Review Report

When the plan goes awry: how to negotiate estrus synchronization errors in beef cattle

Jessica Klabnik,^a Emma Horn^b

^aDepartment of Veterinary Clinical Sciences, Auburn University College of Veterinary Medicine, Auburn, AL, USA, ^bGeneuity Clinical Research Services, Maryville, TN, USA

Abstract

Estrus synchronization protocols vary widely, and errors during implementation are not uncommon. When an error is made, recommendations for resolution must consider the initial purpose of implementing the synchronization protocol in the herd. The ideal solution is to convert the protocol to a different published protocol. When this is not possible, the safest solution for maintaining an acceptable pregnancy rate is often either restarting a synchronization program or potentially foregoing artificial insemination for the season. Knowledge of estrous cycle physiology and how hormone treatments manipulate the cycle are foundational when attempting to modify a protocol based on the specific error that is made. Errors made early in a synchronization protocol tend to be more manageable than those made toward the end of the protocol. The use of less valuable semen and immediate introduction of a clean-up bull should be considered to maximize pregnancy outcomes when attempting to correct an error during estrus synchronization.

Keywords: Artificial insemination, bovine, estrus synchronization

 

Introduction

The goal of estrus synchronization is to create a herd of fertile females that will either express estrus and/or ovulate within an ideal timeframe. If breeding is based on estrus detection, a larger variability of timeframe may be acceptable. However, when fixed-timed artificial insemination (AI) is planned, a tighter synchrony of the group is critical.¹ Ovulation occurs ~ 30 hours after the onset of estrus in Bos taurus, providing a very narrow window in which successful pregnancy can occur as a result of insemination.² Success of synchronization is practically determined by pregnancy outcome, rather than estrus expression or ovulation rates. The determination of an ovulation rate is not practical, and neither the ovulation rate nor estrus expression is directly predictive of pregnancy outcome.

Numerous factors affect success of estrus synchronization, including the selection of a protocol that fits the herd type.³ Protocols often differ for B. taurus versus B. indicus, dairy versus beef, and conventional versus sex-sorted semen. In the presynchronization period, animal health, animal nutrition, liquid-nitrogen tank maintenance, and pharmaceutical storage and date of expiration should be evaluated. Critical factors include proper dosing, route of treatment, and timing of pharmaceuticals during synchronization, and semen handling, AI technique, and AI timing. The ability of the producer to comply with drug treatment and AI timings should be considered when selecting a protocol. After synchronization, animal handling, stress, transport, and nutrition can also impact success.³

Errors during synchronization related to pharmaceutical treatment are not uncommon.⁴ There are numerous published protocols that can be quite complex, particularly if presynchronization or resynchronization protocols are included. Most protocols use several pharmaceuticals, with varying frequency and/or dosage. Unfortunately, this results in seemingly infinite opportunities for error during implementation of a synchronization protocol. Ultimately, this also means that a ‘one-size fits most’ solution is not realistic or reliable.

Recommendations regarding solutions must be made carefully since a previous bad experience may negatively impact perception of estrus synchronization or AI.^5,6 The purpose of this review is to provide useful information for recommending a feasible solution after an error has occurred during estrus synchronization in a herd. This includes the following: 1. a brief review of the purposes of implementing estrus synchronization; 2. four general categories of solutions to consider; and 3. core synchronization theory and the underlying estrous cycle physiology. A strong understanding of synchronization mechanisms and estrous physiology is vital to successfully modify an existing protocol. Superstimulation, superovulation, and estrogen-based protocols are outside the scope of the presented information.

Purposes of implementing estrus synchronization

The intended purpose of estrus synchronization must be considered when deciding on a solution for errors in protocol execution. Cost-prohibitive or time-consuming corrections may jeopardize the intended benefits of synchronization. Estrus synchronization protocols are used to increase economic impact, genetic selection, and overall efficiency of production in cattle herds.⁷ Synchronization can increase pregnancy rates to fixed-time AI, embryo transfer, or live cover insemination,^8,9 which, in turn, increases profitability and decreases cost of rebreeding. More uniform calf crops of superior genetics can be produced when synchronization is combined with AI. This enhances the marketability of calves and increases their market value.^10–12 Some synchronization protocols can also induce cyclicity in noncycling individuals. However, pregnancy rates were significantly lower in females that were not cycling prior to synchronization/AI compared to those that were cycling.¹³

Critical evaluation of errors and determining solutions

With the purpose of estrus synchronization in mind, consideration can be given to 4 general solutions: restart the protocol, forgo AI for the season, convert to a different published protocol, or modify the protocol based on knowledge of synchronization theory and estrous cycle physiology.

Restarting a synchronization protocol is a straightforward option for the producer. If time in the breeding season allows, and the operation can cover the cost of restarting the synchronization protocol, this may be a feasible solution to circumvent errors made in the initial synchronization attempt. It may reduce the risk of another error resulting from lack of familiarity with a new or modified protocol.¹⁴ However, the cost of restarting may render this option less desirable. Costs may include additional pharmaceuticals, extra labor, and loss of production associated with delayed breeding (i.e. lighter calf crop at weaning and shorter or shifted breeding season). These costs are quite considerable in a commercial beef operation but may be overcome in certain seed stock beef operations.¹⁵ Restarting the protocol allows for a fresh start with an already known and prepared-for protocol.

A second general solution is to forgo AI and solely use clean up bull(s) for that breeding season. Ideally, the bull(s) must be able to breed multiple times while exposed to the herd over a window of time, minimizing negative effects if the error resulted in less synchronous ovulation. Bulls with high libido may achieve multiple services in a routine estrus synchronization program.^16,17 This option may reduce labor and pharmaceutical costs. One concern with this option is ensuring sufficient bull power for the synchrony of expected estrus. In a non-synchronized herd, the serving capacity of a fertile bull is expected to be 1 bull to 25 to 60 cows.¹⁸ In a study of synchronized females, pregnancy rates were not affected by either the serving capacity ratio (ranging from 1:7 to 1:51) or the number of females exhibiting estrus when exposed to bulls classified as high or medium libido.¹⁹ However, serving capacity may still be a concern as libido is not routinely tested,²⁰ and newer synchronization protocols may have tighter estrus expression compared to more simple protocols (i.e. Syncro-mate B or ‘two-shot’ prostaglandin F₂α [PGF₂α]).¹⁹ Therefore, when considering this solution, the type of protocol in use and the stage of the protocol in which the error occurred must be considered.

In some cases, errors during estrus synchronization can be corrected. In an ideal situation, the correction changes the planned protocol to another published protocol. A good starting place to find additional protocols is a local extension website. Insemination, semen sexing, and embryo transfer companies have synchronization protocols listed and diagramed for producer use; these companies may also provide consulting, services, and products useful for executing protocols. The multistate Beef Reproduction Task Force is aimed at bringing research and extension together, and their website (https://beefrepro.org/) contains links for downloadable resources and protocol timelines.²¹ Because protocols should be chosen based on the producer’s needs and capabilities, the decision to transform an existing protocol once an error has occurred to a different published protocol requires evaluation of both the producer’s abilities and the timing of the error in the initial protocol.²²

Less ideal situations (i.e. unable to switch to another published protocol) are much more common, and modifications of the protocol in these instances are likely to result in poorer synchrony.²³ If breeding is based on estrus detection, lesser synchrony resulting from normal physiologic variability of the estrous cycle may not be of concern. Estrous behavior may be expressed earlier and for a shorter duration in Bos indicus cattle compared to Bos taurus.^24,25 Ideally, AI should be performed 6 - 24 hours prior to ovulation, which is ~ 2 - 14 hours after the onset of standing heat.^26,27 In a fixed-time AI protocol in which the window of insemination is narrow (± 2 - 4 hours), variability in the synchronization of ovulation is more detrimental.

Regardless of the program type, poorer synchrony that results from either a poorly timed luteinizing hormone (LH) surge or prolonged periods of follicular dominance is of particular concern. A narrow window of oocyte viability occurs after oocyte maturation is triggered by the LH surge.²⁸ Prolonged periods of follicular dominance reduce embryonic development and quality due to prolonged exposure of the oocyte to follicular estradiol, which has been correlated with delayed meiotic progression and blastocyst rate.^29–31 Because the LH surge and follicular dominance occur toward the end of the protocol, it is easier to correct errors occurring at the beginning of the protocol before these events are underway. Errors toward the end of synchronization tend to be more challenging to overcome. Fertility of the oocyte within a late atretic dominant follicle declines rapidly as crucial estradiol production declines and progesterone production slowly increases with granulosa cell aging.³² The ovulated oocyte, arrested at metaphase II, is highly susceptible to postovulatory aging, resulting in decreased viability.^33,34 Decreased developmental competence, or the oocyte’s ability to be fertilized and give rise to a healthy embryo, is significantly decreased due to postovulatory oocyte aging.³⁵ Decreasing estradiol concentrations due to the decline of the dominant follicle, and potential postovulatory oocyte aging can both compromise fertility near the end of the estrous cycle. Even females displaying estrus may have aged oocytes and be subfertile.³⁶ In these cases, modification of synchronization is highly situational, depending on the protocol implemented and the error that was made. Critical evaluation of what was given, not given, and the timing of these events in light of their physiologic purpose is crucial and complex. Therefore, the authors highly recommend use of less valuable semen and immediate introduction of a clean-up bull to attempt to maximize pregnancy outcomes if restarting synchronization and foregoing AI are not options. The only relatively simple error to overcome is when gonadotropin releasing hormone (GnRH) treatment at AI is forgotten. GnRH should be given as soon as possible to help ensure ovulation as close to insemination as possible, but it is likely that the majority of well-synchronized females will ovulate in an acceptable time frame, even if ovulation is less synchronous than initially desired.³⁷

Integration of estrous cycle physiology and synchronization protocol

A strong knowledge base of synchronization theory and underlying estrous cycle physiology is critical in cases where modification of the protocol is the reasonable solution. Estrous cycle reviews are available that detail information beyond core concepts related to synchronization.^24,38 As the bovid has a continuous, monoovulatory, and nonseasonal estrous cycle,^24,38 synchronization protocols are designed to manipulate a group of females at variable stages of their cycle into 1 particular part of the cycle (estrus) at the same time. In a synchronization protocol, all individuals receive the same pharmaceutical at the same time, regardless of their status in the estrous cycle. Producers do not keep track of each animal’s stage in the estrous cycle for practical reasons, and therefore, the entire herd receives uniform treatment protocol to minimize labor for producers and increase herd synchrony. Because the bovid is continually cycling, certain synchronization protocols may alter hormonal events midcycle and initiate the next cycle in which breeding will occur in an optimal timeframe.^39,40

At synchronization, the ‘ideal’ cow (Figure. Cow A: Panels C and D) would already be cycling and nearing the end of her 21-day cycle, such that she would ovulate 6 to 24 hours^26,27 after planned insemination without manipulation. This cow would be ‘ideal’ because her cycle already aligns with the scheduled breeding at the end of synchronization. She would arrive at the planned breeding date and ovulate, without need for interference. It is impossible, however, to determine which cows, if any, are the ‘ideal’ member of the herd before synchronization. This means that the ‘ideal’ cow still received synchronization drugs along with her herdmates. However, most individuals in the herd will not be in this ‘ideal’ category, and their cycle will need to be manipulated to ensure that they ovulate at the appropriate time. When progesterone is low, ovulation results from a LH surge acting on the dominant follicle.⁴¹ Synchronization of herd-mates (Figure. Cows B through E) is aimed at manipulating cycles to align 3 key requirements: a low progesterone environment, LH surge, and the existence of a dominant follicle containing a fertile cumulus-oocyte-complex.^42,43

Figure. Use of estrus synchronization to modify cows at various stages of the estrous cycle in an example herd. A herd of unsynchronized females (Panels A, C, E, G, I, and K) may be at different stages of their estrous cycle at the start of a given protocol. Cow A is considered the ideal cow because no modification of her cycle is needed for her to ovulate near the planned breeding date (Panels C and D). In a herd of cycling females, the stage of the cycle in all cows is unknown, and therefore, all cows will be enrolled in an estrus synchronization program. Implementing an estrus synchronization program (e.g. 7-day CO-Synch + CIDR^68,79 [Panel B]), will modify the cycles of herd mates (Panels F, H, J, and L) to eventually coincide with the ideal cow by the time of the planned breeding date (Panel D). Note timeline presented is approximate, and cycles are not exactly to scale.

Low progesterone

A low progesterone environment is required for the dominant follicle to ovulate.⁴¹ This environment is necessary for ovulation because high progesterone environments prevent an LH surge from occurring. Progesterone inhibits episodic GnRH secretion from the hypothalamus. Conversely, GnRH exerts a positive effect on the pulsatile release of LH from the anterior pituitary, eventually culminating in a preovulatory LH surge and subsequent ovulation. Under a high progesterone environment, the dominant follicle can become atretic, regress, and a new follicular wave begins.⁴¹

Progesterone is produced by the corpus luteum.³⁸ Circulating progesterone concentrations in cattle with a functional corpus luteum must reach serum concentrations > 1 ng/ml to be considered physiologically relevant.⁴⁴ In the natural estrous cycle, progesterone concentrations decrease (< 1 ng/ml) due to lysis of the corpus luteum that results from the luteolytic activity of PGF₂αas it binds to its receptors. The number of PGF₂α receptors on the corpus luteum increases from the early to late luteal phase of the cycle, peaking around day 16 when luteolysis typically occurs.⁴⁵ PGF₂α is produced by the uterus.³⁸

To synchronize a low progesterone environment, PGF₂α treatment may be given to cattle. If the number of PGF₂α receptors is sufficient, lysis will occur and progesterone concentrations will decline.^46,47 Concentrations of serum progesterone decline to < 2 ng/ml within 12 hours after 30 mg of intramuscular PGF₂α.⁴⁸ Typically, a corpus luteum will be capable of responding to a single dose of exogenous PGF₂α by days 5 - 7 of the cycle (if day 0 is the day when ovulation last occurred).^49,50 Although the majority of females in an unsynchronized herd will have a corpus luteum capable of responding (Figure. Panel E and F), a significant remainder will not (Figure Panel G).⁵¹ Therefore, PGF₂α may also be used to presynchronize the herd in some protocols. The simplest example of this is a ‘two-shot’ PGF₂α protocol. In this protocol, the first PGF₂α treatment synchronizes the majority of females. After enough time has passed to allow their new corpus luteum to mature and be responsive to PGF₂α, a second PGF₂α treatment is given. Females with a less mature corpus luteum at the first treatment likely did not respond. It is expected that these individuals’ corpus luteum will be mature enough by the second PGF₂α treatment to respond and lyse or be close to natural lysis.^51,52

Another way to create a low progesterone environment is the timely removal of progesterone treatment. Progesterone treatment for a short period prevents the female bovid from ovulating and subsequently developing a young corpus luteum. This period in which luteolysis is expected to occur in the protocol is depicted (Figure, Panel H). The source of progesterone is via a controlled internal drug release device (CIDR) and removal at PGF₂α treatment with the intent of creating a low progesterone environment in all animals at the same time.⁵³ Notably, Bos indicus-influenced cattle may be more sensitive to progesterone’s effects during earlier aspects of the protocols. Improved pregnancy rates to fixed-time AI result when a functional corpus luteum is eliminated at the beginning of the protocol.²⁵

Luteinizing hormone surge

The hypothalamic-pituitary-gonadal axis is differentially regulated by progesterone and estrogen. The hypothalamic surge center is responsible for inducing a LH surge in low progesterone environments when progesterone’s negative inhibition on GnRH release is absent. Progesterone also negatively inhibits follicle stimulating hormone production, yet cohorts of small follicles are recruited due to transient rises in circulating follicle stimulating hormone, even in a high progesterone environment. Eventually, 1 follicle is selected, and the growing dominant follicle produces increasing concentrations of estradiol during proestrus. After corpus luteum regression, and without the negative feedback of progesterone on GnRH, LH then increases in a pulsatile fashion.⁵⁴ These changes culminate in the LH surge and subsequently ovulation.⁵⁵ After ovulation, the estradiol producing follicle becomes a progesterone producing corpus luteum.⁵⁶ Detailed information on the hypothalamic-pituitary-gonadal axis and effects of progesterone are available.^{25,53,57–59}

Although an LH surge may occur within estrus synchronization programs, the timing may be too variable for fixed-time AI programs (Figure. Panel I).^23,28 Theoretically, ovulation of a dominant follicle could be induced by stimulation of various aspects of the hypothalamic-pituitary-gonadal axis. This may include treatment with estrogens, GnRH, LH, or their analogues.⁶⁰ As estrogens are illegal in food producing species in the United States, a GnRH analogue is typically given. Bos indicus-influenced cattle have a highly variable and relatively low rate of ovulation to exogenous GnRH compared to Bos taurus.^61,62 An LH surge is expected to occur ~ 2 hours after exogenous GnRH treatment.^63,64 Ovulation occurs ~ 22 - 34 hours (in Bos taurus^63,64) or 26 - 28 hours (in Bos indicus⁶⁵) after the LH surge. Sperm must travel from the site of deposition (i.e. uterus during AI) to the site of fertilization (i.e. ampullary-isthmic junction of the uterine tube). To coordinate travel efforts of both gametes, GnRH is commonly given 16 - 24 hours prior to AI.^64,66,67 However, to reduce labor costs, GnRH is given at AI in some protocols (Figure. Panel J).^21,68,69 The majority of inseminations in North America utilize conventionally frozen semen. Protocols designed for insemination using other semen types (i.e. fresh, chilled, or sex-sorted), number of sperm (i.e. sex-sorted), or in other anatomic locations (i.e. uterine horn breeding) impact the timing of insemination and GnRH treatment.

Dominant follicle

In the natural estrous cycle, Bos taurus cattle can have either 2 or 3 follicular waves. The 2 wave cycle produces a follicular wave ~ every 10 days, whereas the 3 wave cycle produces a wave ~ every 7 - 9 days.⁷⁰ The average length of the estrous cycle is 21 days ± 2 days.⁷¹ However, cattle with 3 wave estrous cycles tend to have longer cycles, landing in the 22+ day range overall.⁷⁰ Bos indicus cattle estrous cycle average length is 21 days for 2 wave and 22 days for 3 wave cycles. Four and 5 wave cycles have also been reported.²⁴ Each wave contains 3 stages: emergence/recruitment, selection, and dominance.³⁸ A dominant follicle is lined with mural granulosa cells possessing sufficient LH receptors to respond to LH surge.⁷² Dominant follicles also produce inhibin, a hormone that prevents progression of other follicles.³² As previously reviewed, a dominant follicle will become atretic in a high progesterone environment, as it is unable to ovulate.⁵³ In a low progesterone environment, the growing dominant follicle produces increasing concentrations of estradiol during proestrus, triggering the LH surge, and eventual ovulation.⁵⁵

The natural variability in wave length^24,70 results in considerable challenges when attempting to synchronize a group of females. If wave timing is left unmanaged, some cattle may have significantly delayed ovulation.⁷³ This could result in subfertility due to aging of the sperm.²⁷ Currently, no therapies exist to change the rate of follicle development (i.e. speed it up or slow it down). Ovulation of the first wave dominant follicle results in reduced pregnancy per AI compared to the second wave. However, when progesterone was given, similar pregnancies per AI resulted.⁷⁴ For practical purposes, wave number is not a consideration related to estrus synchronization, particularly when the protocol includes progesterone treatment.

Altogether, current synchronization protocols rely on inducing a wave at the same time in all herd members, so that dominant follicles, capable of responding to LH surge, will be present at the same time.⁷⁵ Variability in ovulation timing due to wavelength can be managed by utilizing an induced LH surge. Induction of LH surge was described in the previous section (Figure. Panel J). It is worth reiterating that only a dominant follicle, with sufficient LH receptors, will ovulate in response to induced LH surge.⁷² If the present follicle is much younger and, therefore, has not acquired dominance or the appropriate number of LH receptors, it is not capable of ovulation (Figure. Panel K).

Eliminating a large, inhibin-producing follicle helps to synchronize the next follicular wave in cattle.³² Initially, this was performed by follicle ablation,⁷⁶ but most protocols now use a GnRH analogue for this purpose. GnRH exposure results in the induction of an LH surge and the regression (if high progesterone) or ovulation (if low progesterone) of any existing follicles.^64,77,78 After a new wave begins, sufficient time is allowed for the follicle to progress toward dominance and any young corpus luteum to mature. The corpus luteum is then lysed, followed by a second dose of a GnRH analogue treatment near the time of AI to cause ovulation of the dominant follicle (Figure. Panel L). Because exogenous GnRH induction of a new wave may result in a new corpus luteum,^64,77 subsequent use of PGF₂α is mandatory if the cow is to be bred before natural luteolysis would occur.⁸⁰

A female bovid will not respond to the initial dose of GnRH analogue if a dominant follicle is not present at treatment. Therefore, some protocols include initial measures to create a persistent dominant follicle prior to this initial GnRH dose. Long-term (i.e. ~ 14 day) progesterone treatment is used.⁸¹ During this period, the female will lyse her own corpus luteum but may not ovulate due to high concentrations of progesterone. Lysis of the mature corpus luteum may be induced by uterine release of PGF₂α.^31,49 Under natural circumstances, the lysis of the corpus luteum will cease progesterone production and remove the feedback of progesterone on hypothalamic GnRH release, allowing for eventual ovulation and subsequent LH surge.⁸² In cases where progesterone is given and a corpus luteum is not present, circulating progesterone concentrations are at physiologically relevant concentrations that are high enough to prevent LH surge but lower than the concentrations produced by a mature corpus luteum. These lower progesterone concentrations result in persistence of a dominant follicle, rather than follicle turnover. In the absence of LH surge (natural or induced), the dominant follicle persists.⁸³ When progesterone treatment is terminated, ovulation will occur, a corpus luteum will form, and a new wave will start. Synchrony of later waves is then used for breeding purposes. With these protocols, the initial oocyte in the persistent dominant follicle ages, and the herd will express unfertile estrus after the termination of progesterone treatment. Producers must wait to breed according to protocol for acceptable pregnancy outcomes.¹⁴ A similar concept is used in the relatively new 7 & 7 protocol. Initially, exogenous progesterone and prostaglandin are given to mimic natural corpus luteum lysis and stalling of a dominant follicle for up to 7 days. The GnRH analogue in the middle of the 14 days of progesterone treatment ultimately results in a new wave that is then utilized for breeding.⁸⁴

Conclusion

Errors during synchronization related to pharmaceuticals’ treatment are not uncommon. The opportunities for error during implementation of a synchronization protocol can appear quite infinite and ultimately suggest that a ‘one-size fits most’ solution is not realistic. Recommendations for solutions must be made carefully, considering both the producer and the affected herd. With the purpose of estrus synchronization in mind, 4 general solutions can be considered: restart the protocol, forgo AI for the season, convert to a different published protocol, or modify the protocol. A strong understanding of synchronization theory and estrous cycle physiology is vital if the chosen solution is to modify an existing protocol. Synchronization protocols manipulate cycles to align 3 key requirements: low progesterone environment, LH surge, and the existence of a dominant follicle containing a fertile cumulus-oocyte-complex.

Acknowledgment

Authors thank Herris Maxwell and Chance Armstrong for intellectual discussions regarding manuscript content and J. Lannett Edwards for the use of her dominant follicle image.

References

1.	Pursley JR, Silcox RW, Wiltbank MC: Effect of time of artificial insemination on pregnancy rates, calving rates, pregnancy loss, and gender ratio after synchronization of ovulation in lactating dairy cows. J Dairy Sci 1998;81:2139–2144. doi: 10.3168/jds.S0022-0302(98)75790-X
2.	Roelofs JB, van Eerdenburg FJ, Soede NM, et al: Various behavioral signs of estrous and their relationship with time of ovulation in dairy cattle. Theriogenology 2005;63:1366–1377. doi: 10.1016/j.theriogenology.2004.07.009
3.	Lamb GC: Overcoming compliance issues to ensure success of a TAI project. In: Proceedings of the 2021 Virtual Applied Reproductive Strategies in Beef Cattle, September 15–17, Beef Reproduction Task Force: 2021. Available from: https://www.youtube.com/watch?v=xqTceTU5vpY
4.	Perry GA, Smith MF: Management factors that impact the efficiency of applied reproductive technologies. In: Proceedings of the Applied Reproductive Strategies in Beef Cattle, August 17–18, Beef Reproduction Task Force: 2015, Davis, CA: 2015. p. 1–29.
5.	Kibre D, Ashebir G, Gebrekidan B, et al: Study on factors affecting estrus synchronization in smallholder dairy farming systems of Tigray, Northern Ethiopia. Vet Med Int 2022;2022:1–8. doi: 10.1155/2022/2435696
6.	Patterson DJ, Mallory DA, Nash JM, et al: Estrus synchronization protocols for heifers. In: Proceedings of the Applied Reproductive Strategies in Beef Cattle, January 28–29, Beef Reproduction Task Force: 2010, San Antonio, TX: 2010. p. 1–39.
7.	Larson LL, Ball PJH: Regulation of estrous cycles in dairy cattle: a review. Theriogenology 1992;38:255–267. doi: 10.1016/0093-691X(92)90234-I
8.	Beal W: Current estrus synchronization and artificial insemination programs for cattle. J Anim Sci 1998;76:30–38. doi: 10.2527/1998.76suppl_330x
9.	Tegegne A, Warnick AC, Mukasa-Mugerwa E, et al: Fertility of Bos indicus and Bos indicus x Bos taurus crossbreed cattle after estrus synchronization. Theriogenology 1989;31:361–370. doi: 10.1016/0093-691X(89)90542-6
10.	Griffith AP, Boyer CN, Rhinehart JD, et al: Cost-Benefit Analysis of Timed AI and Natural Service in Beef Cattle. St. Paul, AgEcon Search UMN: 2020. p. 1–11.
11.	Moore K: Cloning and the beef cattle industry. In: Michael JF, Robert SS, Joel VY. Factors Affecting Calf Crop: Biotechnology of Reproduction. Boca Raton, FL; CRC Press: 2002. p. 219–229.
12.	Perry G, Daly R, Melroe T: Increasing your calf crop by management, pregnancy testing, and breeding soundness examination of bulls. SDSU Extens Extra Arch 2009;91:1–6.
13.	Odde KG: A review of synchronization of estrus in postpartum cattle. J Anim Sci 1990;68:817–830. doi: 10.2527/1990.683817x
14.	Perry GA, Smith MF: Keys to successful estrus synchronization and artificial insemination programs. Proceedings of the Applied Reproduction Strategies in Beef Cattle Symposium. Stillwater, OK: 2014. p. 47–74.
15.	Lardner H, Damiran D, Larson K: Comparison of FTAI and natural service breeding programs on beef cow reproductive performance, program cost and partial budget evaluation. J Agric Sci 2020;12:1–13. doi: 10.5539/jas.v12n9p1
16.	Farin PW, Chenoweth PJ, Mateos ER, et al: Beef bulls mated to estrus synchronized heifers – single vs multi-sire breeding groups. Theriogenology 1982;17:365–372. doi: 10.1016/0093-691X(82)90016-4
17.	Farin PW, Chenoweth PJ, Tomky DF, et al: Breeding soundness, libido and performance of beef bulls mated to estrus synchronized females. Theriogenology 1989;32:717–725. doi: 10.1016/0093-691X(89)90460-3
18.	Petherick J: A review of some factors affecting the expression of libido in beef cattle, and individual bull and herd fertility. Appl Anim Behav Sci 2005;90:185–205. doi: 10.1016/j.applanim.2004.08.021
19.	Pexton J, Farin P, Rupp G, et al: Factors affecting mating activity and pregnancy rates with beef bulls mated to estrus synchronized females. Theriogenology 1990;34:1059–1070. doi: 10.1016/S0093-691X(05)80005-6
20.	Koziol JH, Armstrong CL: Manual for Breeding Soundness Examination of Bulls. Montgomery, AL; Society for Theriogenology: 2018.
21.	Johnson SK, Funston RN, Hall JB, et al: Multi-state beef reproduction task force provides science-based recommendations for the application of reproductive technologies. J Anim Sci 2011;89:2950–2954. doi: 10.2527/jas.2010-3719
22.	Islam R: Synchronization of estrus in cattle: a review. Vet World 2011;4:136–141. doi: 10.5455/vetworld.2011.136-141
23.	Lucy MC, McDougall S, Nation DP: The use of hormonal treatments to improve the reproductive performance of lactating dairy cows in feedlot or pasture-based management systems. Anim Reprod Sci 2004;82–83:495–512. doi: 10.1016/j.anireprosci.2004.05.004
24.	Sartori R, Barros CM: Reproductive cycles in Bos indicus cattle. Anim Reprod Sci 2011;124:244–250. doi: 10.1016/j.anireprosci.2011.02.006
25.	Williams GL, Stanko RL: Pregnancy rates to fixed-time AI in Bos indicus-influenced beef cows using PGF2alpha with (Bee Synch I) or without (Bee Synch II) GnRH at the onset of the 5-day CO-Synch + CIDR protocol. Theriogenology 2020;142:229–235. doi: 10.1016/j.theriogenology.2019.09.047
26.	Trimberger GW, Hansel W: Conception rate and ovarian function following estrus control by progesterone injections in dairy cattle. J Anim Sci 1955;14:224–232. doi: 10.2527/jas1955.141224x
27.	Roelofs JB, Graat EAM, Mullaart E, et al: Effects of insemination–ovulation interval on fertilization rates and embryo characteristics in dairy cattle. Theriogenology 2006;66:2173–2181. doi: 10.1016/j.theriogenology.2006.07.005
28.	Pohler KG, Geary TW, Atkins JA, et al: Follicular determinants of pregnancy establishment and maintenance. Cell Tissue Res 2012;349:649–664. doi: 10.1007/s00441-012-1386-8
29.	Ahmad N, Neal Schrick F, Butcher RL, et al: Effect of persistent follicles on early embryonic losses in beef cows. Biol Reprod 1995;52:1129–1135. doi: 10.1095/biolreprod52.5.1129
30.	Bourdon M, Santulli P, Kefelian F, et al: Prolonged estrogen (E2) treatment prior to frozen-blastocyst transfer decreases the live birth rate. Hum Reprod 2018;33:905–913. doi: 10.1093/humrep/dey041
31.	Cerri RL, Rutigliano HM, Chebel RC, et al: Period of dominance of the ovulatory follicle influences embryo quality in lactating dairy cows. Reproduction 2009;137:813–823. doi: 10.1530/REP-08-0242
32.	Mihm M, Crowe M, Knight P, et al: Follicle wave growth in cattle. Reprod Domest Anim 2002;37:191–200. doi: 10.1046/j.1439-0531.2002.00371.x
33.	Fissore RA, Kurokawa M, Knott J, et al: Mechanisms underlying oocyte activation and postovulatory ageing. Reproduction 2002;124:745–754. doi: 10.1530/rep.0.1240745
34.	Sirard M-A, Richard F, Blondin P, et al: Contribution of the oocyte to embryo quality. Theriogenology 2006;65:126–136. doi: 10.1016/j.theriogenology.2005.09.020
35.	Lord T, Nixon B, Jones KT, et al: Melatonin prevents postovulatory oocyte aging in the mouse and extends the window for optimal fertilization in vitro. Biol Reprod 2013;88:67. doi: 10.1095/biolreprod.112.106450
36.	Funston RN, Ansotegui RP, Lipsey RJ, et al: Synchronization of estrus in beef heifers using either melengesterol acetate (MGA)/prostaglandin or MGA/Select Synch. Theriogenology 2002;57:1485–1491. doi: 10.1016/S0093-691X(02)00654-4
37.	Cruppe L, Day M, Abreu F, et al: The requirement of GnRH at the beginning of the five-day CO-Synch+ controlled internal drug release protocol in beef heifers. J Anim Sci 2014;92:4198–4203. doi: 10.2527/jas.2014-7772
38.	Forde N, Beltman ME, Lonergan P, et al: Oestrous cycles in Bos taurus cattle. Anim Reprod Sci 2011;124:163–169. doi: 10.1016/j.anireprosci.2010.08.025
39.	DeJarnette JM, Marshall CE: Effects of pre-synchronization using combinations PGF(2 alpha) and (or) GnRH on pregnancy rates of Ovsynch- and Cosynch-treated lactating Holstein cows. Anim Reprod Sci 2003;77:51–60. doi: 10.1016/S0378-4320(03)00033-2
40.	Stevenson JL, Dalton JC, Santos JEP, et al: Effect of synchronization protocols on follicular development and estradiol and progesterone concentrations of dairy heifers. J Dairy Sci 2008;91:3045–3056. doi: 10.3168/jds.2007-0625
41.	Sunderland SJ, Crowe MA, Boland MP, et al: Selection, dominance and atresia of follicles during the oestrous cycle of heifers. Reproduction 1994;101:547–555. doi: 10.1530/jrf.0.1010547
42.	Fortune J, Hansel W: Concentrations of steroids and gonadotropins in follicular fluid from normal heifers and heifers primed for superovulation. Biol Reprod 1985;32:1069–1079. doi: 10.1095/biolreprod32.5.1069
43.	Santos J, Narciso C, Rivera F, et al: Effect of reducing the period of follicle dominance in a timed artificial insemination protocol on reproduction of dairy cows. J Dairy Sci 2010;93:2976–2988. doi: 10.3168/jds.2009-2870
44.	Starbuck G, Gutierrez C, Peters A, et al: Timing of follicular phase events and the postovulatory progesterone rise following synchronisation of oestrus in cows. Vet J 2006;172:103–108. doi: 10.1016/j.tvjl.2005.02.006
45.	Skarzynski DJ, Jaroszewski JJ, Okuda K: Luteotropic mechanisms in the bovine corpus luteum: role of oxytocin, prostaglandin F2 α, progesterone and noradrenaline. J Reprod Dev 2001;47:125–137. doi: 10.1262/jrd.47.125
46.	Rao CV, Estergreen VL, Carman FR, Jr., et al: Receptors for gonadotrophin and prostaglandin F2α in bovine corpora lutea of early, mid and late luteal phase. Acta Endocrinol 1979;91:529–537. doi: 10.1530/acta.0.0910529
47.	Shirasuna K, Akabane Y, Beindorff N, et al: Expression of prostaglandin F2α (PGF2α) receptor and its isoforms in the bovine corpus luteum during the estrous cycle and PGF2α-induced luteolysis. Domest Anim Endocrinol 2012;43:227–238. doi: 10.1016/j.domaniend.2012.03.003
48.	Hafs HD, Louis TM, Noden PA, et al: Control of the estrous cycle with prostaglandin F2α in cattle and horses. J Anim Sci 1974;38:10–21. doi: 10.1093/ansci/38.suppl_10.38
49.	Miyamoto A, Shirasuna K, Wijayagunawardane MPB, et al: Blood flow: a key regulatory component of corpus luteum function in the cow. Domest Anim Endocrin 2005;29:329–339. doi: 10.1016/j.domaniend.2005.03.011
50.	Tsai SJ, Wiltbank MC: Prostaglandin F-2 alpha regulates distinct physiological changes in early and mid-cycle bovine corpora lutea. Biol Reprod 1998;58:346–352. doi: 10.1095/biolreprod58.2.346
51.	Adeyemo O, Akpokodje UU, Odili PI: Control of estrus in Bos indicus and Bos taurus heifers with prostaglandin-F2-alpha. Theriogenology 1979;12:255–262. doi: 10.1016/S0093-691X(79)80005-9
52.	Kaneko K, Mungthong K, Noguchi M: Day of prostaglandin F-2 alpha administration after natural ovulation affects the interval to ovulation, the type of ovulated follicle, and the failure to induce ovulation in cows. J Vet Med Sci 2020;82:590–597. doi: 10.1292/jvms.19-0674
53.	Wiltbank MC, Souza AH, Carvalho PD, et al: Improving fertility to timed artificial insemination by manipulation of circulating progesterone concentrations in lactating dairy cattle. Reprod Fert Dev 2011;24:238–243. doi: 10.1071/RD11913
54.	Rossmanith WG, Liu CH, Laughlin GA, et al: Relative changes in LH pulsatility during the menstrual-cycle – using data from hypogonadal women as a reference point. Clin Endocrinol 1990;32:647–660. doi: 10.1111/j.1365-2265.1990.tb00909.x
55.	Thompson K, Stevenson J, Lamb G, et al: Follicular, hormonal, and pregnancy responses of early postpartum suckled beef cows to GnRH, norgestomet, and prostaglandin F2α. J Anim Sci 1999;77:1823–1832. doi: 10.2527/1999.7771823x
56.	Sinchak K, Wagner EJ: Estradiol signaling in the regulation of reproduction and energy balance. Front Neuroendocrinol 2012;33:342–363. doi: 10.1016/j.yfrne.2012.08.004
57.	Carruthers TD, Convey EM, Kesner JS, et al: The hypothalamo-pituitary gonadotrophic axis of suckled and nonsuckled dairy cows postpartum. J Anim Sci 1980;51:949–957. doi: 10.2527/jas1980.514949x
58.	Nett T: Function of the hypothalamic-hypophysial axis during the post-partum period in ewes and cows. J Reprod Fertil Suppl 1987;34:201–213.
59.	Okamura H, Yamamura T, Wakabayashi Y: Kisspeptin as a master player in the central control of reproduction in mammals: an overview of kisspeptin research in domestic animals. Anim Sci J 2013;84:369–381. doi: 10.1111/asj.12056
60.	Pereira JFS, Hartmann W: Regulation of the hypothalamic-pituitary-gonadal axis and the manipulation of the estrous cycle of bovine females. In: Bergstein-Galan TG: editor. Reproduction Biotechnology in Farm Animals. Hyderabad; Avid Science: 2018. p. 75–96.
61.	Saldarriaga JP, Cooper DA, Cartmill JA, et al: Ovarian, hormonal, and reproductive events associated with synchronization of ovulation and timed appointment breeding of Bos indicus-influenced cattle using intravaginal progesterone, gonadotropin-releasing hormone, and prostaglandin F2α1. J Anim Sci 2007;85:151–162. doi: 10.2527/jas.2006-335
62.	Zuluaga JF, Saldarriaga JP, Cooper DA, et al: Presynchronization with gonadotropin-releasing hormone increases the proportion of Bos indicus-influenced females ovulating at initiation of synchronization but fails to improve synchronized new follicular wave emergence or fixed-time artificial insemination conception rates using intravaginal progesterone, gonadotropin-releasing hormone, and prostaglandin F2α1. J Anim Sci 2010;88:1663–1671. doi: 10.2527/jas.2009-2480
63.	Martinez MF, Adams GP, Bergfelt DR, et al: Effect of LH or GnRH on the dominant follicle of the first follicular wave in beef heifers. Anim Reprod Sci 1999;57:23–33. doi: 10.1016/S0378-4320(99)00057-3
64.	Pursley JR, Mee MO, Wiltbank MC: Synchronization of ovulation in dairy cows using PGF2α and GnRH. Theriogenology 1995;44:915–923. doi: 10.1016/0093-691X(95)00279-H
65.	Pinheiro OL, Barros CM, Figueiredo RA, et al: Estrous behavior and the estrus-to-ovulation interval in Nelore cattle (Bos indicus) with natural estrus or estrus induced with prostaglandin F2α or norgestomet and estradiol valerate. Theriogenology 1998;49:667–681. doi: 10.1016/S0093-691X(98)00017-X
66.	Binversie J, Pfeiffer K, Larson J: Modifying the double-Ovsynch protocol to include human chorionic gonadotropin to synchronize ovulation in dairy cattle. Theriogenology 2012;78:2095–2104. doi: 10.1016/j.theriogenology.2012.08.004
67.	Stevenson JS, Pursley JR, Garverick HA, et al: Treatment of cycling and noncycling lactating dairy cows with progesterone during Ovsynch1. J Dairy Sci 2006;89:2567–2578. doi: 10.3168/jds.S0022-0302(06)72333-5
68.	Geary T, Whittier J, Hallford D, et al: Calf removal improves conception rates to the Ovsynch and CO-Synch protocols. J Anim Sci 2001;79:1–4. doi: 10.2527/2001.7911
69.	Geary TW, Whittier JC, Thrift FA, et al: Effects of a timed insemination following synchronization of ovulation using the Ovsynch or CO-Synch protocol in beef cows. Prof Anim Sci 1998;14:217–220. doi: 10.15232/S1080-7446(15)31832-5
70.	Ginther O, Knopf L, Kastelic J: Temporal associations among ovarian events in cattle during oestrous cycles with two and three follicular waves. Reproduction 1989;87:223–230. doi: 10.1530/jrf.0.0870223
71.	Crowe MA: Reproduction, events and management| estrous cycles: characteristics. In: McSweeney P, McNamara J: editors. Encyclopedia of Dairy Sciences. 3rd edition, Oxford; Academic Press: 2011. p. 948–953.
72.	Colazo MG, Kastelic JP, Davis H, et al: Effects of plasma progesterone concentrations on LH release and ovulation in beef cattle given GnRH. Domest Anim Endocrinol 2008;34:109–117. doi: 10.1016/j.domaniend.2006.11.004
73.	Taponen J, Katila T, Rodrıguez-Martınez H: Induction of ovulation with gonadotropin-releasing hormone during proestrus in cattle: influence on subsequent follicular growth and luteal function. Anim Reprod Sci 1999;55:91–105. doi: 10.1016/S0378-4320(99)00011-1
74.	Denicol AC, Lopes G, Mendonça LGD, et al: Low progesterone concentration during the development of the first follicular wave reduces pregnancy per insemination of lactating dairy cows. J Dairy Sci 2012;95:1794–1806. doi: 10.3168/jds.2011-4650
75.	Wolfenson D, Thatcher WW, Savio JD, et al: The effect of a GnRH analogue on the dynamics of follicular development and synchronization of estrus in lactating cyclic dairy cows. Theriogenology 1994;42:633–644. doi: 10.1016/0093-691X(94)90380-2
76.	Bergfelt DR, Lightfoot KC, Adams GP: Ovarian synchronization following ultrasound-guided transvaginal follicle ablation in heifers. Theriogenology 1994;42:895–907. doi: 10.1016/0093-691X(94)90113-W
77.	Macmillan K, Thatcher W: Effects of an agonist of gonadotropin-releasing hormone on ovarian follicles in cattle. Biol Reprod 1991;45:883–889. doi: 10.1095/biolreprod45.6.883
78.	Thatcher W, Drost M, Savio J, et al: New clinical uses of GnRH and its analogues in cattle. Anim Reprod Sci 1993;33:27–49. doi: 10.1016/0378-4320(93)90105-Z
79.	Lamb G, Stevenson J, Kesler D, et al: Inclusion of an intravaginal progesterone insert plus GnRH and prostaglandin F2α for ovulation control in postpartum suckled beef cows. J Anim Sci 2001;79:2253–2259. doi: 10.2527/2001.7992253x
80.	Diskin MG, Austin EJ, Roche JF: Exogenous hormonal manipulation of ovarian activity in cattle. Domest Anim Endocrinol 2002;23:211–228. doi: 10.1016/S0739-7240(02)00158-3
81.	McDowell CM, Anderson LH, Lemenager RP, et al: Development of a progestin-based estrus synchronization program: II. Reproductive response of cows fed melengestrol acetate for 14 days with injections of progesterone and prostaglandin F2α. J Anim Sci 1998;76:1273–1279. doi: 10.2527/1998.7651273x
82.	Christensen A, Bentley GE, Cabrera R, et al: Hormonal regulation of female reproduction. Horm Metab Res 2012;44:587–591. doi: 10.1055/s-0032-1306301
83.	Custer E, Beal W, Wilson S, et al: Effect of melengestrol acetate (MGA) or progesterone-releasing intravaginal device (PRID) on follicular development, concentrations of estradiol-17 β and progesterone, and luteinizing hormone release during an artificially lengthened bovine estrous cycle. J Anim Sci 1994;72:1282–1289. doi: 10.2527/1994.7251282x
84.	Andersen CM, Bonacker RC, Smith EG, et al: Evaluation of the 7 & 7 Synch and 7-day CO-Synch plus CIDR treatment regimens for control of the estrous cycle among beef cows prior to fixed-time artificial insemination with conventional or sex-sorted semen. Anim Reprod Sci 2021;235:106892. doi: 10.1016/j.anireprosci.2021.106892