Synchronizing ovulation with progesterone and estradiol.

Keywords: P&E, progesterone, ET, estradiol, synchronize, estrus, estrous, embryo, transfer, bovine, cattle

The use of progesterone and estradiol (P&E) paired with follicle stimulating hormone (FSH) for embryo transfer (ET) has gained widespread use in cattle. Alternatives exists, including Ovsynch treatment paired with FSH. In general however, results are superior when P&E is used.

The image in this entry shows a popular method of synchronizing ovulation with P&E in preparation for ET.

Its should be emphasized that there there is no "one size fits all" for superovulation and embryo transfer. This is a highly specialized area of practice; complex, constantly evolving, and differing from one subspecies of cattle to another, even one breed to another. The image shown here would be most appropriate for a high producing dairy cow (Bos taurus ss taurus). Treatments for beef cows differ considerably from this treatment mainly because of management considerations but also because of physiological differences, especially between Bos taurus ss taurus and Bos taurus ss indicus. These differences are discussed in: Historical perspectives and recent research on superovulation in cattle. Bó GA and Mapletoft, R.J. 2014. Theriogenology 81:38–48

Before reading further, visitors new to LORI are encouraged to visit the entries on follicle waves and luteal function.  The following summary may also help the reader to understand the physiology and pharmacology represented in the image.

Cardinal points in the applied physiology of controlling the growth of follicle in cattle include: Large growing follicles (> 0.5 mm to 18 mm in diameter) are, or soon will be, candidates for ovulation. To initiate a new wave of follicle growth, large follicles must be destroyed, induced to ovulate or their growth must be suppressed. The growth rate of the new wave of follicles is predictable; a cornerstone of synchronized ovulation in any group of animals. Follicle growth begins two to three days after complete suppression of a follicle wave. Suppression may be achieved instantaneous if follicles larger than 0.5 mm in diameter are destroyed by aspiration or laser treatment. Alternatively, the growth of follicles can be suppressed over a period three or four days if estrogens are used to inhibit FSH secretion. The latter approach is less invasive than ablation of follicles and is therefore commonly used in practice. Evidence suggests that once follicle suppression (or destruction) is complete, endogenous FSH stimulation is no longer suppressed because of the absence of inhibin from large follicles. Also, anti Müllerian hormone (AMH) is lost from previously growing follicles thereby eliminating the insensitivity of some small follicles to FSH. Collectively, these events allow a new follicle wave to emerge several days after estrogen treatment. Normally, most follicles in a cohort undergo regression because inhibin is production by the larger follicle/s. This is a natural protective mechanism to decrease the incidence of multiple births. In donor cows, the use of exogenous FSH bypasses the effects endogenous inhibin and causes more follicles in the cohort to grow to maturity than otherwise. FSH has a short circulating and effective half life and must be administered twice daily over three or four days as the new follicle wave is being recruited. In the days after FSH treatment, many follicles continue to grow because they have bound enough FSH and are then protected from the effect of inhibin produced by other large follicles. FSH treatment can be given in a slow release formulation. This is done in beef cattle where repeated treatments are inconvenient. It is generally less effective than multiple doses of FSH. Stimulation with gonadotropin releasing hormone (GnRH) in the last stages of follicle development will induce a luteinizing hormone (LH) surge, thereby synchronizing the ovulation of a large group of follicles. In some cases, a purified LH extract is given instead of GnRH. Progesterone in the P&E treatment has no significant effect on the growth and regression of follicles waves (recall that large follicles are often present during the mid-luteal phase). It will however, suppresses estrus and block maturation and ovulation of the largest follicles.

Discussion of the image
The donor cow is represented in the top half of the image, the recipient(s) below.
At far left, the ovarian dynamics of both donor and recipients are shown, the synchrony of their follicle waves unknown (see question marks) as they approach the period of treatment.
The days of treatment are shown in phases of darkness as light on the X axis of the graph. In reality there is no such thing as day 0, only time 0; a source of confusion in physiological publications. Therefore the author habitually refers to day 0 as day 1 in discussions of this nature.

Image size: 3057 x 2601px
At the start of treatment, a progesterone releasing intravaginal device (PRID) is inserted into both donor and recipients. The author uses the acronym PRID and the "T" shaped icon in the image to refer to any of the devices commonly used for this purpose (CIDR®, Cue-Mate®, the Triangular PRID-E® or the original coil PRID).

When the PRID is inserted, both donor and recipients are also treated with a dose of 2.5 to 5 mg of estradiol 17 beta (or 2 to 2.5 mg estradiol benzoate) together with 50 to 100 mg of progesterone. This mixture is given by i.m. injection. Estradiol benzoate is given at a lower dose than estradiol 17𝛽 because it is a conjugated estrogen, having a longer circulating half life than the native hormone, estradiol 17𝛽.
Readers are reminded that exogenous estrogens also cause the release of LH via positive feedback effects on the hypothalamic-pituitary axis. This explains the red colored LH surges in the images, shortly after the start of treatment in both donor and recipients.
The PRIDs are removed from both donor and recipients on the morning of the seventh day after its insertion. The circulating progesterone profiles of different age corpora lutea are shown as various shades of yellow in the transparent yellow blocks. Each block is rounded at the top right corner to symbolize the decrease in serum progesterone concentration as prostaglandins (PGs) are administered and the PRIDs are removed. PGs are given at that time to destroy any corpora lutea that may have formed shortly before PRID treatment. In the donor cow, prostaglandin treatment is repeated after 12 hours to ensure complete luteolysis.
The suppression of endogenous FSH during the first two days of treatment is followed by increased FSH secretion as the effect of exogenous estrogen wanes. This explains why a new follicle wave emerges about four days after treatment begins. To augment the effect of endogenous FSH and cause superovulation, exogenous FSH treatments also begin then i.e. four days after the onset of treatment. Obviously FSH treatment is omitted in the recipients.
Estrous behavior is not used as a cue for insemination. Instead, fixed time artificial insemination (FTAI) is used. GnRH is administered in the evening of the second day after PRID removal and the following morning and evening (12 and 24 hours after GnRH treatment) the donor cow is inseminated. Six days later, the donor cow is flushed and embryos are transferred to the recipients.

The author has been assisted in creating the image for this entry by Dr Reuben Mapletoft and in its editing, by Drs Scott Norman and Allan Gunn. For this, he is truly grateful.
Selected references:
Bó G.A. et al. 2002. The control of follicular wave development for self-appointed embryo transfer programs in cattle. Theriogenology. 57:53-72.
Bó, G.A. et al. 2010 New approaches to superovulation in the cow. Reprod Fertil Dev. 22:106-12.
Bó GA and Mapletoft, R.J. 2014. Historical perspectives and recent research on superovulation in cattle. Theriogenology 81:38–48
de Souza, L.B. 2013 Effect of synchronization of follicle-wave emergence with estradiol and progesterone and superstimulation with follicle-stimulating hormone on milk estrogen concentrations in dairy cattle Can J Vet Res : 75–78.
Sowers, A.F. et al. 2008 Anti-Mullerian Hormone and Inhibin B in the Definition of Ovarian Aging and the Menopause Transition. J Clin Endocrinol Metab. 93: 3478–3483.
Stevenson, J.S. et al. 2004. Use of Estradiol Cypionate as a Substitute for GnRHin Protocols for Synchronizing Ovulation in Dairy Cattle. J. Dairy Sci. 87:3298–3305

Synchronizing follicle growth in cattle; Ovsynch

Keywords: synchronize, bovine, ovsync, cow, estrous, estrus, cycle, mechanism

Image size: 5363 x 6393px

This diagram, extrapolated from many sources, shows the mechanism of Ovsynch treatment.

The receptive periods during the estrous cycle when GnRH will cause ovulation  are shown as gray vertical bars. If GnRH is given during any of these periods (about 65% of the duration of a three wave estrous cycle) follicles present at that time will ovulate, causing a drop in inhibin, FSH release, and the start of new wave of follicle growth. It is this wave of of follicle growth that is utilized to provide synchronous ovulations about nine days later.

As mentioned, a synchronous response to the first injection of GnRH occurs in about 65% of cows. Nine days after the first injection of GnRH, some of the non-responding cows will either respond to the injection of prostaglandin and show estrus or they may show estrus spontaneously. In this manner, about 65% of cows will respond to the first injection of GnRH and about 35% of the cows (the non-responders) will be in estrus after the injection of prostaglandin and will respond to to the second injection of GnRH, together with the other cows. Collectively this means that about 85% of all the treated cows could respond to Ovsynch treatment.

These figures assume that all the cattle being treated are actually having estrous cycles. In high producing cows, is seldom the case so the response to Ovsynch treatments may often fall short of these calculations.

Synchronizing follicle growth in cattle.1

Keywords: synchronize, bovine, ovsync, cow

Image size: 5370 x 3534px

This diagram, extrapolated from many sources, shows the receptive periods during the estrous cycle when GnRH will cause ovulation (gray vertical bars). If GnRH is given during any of these periods, follicles present at that time will ovulate, causing a drop in inhibin, FSH release, and the start of new wave of follicle growth. It is this wave of of follicle growth that is utilized to provide synchronous ovulations about nine days later.

Seven days after the initial injection of GnRH, an injection of prostaglandin is given to cause luteolysis of the corpora lutea that were formed seven days earlier. Two days after the injection of prostaglandin, another injection of GnRH is given so that the follicles that have grown in the new wave are induced to ovulate. Sometimes cattle are bred at this time; this is called "Cosync" treatment because the second injection and the time the breeding coincide. Alternatively, they can be bred at a fixed time, usually about the morning after the second injection of GnRH (about 16 hours later). This is referred to as "Ovsync" because ovulations are closely grouped around this period of time. Finally, cows may only be selected for breeding if they show heat; hence the name "Selectsync".

In some instances, injections of prostaglandin are given (even if "Ovsync" itself may be used) to increase the chances that cows will be in the receptive periods (gray vertical bars) at the time the first GnRH is given for Ovsync. The economic value of these pre-synchronization ("Presync") treatments is questionable.

Sensitive periods for Ovsynch treatment

Keywords: cow, synchronize, bovine, estrous, estrus, cycle, follicle, wave, Ovsynch

A diagram modified from several sources, showing an estrous cycle with three follicle waves. The physiology of two or three follicle waves is important during “Ovsych” treatment (GnRH-Prostaglandin-GnRH) because it is only those follicles that have already been recruited and already growing in a follicle wave that will ovulate after GnRH treatment; an essential step in re-setting the recruitment of follicles so that a mature follicle will be available for ovulation about 8 to 9 days later. This image shows the periods of time (vertical grey bars) during which follicles can be induced to ovulate by GnRH treatment.