use Clarification Act (AMDUCA). Enforcement of restrictions seems likely to 185 increase, driven by food safety and consumer demands. 186 Available products meeting these criteria and commonly utilized in clinical 187 settings are limited to progesterone and progestins, prostaglandin F2ά (PGF2ά) and its 188 analogs, and GnRH agonists. Other potentially useful products such as FSH, LH, or 189 human chorionic gonadotropin (hCG), are uncommonly utilized in synchronization 190 protocols. Noticeably, and deliberately absent from this list are injectable estrogenic 191 compounds, which although valuable and historically widely used, are not currently 192 approved for estrus control in food animals, and not commercially available in the United 193 States. As mentioned earlier, compounded products, including estrogen, must be 194 avoided. Table 1 provides a list of some commercially available products. 195 Control of the length of the luteal phase 196 The length of the luteal phase may be extended by administration of exogenous 197 progesterone or progestins, or truncated by the administration of luteolytic doses of 198 prostaglandin. 199 Extending the luteal phase with progestins 200 Early attempts at synchronizing estrus focused on administration of progesterone 201 or progestins, followed by acute withdrawal. In this manner, the period of progesterone 202 dominance is extended beyond the normal lifespan of the corpus luteum. Exogenous 203 progesterone inhibits release of LH, preventing development of ovarian follicles to 204 203 preovulatory size. Removal of the exogenous source or progesterone from groups of 205 cattle results in a relatively synchronized estrus followed by ovulation. An additional 206 benefit of progestin therapy is the ability to hasten the onset of estrous cyclicity in 207 animals that may be anestrous at the beginning of the protocol. 208 Melengestrol acetate. Melengestrol acetate (MGA), an orally active 209 progestational steroid, was developed in the 1960s and first marketed to both improve 210 feed efficiency and rate of gain in feedlot heifers and suppress estrus behavior. 211 Suppression of ovulation and estrus behavior occurs when consumption was 212 approximately 0.5 mg per head per day.10 213 Early efforts at synchronization with MGA in cycling cattle relied on feeding 0.5 214 to 1 mg daily for 14 to 18 days, a period sufficient to allow spontaneous luteal regression 215 in all animals within a group. Because the luteal phase is extended past the time of 216 luteolyis, follicular development and ovulation is suppressed. Following withdrawal of 217 MGA from the diet, follicular development resumes and a synchronized estrus follows. 218 A majority of animals treated in this manner exhibit signs of estrus and ovulate 3 to 7 219 days following MGA withdrawal. Pregnancy rates to this synchronized estrus are 220 variable and generally disappointing. The reduction in fertility, however, is not apparent 221 in subsequent cycles. Because the reduction in fertility is confined to the first post 222 treatment estrus, programs have developed to take advantage of the synchrony of the 223 second post treatment cycle. These programs are plagued by decreasing synchrony of the 224 second post treatment estrus due to the inherent variability of estrous cycle length. These 225 protocols require that the synchronization be planned well in advance of the onset of the 226 breeding season. 227 204 The reduced fertility of the first synchronized estrus following MGA withdrawal 228 has been attributed to altered development of ovarian follicles. Dominant follicles which 229 would either undergo atresia or ovulate during a normal ovarian cycle persist beyond 230 their normal lifespan in the sub-luteal progesterone environment provided by the 231 exogenous MGA. Following withdrawal of the progestin, these persistent follicles grow, 232 produce sufficient estradiol to induce estrus and the LH surge, and ovulate, but the 233 oocytes associated with these follicles are often developmentally compromised. 234 Administrating MGA in the feed for shorter intervals partially overcomes the 235 negative effects of persistent follicles, but synchrony of estrus is decreased. 236 Incorporating higher doses of MGA has not been effective in overcoming problems 237 associated with development of persistent follicles.11 Although some reports indicate 238 acute administration of progesterone late in the artificially lengthened luteal phase will 239 induce follicular turnover,12 the lack of FDA approval for injectable progesterone 240 products precludes use of this strategy. 241 Several variations of the initial MGA protocol utilizing PGF2ά have been 242 developed to overcome problems associated with lack of synchrony of the second post 243 treatment estrus, and will be discussed in a later section. 244 Intra-vaginal progesterone. Intra-vaginal delivery of progesterone as a method of 245 extending or controlling the length of the luteal phase of the cycle has been available in 246 the United States since 2002. Currently the only FDA approved device is the Eazi-Breed 247 CIDR® (Pfizer Animal Health, New York, NY, USA). The Eazi-Breed CIDR® has 248 been available for many years in other countries prior to introduction to the U.S. and a 249 205 large number of clinical and research trials have demonstrated the efficacy of 250 progesterone delivery by this route. 251 Labeled protocols for Eazi-Breed CIDR® specify that the pessary be placed intra-252 vaginally for 7 days, with administration of PGF2ά either one day prior to pessary 253 removal, or on the day of removal. The protocols yield similar results.13 This 254 combination approach allows precise control of the length of the luteal phase, and 255 synchronizes estrus. Because of inherent variations in follicular wave emergence, estrus 256 activity following this protocol is not synchronized sufficiently to permit appointment 257 breeding. The range of estrus activity is often much more tightly controlled than in 258 programs which use prostaglandins as the sole agent. 259 Progesterone-releasing pessaries are often incorporated into other synchronization 260 programs to improve the degree of control of the luteal phase, and like other progestin 261 based synchronization protocols, may hasten the onset of cyclicity in anovular cattle.14,15 262 Truncating the luteal phase with prostaglandin F2ά 263 Almost 3 decades ago, PGF2 ά and its analogs were introduced as the first drugs 264 approved to control the estrous cycle in cycling cows. Prostaglandin is released from the 265 endometrium after mid-cycle in non-pregnant cattle, and result in regression of the CL, 266 removing the progesterone mediated inhibition of LH pulsatility. Although the 267 developing CL is resistant to the effects of prostaglandin, sensitivity increases as the CL 268 matures, and by day 6 post-estrus, the luteal phase can be terminated in virtually all cows 269 administered exogenous PGF2ά. 270 Administration of PGF2ά or its analogs after day 5-6 of the estrous cycle results 271 in CL regression and loss of progesterone dominance. When administered to groups of 272 206 cycling cattle, the synchronized end of the luteal phase is followed by a synchronized 273 estrus. 274 Following the commercial introduction, PGF2ά protocols rapidly became popular 275 in estrus synchronization protocols both for artificial insemination and in synchronizing 276 estrous cycles for recipients in embryo transfer programs. The two commercially 277 available products, dinoprost (Lutalyse®; Pfizer Animal Health, New York, NY, USA) 278 and cloprostenol (Estrumate®;Intervet/Schering–Plough animal Health, Summit, NJ, 279 USA), are in similar in their action, and while cloprostenol has a longer duration of 280 action, their effects are clinically comparable.16 281 Following a single injection of PGF2ά to randomly cycling groups of cows or 282 heifers, 60 to 80% can be expected to be in estrus within a few days following luteolysis. 283 Poor to no response is expected in cattle which have ovulated recently. Cattle which have 284 undergone spontaneous luteolysis would typically exhibit estrus coincidentally with their 285 “synchronized” herd mates. Single injection protocols can be tailored to a producer’s 286 needs is several ways. 287 Traditional estrus detection with artificial insemination for 6 to 7 days, followed 288 by PGF2ά administration to those not previously inseminated increases the percent 289 response by removing those with immature CLs from the pool. Animals injected are 290 expected to exhibit estrus in 2 to 5 days. Drug costs are very low in this protocol, but the 291 number of days during which animals need to be observed and bred is fairly high. These 292 single injection programs are useful and widely practiced in many farm settings, but 293 many management situations exist in which a shorter window of estrus synchronization is 294 207 desired. In these cases, group synchronization utilizing 2 injections of prostaglandin may 295 offer advantages.17 296 In two injection schemes, animals which respond to the first injection and animals 297 in the group which have undergone spontaneous luteolysis just prior to administration of 298 the drug are expected to be in heat in from 2 to 5 days. Those animals with an immature 299 CL, (~ day 1-5 of the cycle) are not be expected to respond to the injection of PGF2ά, and 300 luteal development will continue. At the time of the second injection, all animals in the 301 group should be an appropriate stage of the luteal phase and expected to exhibit a 302 synchronized estrus. 303 Although label indications for dinoprost suggest a two injection scheme with the 304 injections separated by 10 to 12 days, 14 day intervals are more commonly used and 14 305 day programs are considered equally effective if not superior to the shorter intervals 306 specified on the product label. 307 Strategies aimed at identifying cows at the appropriate stage of the cycle to 308 respond to PGF2ά injections eliminate injections in animals that will not respond to 309 exogenous prostaglandin. Transrectal palpation for the presence of a CL, determination 310 of milk progesterone concentrations,18 or identification of the CL with ultrasound have 311 been investigated as methods to identify cattle with functional luteal tissue. Although 312 these techniques can be effective, routine administration of PGF2ά to non-inseminated 313 animals at random stages of the estrous cycle has remained the most common, and 314 perhaps most economically justifiable, procedure. 315 Estrus synchronization protocols which use PGF2ά as the sole agent are 316 characterized by an inherent variability in the time of the onset of estrus, typically 317 208 exhibiting a bell shaped curve with estrus activity beginning 2 days post injection, 318 peaking in 3 to 4 days, and declining rapidly after day 5. While this synchronization of 319 estrus is useful and advantageous compared to observing and handling cattle over an 320 entire estrous cycle, synchrony of ovulation is too variable to allow timed artificial 321 insemination protocols. 322 MGA plus prostaglandin F2ά 323 The availability of PGF2ά products led to estrus synchronization protocols which 324 combined feeding MGA for various lengths of time with injections of prostaglandin. 325 Feeding MGA for 14 to 18 days, as described above, results in synchrony of estrus, but 326 poor fertility is associated with the first estrus following prolonged feeding of the 327 progestational agent. Because estrous cycles in cattle fed MGA are synchronized 328 following withdrawal of the progestin, there is an opportunity to administer PGF2ά to a 329 group of animals with a synchronized luteal phase following MGA withdrawal. 330 Treatment with prostaglandin late in the luteal phase reduces the variability in the interval 331 from PGF2ά injection to estrus. Following treatment with PGF2ά, synchrony of the 332 second estrus following MGA withdrawal is better than in MGA protocols which do not 333 control the luteal phase length prior to the second synchronized estrus, and fertility to the 334 synchronized estrus is not compromised. Initial protocols injected prostaglandins at 17 335 days after withdrawal of MGA, and more recently protocols in which prostaglandin 336 injection has been delayed until 19 days after MGA withdrawal have shown tighter estrus 337 synchrony.19 Detection of estrus and breeding based on the signs of estrus are 338 recommended. These combination programs are quite effective and economical in 339 209 situations where feed intake is adequately controlled. The length of treatment in these 340 MGA protocols remains a disadvantage, requiring a long lead time prior to insemination. 341 Shorter MGA programs combined with prostaglandin injections at the time of MGA 342 withdrawal either did not overcome the infertility associated with longer feeding 343 periods,20 or did not show improvement over PGF2ά alone.21 344 Synchronization of the emergence of the follicular wave 345 Following atresia of the dominant follicle, a new cohort of follicles emerges in 346 response to FSH, and the transition from the dominance of one follicle to emergence of a 347 new cohort of developing ovarian follicles is termed follicular turnover. Treatments 348 aimed at ending the period of follicular dominance will induce follicular turnover, 349 allowing more precise control of the events and timing of the estrous cycle. 350 At least three methods to induce synchronous emergence of a new follicular wave 351 are practiced, but only one lends itself to mass synchronization schemes.22 Aspiration or 352 ablation a mid-cycle dominant follicle removes the inhibitory effects of estradiol and 353 inhibin on pituitary FSH release, and is followed by wave emergence in 1 to 2 days. 354 Administration of an acute dose of exogenous estradiol or a combination of estradiol and 355 progesterone results in emergence of a new follicular wave in approximately 3-4 days. 356 Administration of GnRH can induce ovulation or luteinization of a dominant follicle, 357 removing the source of inhibin and estradiol, followed by emergence of a new follicular 358 wave in 1.5-2 days.22,23 359 Follicular ablation is not practical in on farm settings involving synchronization 360 of the estrous cycle of groups of cattle. As previously noted, the lack of commercial 361 sources of injectable estrogens and the legal atmosphere surrounding the use of 362 210 compounded pharmaceuticals in food animals effectively removes administration of 363 estrogen or estrogen/progesterone injections as an option for synchronizing follicular 364 wave emergence. 365 Although not specifically approved by the Food and Drug Administration for this 366 purpose, GnRH is available in the United States and licensed for use in cattle. 367 Administration of GnRH is currently the basis for control of follicular wave emergence 368 and ovulation in many synchronization programs. 369 Gonadotropin-releasing hormone 370 GnRH, a decapeptide hormone produced in the hypothalamus and transported to 371 the anterior pituitary, influences the secretion of the hormones FSH and LH. Commercial 372 formulations have been available in the United States and worldwide for nearly 3 373 decades, and are approved for treatment of cystic ovarian disease. Currently, no meat or 374 milk withdrawal periods are required for use of this product in cattle. 375 Endogenous GnRH stimulates release of LH and FSH, with a varying magnitude 376 of response and ratio depending on the ovarian structures and resultant hormonal mix 377 present. During the luteal phase of the cycle, high progesterone concentrations limit the 378 release of GnRH, and subsequently LH pulse frequency. Conversely, during the 379 follicular phase, removal of the negative influence of progesterone is followed by 380 continued growth of the dominant follicle, and increasing production of estradiol by the 381 follicle triggers increasing pulsatile release of GnRH from the hypothalamus. This 382 feedback loop culminates in the pre-ovulatory LH surge, which induces ovulation and 383 initiates the onset of another luteal phase. 384 211 Exogenous GnRH administered in the luteal phase during the period of follicular 385 dominance results in ovulation or luteinization of dominant follicles, and is followed by 386 emergence of a new follicular wave. Administration shortly after follicular wave 387 emergence is less likely to result in initiation of a new follicular wave. Administration 388 of exogenous GnRH following spontaneous or induced luteolysis can be used to induce 389 an LH surge and control the time of ovulation. The actions of GnRH have been 390 incorporated into synchronization protocols which control the time of follicular wave 391 emergence and ovulation. 392 Ovsynch and related protocols 393 In 1995, a protocol for TAI using sequential administration of GnRH, PGF2ά, 394 and GnRH to synchronize follicular wave emergence, luteolysis, and ovulation was 395 introduced.24 This protocol, widely known as Ovsynch, was the first allowing 396 appointment breeding to gain widespread acceptance. The basic program with numerous 397 modifications is the basis for many of the successful ovulation synchronization protocols 398 used today. 399 Figure 2a illustrates a timeline schematic for the Ovsynch protocol. Figure 2b 400 outlines an injection scheduling calendar for Ovsynch. On day 0, all eligible cattle 401 receive GnRH, followed 7 days later by PGF2ά. Forty eight hours later, a second 402 injection of GnRH is administered, and TAI, without regard to heat detection, is carried 403 out 16 to 20 hours following the second GnRH. The initial injection of GnRH is intended 404 to synchronize emergence of a new follicular wave following induction of ovulation of a 405 dominant follicle. The corpus luteum formed following this diestrus ovulation will not 406 become susceptible to the luteolytic effects of prostaglandin for several days, and will 407 212 provide a source of progesterone should the CL which developed following the previous 408 ovulation be destroyed. The prostaglandin injection 7 days after initiation of the protocol 409 will initiate luteolysis and allow continued development of the dominant follicle which 410 arose from the induced follicular wave to pre-ovulatory size. The second injection of 411 GnRH will induce an LH surge, with ovulation following in approximately 28 hours.24 412 Not all randomly cycling cattle have equivalent ovarian structures. Synchrony of 413 ovulation is improved if a dominant follicle is present at the time of the first GnRH 414 injection compared to initiation prior to the time of follicular deviation. The day of the 415 cycle at which Ovsynch is initiated influences pregnancy rates to timed artificial 416 insemination. Cows in which Ovsynch was initiated near mid-cycle had greater rates of 417 synchronous ovulation following the first injection of GnRH, and programs beginning on 418 day 5 to 12 of the estrous cycle resulted in greater pregnancy rates.25,26 419 Many modifications to the initial Ovsynch protocol have been developed with the 420 goal of increasing rate of synchronous ovulation and pregnancy. The most common have 421 utilized 2 prostaglandin injections, approximately 2 weeks apart, with the last injection 12 422 or 14 days prior to the first GnRH injection of Ovsynch. These programs increase the 423 percentage of animals at the optimum stage of the cycle when the first injection of GnRH 424 is administered, and are collectively referred to as Pre-Synch Ovsynch programs, or 425 Prostaglandin Pre-Synch programs. Pregnancy rates to TAI are improved compared to 426 Ovsynch alone.27 A sample schedule for a Pre-Synch Ovsynch protocol is shown in 427 Figure 3. The long lead time (initiation of hormone injections 36-38 days prior to 428 insemination) present an obstacle to use of Pre-Synch, which is minimized in dairy herds 429 213 by initiating the protocol during the voluntary waiting period during which breeding is 430 commonly withheld for 45 to 70 days following parturition. 431 More recently, protocols utilizing both GnRH and prostaglandin to increase the 432 percentage of cows that ovulate following the first GnRH injection have been developed 433 with improvement in pregnancy rate following timed artificial insemination similar to or 434 better than that seen with Pre-Synch Ovsynch programs.28,29 The injection schedule for 435 the G6G protocol described by Bello is illustrated in Figure 4. The injection of PGF2ά 436 which initiates the protocol induces luteolysis of mid- and late-cycle CL’s. Two days 437 later, an injection of GnRH synchronizes ovulation following luteolysis, and initiates a 438 new follicular wave in animals which have a dominant follicle. An Ovsynch protocol is 439 initiated six days later. The goal is to optimize the number of animals with a dominant 440 follicle at the onset of Ovsynch, and ultimately synchronization of ovulation at the 441 completion of Ovsynch.28 The G6G protocol has a shorter lead time than Pre-Synch, but 442 has scheduling disadvantages. Increased pregnancy rates to timed artificial insemination 443 compared to Ovsynch alone are reported. 444 MGA plus PGF2ά plus GnRH protocols 445 Estrus synchronization protocols incorporating oral administration of MGA, 446 followed with administration of PGF2ά have been modified by incorporating GnRH near 447 the time of the first post MGA estrus to synchronize the first follicular wave and 448 ovulation.30-32 These programs generally allow for shorter duration of MGA feeding, and 449 offer the potential advantage of hastening the onset of cyclicity in the late pre-pubertal 450 period or post partum period. Adaptations allowing fixed-time artificial insemination33 451 214 or heat detection are utilized. Figure 5 illustrates a treatment schedule for the MGA 7-11 452 Synch modification with fixed time insemination. 453 Synchronization of ovulation and timing of insemination 454 GnRH administration in the low progesterone environment following luteolysis 455 results in an LH surge followed by ovulation in 24 to 32 hours. Protocols which used 456 estrogen to induce ovulation enjoyed some popularity,34 but no approved product is 457 available for veterinarians at this time. Agents with LH activity, such as hCG, could be a 458 suitable substitute for GnRH, but protocols utilizing this strategy are not common. 459 Timing of insemination should include ample opportunity for sperm capacitiation 460 prior to ovulation, and insemination at 16 to 20 hours following the ovulation-inducing 461 dose of GnRH is the most common recommendation.35 Earlier insemination, including 462 at the time of administration of GnRH (Co-Synch protocol), may result in acceptable 463 pregnancy rates, while insemination later than 24 hours after GnRH leads to less 464 favorable outcomes. 465 In the original Ovsynch protocol, the second GnRH injection, administered to 466 synchronize ovulation, was given 48 hours following induction of luteolysis with PGF2ά. 467 This timing was convenient for dairies, and resulted in grouping of chores associated with 468 the protocol around times when cows were handled or locked up. Recent studies suggest 469 that delaying the administration of the second GnRH injection until 56 hours after 470 induced luteolysis will improve pregnancy rates.36 Modification of any protocol should 471 take into consideration farm schedules to insure compliance, particularly if handling or 472 restraint occurs outside the normal daily or weekly routine. 473 Choosing and implementing the right protocol 474 215 Many estrus synchronization and TAI programs are available, and when 475 implemented properly, most are effective. Selecting a protocol for a particular herd will 476 depend on the ability of herd management to comply with the time lines inherent in each 477 protocol. A lack of commitment or cavalier attitude concerning the timing of injections 478 and breeding will negatively impact the ultimate success any program. 479 An old saying in the dairy business is “When heat detection is everybody’s job, it 480 usually means it is nobody’s job”. This wisdom is applicable to scheduling and 481 administration of protocols. Unless a job is a priority, compliance will suffer. 482 Accurate record keeping and proper administration of all hormones are essential. 483 Injection schedules can become very complicated when dealing with large herds and 484 when multiple protocols used within a herd. Both PCDART and DAIRYCOMP 305 485 have routines which will generate action lists to facilitate scheduling of injections. 486 Timed artificial insemination programs may overcome some management 487 problems associated with estrus detection, but management problems with nutrition, 488 housing, semen handling, and concurrent disease are not eliminated. 489 Drug cost, availability of trained technicians, class of livestock and the facilities 490 available for repeatedly handling cattle will influence the choice of protocols for 491 individual farms. Selection of the appropriate protocol for a particular herd or 492 management system means selection of the protocol herd management will be most likely 493 to efficiently and consistently implement, rather than the newest or most popular protocol 494 available. 495 Discussion 496 216 Today, theriogenologists have an increasing number of options to synchronize 497 estrus and ovulation. These systems developed in concert with an increased 498 understanding of reproductive endocrinology and the proper selection and 499 implementation of any of the available protocols requires an understanding of follicular 500 dynamics, luteolysis and ovulation. Programs which allow for the successful utilization 501 of TAI require much more precise control of the events of the estrous cycle than those 502 which incorporate estrus detection. Although the legal and regulatory environment 503 restricts or forbids access to certain potentially useful pharmacologic agents, successful 504 strategies utilizing available agents have been devised, and offer adequate control of the 505 cycle. 506 References 507 508 1. Bazer FW, Spencer TE, Ott TL: Interferon tau: a novel pregnancy recognition 509 signal. Am J Reprod Immunol 1997;37:412-420. 510 2. Adams GP, Matteri RL, Kastelic JP, et al: Association between surges of follicle-511 stimulating hormone and the emergence of follicular waves in heifers. J Reprod 512 Fertil 1992;94:177-188. 513 3. Lucy MC: The bovine dominant ovarian follicle. J Anim Sci 2007;85:E89-E99. 514 4. Adams GP, Jaiswal R, Singh J, Malhi P. Progress in understanding ovarian 515 follicular dynamics in cattle. Theriogenology 2008;69:72-80. 516 5. Kaneko H, Terada T, Taya K, et al: Ovarian follicular dynamics and 517 concentrations of oestradiol-17 beta, progesterone, luteinizing hormone and 518 217 follicle stimulating hormone during the periovulatory phase of the oestrous cycle 519 in the cow. Reprod Fertil Dev 1991;3:529-535. 520 6. Ginther OJ, Kot K, Kulick LJ, et al: Emergence and deviation of follicles during 521 the development of follicular waves in cattle. Theriogenology 1997;48:75-87. 522 7. Hampton JH, Bader JF, Lamberson WR, et al: Gonadotropin requirements for 523 dominant follicle selection in GnRH agonist-treated cows. Reproduction 524 2004;127:695-703. 525 8. Beg MA, Bergfelt DR, Kot K, et al: Follicular-fluid factors and granulosa-cell 526 gene expression associated with follicle deviation in cattle. Biol Reprod 527 2001;64:432-441. 528 9. Adams GP: Comparative patterns of follicle development and selection in 529 ruminants. J Reprod Fertil (Suppl) 1999;54:17-32. 530 10. Patterson DJ, Kiracofe GH, Stevenson JS, et al: Control of the bovine estrous 531 cycle with melengestrol acetate (MGA): a review. J Anim Sci 1989;67:1895-532 1906. 533 11. Kojima FN, Chenault JR, Wehrman et al: Melengestrol acetate at greater doses 534 than typically used for estrous synchrony in bovine females does not mimic 535 endogenous progesterone in regulation of secretion of luteinizing hormone and 536 17beta-estradiol. Biol Reprod 1995;52:455-463. 537 12. Anderson LH, Day ML: Acute progesterone administration regresses persistent 538 dominant follicles and improves fertility of cattle in which estrus was 539 synchronized with melengestrol acetate. J Anim Sci 1994;72:2955-2961. 540 218 13. Ambrose DJ, Emmanuel DG, Colazo MG, et al: Pregnancy rates to timed 541 artificial insemination in Holstein heifers given prostaglandin F2alpha twenty-542 four hours before or concurrent with removal of an intravaginal progesterone-543 releasing insert. J Dairy Sci 2008;91:2678-2683. 544 14. Yavas Y, Walton JS: Induction of ovulation in postpartum suckled beef cows: a 545 review. Theriogenology 2000;54:1-23. 546 15. Chebel RC, Santos JE, Cerri RL, et al: Reproduction in dairy cows following 547 progesterone insert presynchronization and resynchronization protocols. J Dairy 548 Sci 2006;89:4205-4219. 549 16. Salverson RR, DeJarnette JM, Marshall CE, et al: Synchronization of estrus in 550 virgin beef heifers using melengestrol acetate and PGF2alpha: an efficacy 551 comparison of cloprostenol and dinoprost tromethamine. Theriogenology 552 2002;57:853-858. 553 17. Thatcher WW, Chenault JR: Reproductive physiological responses of cattle to 554 exogenous prostaglandin F2alpha. J Dairy Sci 1976;59:1366-1375. 555 18. Peters AR, Riley GM, Rahim SE, et al: Milk progesterone profiles and the double 556 injection of cloprostenol in post partum beef cows. Vet Rec 1980;107:174-177. 557 19. Lamb GC, Nix DW, Stevenson JS, et al: Prolonging the MGA-prostaglandin 558 F2alpha interval from 17 to 19 days in an estrus synchronization system for 559 heifers. Theriogenology 2000;53:691-698. 560 20. Beal WE, Chenault JR, Day ML, et al: Variation in conception rates following 561 synchronization of estrus with melengestrol acetate and prostaglandin F2alpha. J 562 Anim Sci 1988;66:599-602. 563 219 21. Chenault JR, McAllister JF, Kasson CW: Synchronization of estrus with 564 melengestrol acetate and prostaglandin F2alpha in beef and dairy heifers. J Anim 565 Sci 1990;68:296-303. 566 22. Martinez MF, Adams GP, Kastelic JP, et al: Induction of follicular wave 567 emergence for estrus synchronization and artificial insemination in heifers. 568 Theriogenology 2000;54:757-769. 569 23. Roche JF, Austin EJ, Ryan M, et al: Regulation of follicle waves to maximize 570 fertility in cattle. J Reprod Fertil (Suppl) 1999;54:61-71. 571 24. Pursley JR, Mee MO, Wiltbank MC: Synchronization of ovulation in dairy cows 572 using PGF2alpha and GnRH. Theriogenology 1995;44:915-923. 573 25. Vasconcelos JL, Silcox RW, Rosa GJ, et al: Synchronization rate, size of the 574 ovulatory follicle, and pregnancy rate after synchronization of ovulation 575 beginning on different days of the estrous cycle in lactating dairy cows. 576 Theriogenology 1999;52:1067-1078. 577 26. Moreira F, de la Sota RL, Diaz T, et al: Effect of day of the estrous cycle at the 578 initiation of a timed artificial insemination protocol on reproductive responses in 579 dairy heifers. J Anim Sci 2000;78:1568-1576. 580 27. Moreira F, Orlandi C, Risco CA, et al: Effects of presynchronization and bovine 581 somatotropin on pregnancy rates to a timed artificial insemination protocol in 582 lactating dairy cows. J Dairy Sci 2001;84:1646-1659. 583 28. Bello NM, Steibel JP, Pursley JR: Optimizing ovulation to first GnRH improved 584 outcomes to each hormonal injection of ovsynch in lactating dairy cows. J Dairy 585 Sci 2006;89:3413-3424. 586 220 29. Souza AH, Ayres H, Ferreira RM, et al: A new presynchronization system 587 (double-ovsynch) increases fertility at first postpartum timed AI in lactating dairy 588 cows. Theriogenology 2008;70:208-215. 589 30. Kojima FN, Salfen BE, Bader JF, et al: Development of an estrus synchronization 590 protocol for beef cattle with short-term feeding of melengestrol acetate: 7-11 591 synch. J Anim Sci 2000;78:2186-2191. 592 31. Funston RN, Ansotegui RP, Lipsey RJ, et al: Synchronization of estrus in beef 593 heifers using either melengesterol acetate (MGA)/prostaglandin or MGA/select 594 synch. Theriogenology 2002;57:1485-1491. 595 32. Wood SL, Lucy MC, Smith MF, et al: Improved synchrony of estrus and 596 ovulation with the addition of GnRH to a melengestrol acetate-prostaglandin 597 F2alpha synchronization treatment in beef heifers. J Anim Sci 2001;79:2210-598 2216. 599 33. Bader JF, Kojima FN, Schafer DJ, et al: A comparison of progestin-based 600 protocols to synchronize ovulation and facilitate fixed-time artificial insemination 601 in postpartum beef cows. J Anim Sci 2005;83:136-143. 602 34. Stevenson JS, Tiffany SM, Lucy MC: Use of estradiol cypionate as a substitute 603 for GnRH in protocols for synchronizing ovulation in dairy cattle. J Dairy Sci 604 2004;87:3298-3305. 605 35. Pursley JR, Silcox RW, Wiltbank MC: Effect of time of artificial insemination on 606 pregnancy rates, calving rates, pregnancy loss, and gender ratio after 607 synchronization of ovulation in lactating dairy cows. J Dairy Sci 1998;81:2139-608 2144. 609 221 36. Brusveen DJ, Cunha AP, Silva CD, et al: Altering the time of the second 610 gonadotropin-releasing hormone injection and artificial insemination (AI) during 611 Ovsynch affects pregnancies per AI in lactating dairy cows. J Dairy Sci 612 2008;91:1044-1052. 613 614 222 Figure 1. 615 616 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Bovine Estrous Cycle Corpus Hemorrhagicum Functional Corpus Luteum Uterus releases PGF2α Follicular Development in Absence of CL Estrus 617 618 Figure 1: Development of successive waves of ovarian follicles during the estrous cycle. 619 Shortly after ovulation, a cohort of follicles emerges; a single follicle gains an advantage 620 that allows it to become the dominant follicle, only to undergo atresia in the high 621 progesterone environment of the luteal phase. A second follicular wave emerges around 622 day 8-9 of the cycle, and the dominant follicle that follows meets the same fate. A third 623 wave emerges on day 16-17 following demise of the second wave dominant follicle. 624 Prostaglandin-induced luteolysis destroys the source of progesterone, the inhibition of 625 LH pulses is removed, and this third wave dominant follicle develops to preovulatory 626 size. Estradiol produced by the dominant follicle increases past the threshold necessary 627 223 to induce the LH surge, and ovulation follows. Image courtesy of Dr. M. Daniel Givens, 628 Auburn University 629 630 631 224 632 Figure 2a. 633 634 Ovsynch Day 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 GnRH PGF2α 5. Inseminate 16-20 hours after GnRH injection. GnRH 1. Ovulation of dominant follicle 2. New Follicular Wave 3. Luteolysis 4. Induce LH surge and ovulation 635 636 Figure 2a: OVSYNCH PROTOCOL. An injection of GnRH on day 0 induces ovulation 637 or luteinization of a dominant follicle, and a new follicular wave emerges 2 days later. 638 Injection of prostaglandin F2alpha on day 7 induces luteolysis, and injection GnRH on 639 day 9 induces an LH surge, with ovulation 28 hours later. Insemination at 16-20 hours 640 after the second GnRH injection (~8 hours ahead of ovulation) allows time for sperm 641 capacitation prior to ovulation. 642 643 225 Figure 2b. SUNDAY MONDAY TUESDAY WEDNESDAY THURSDAY FRIDAY SAT Week 1 GnRH Week 2 PGF2ά GnRH TAI (16-20 hours post GnRH) Figure 2b: Injection calendar for a Pre-Synch Ovsynch protocol that avoids weekend chores. PGF2ά = prostaglandin F2alpha, GnRH = gonadotropin-releasing hormone, TAI = timed artificial insemination. 226 Figure 3. SUNDAY MONDAY TUESDAY WEDNESDAY THURSDAY FRIDAY SAT Week 1 PGF2ά Week 2 Week 3 PGF2ά Week 4 Week 5 GnRH Week 6 PGF2ά GnRH TAI (16-20 hours post GnRH) Figure 3: Injection calendar for a Pre-Synch Ovsynch protocol which utilizes 2 prostaglandin injections, 14 days apart, with the second injection given 14 days prior to the start of Ovsynch. This protocol minimizes the number of days per week on which injections are given, avoids weekend chores, and places all prostaglandin injection on the same day of the week. A modification in which the first two prostaglandin injections are given on Wednesday of week 1 and 3 could be utilized and may have advantages. PGF2ά = prostaglandin F2alpha, GnRH = gonadotropin-releasing hormone, TAI = Ttmed artificial insemination. 227 Figure 4. SUNDAY MONDAY TUESDAY WEDNESDAY THURSDAY FRIDAY SAT Week 1 PGF2ά GnRH Week 2 GnRH Week 3 PGF2ά GnRH TAI (16- 20 hours post GnRH) Figure 4: Injection calendar for a modified G6G protocol. G6G shortens the lead time prior to insemination compared to Pre-Synch Ovsynch, but has the disadvantage of having assigned chores related to the protocol on more days of the week. Scheduling to avoid weekend chores may be more complicated with this protocol. PGF2ά = prostaglandin F2alpha, GnRH = gonadotropin-releasing hormone, TAI = timed artificial insemination. 228 Figure 5. SUNDAY MONDAY TUESDAY WEDNESDAY THURSDAY FRIDAY SAT Week 1 MGA (first day) MGA Week 2 MGA MGA MGA MGA MGA (last day) PGF2ά Week 3 GnRH Week 4 PGF2ά GnRH and TAI (60 hours after PGF2ά) Week 5 Week 6 Figure 5: Treatment calendar for MGA 7-11 Synch with TAI. MGA feeding begins on day 0, and is discontinued on day 7. Prostaglandin injections are given on day 7, and followed on day 11 with GnRH injections. On day 18, prostaglandin is injected to end the luteal phase, and GnRH is injected and cows are inseminated at 60 hours following the prostaglandin injection. Variations eliminating the last GnRH injection utilize heat detection and breeding based on the signs of estrus. 229 MGA = melengestrol acetate incorporated into feed, PGF2ά = prostaglandin F2alpha, GnRH = gonadotropin-releasing hormone, TAI = timed artificial insemination. 230 Table 1. Class Drug Trade Name Route of Adminstration Manufacturer Progestatational agents Melengestrol acetate Progesterone MGA® Eazi-Breed CIDR® Oral Intravaginal pessary Pfizer Pfizer Prostaglandin F2ά and analogs Dinoprost tromethamine Cloprostonol Lutalyse® Estrumate ® Intramuscular injection Intramuscular injection Pfizer GnRH agonists Gonadorelin diacetate tetrhydrate Cystorelin® Ovacyst® Intramuscular injection Intramuscular injection Merial 231 Gonadorelin hydrochloride Factrel® Intramuscular injection Fort Dodge Animal Health Table 1: Commercially available products used to manipulate the estrous cycle of cattle. 232 << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /All /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Warning /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages false /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /CMYK /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness false /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages false /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages false /ColorImageDownsampleType /Average /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages false /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.76 /HSamples [2 1 1 2] /VSamples [2 1 1 2] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages false /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages false /GrayImageDownsampleType /Average /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages false /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.76 /HSamples [2 1 1 2] /VSamples [2 1 1 2] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages false /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages false /MonoImageDownsampleType /Average /MonoImageResolution 300 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages false /MonoImageFilter /FlateEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ENU ([Based on 'No Compression'] [Based on 'No Compression wbleeed'] [Based on '[High Quality Print]'] Use these settings to create Adobe PDF documents for quality printing on desktop printers and proofers. 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