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

Embryonic/fetal membrane slipping

Keywords: membrane, slip, fetus, embryo, diagnosis, pregnancy, ruminant, bovine, ovine, caprine, staging.

Membrane slip is commonly used to diagnose pregnancy in cattle, especially in dairy cattle where early pregnancy diagnosis is important. Membrane slip manifests itself when gentle pressure is applied to a fold of the uterine wall. If a conceptus is present, its free allantois or allantochorion or even the free chorion itself, slips away from the constricted section of the uterus. This creates a distinct, palpable "click" between the operator's fingers. 

The composite image below shows how a membrane slip is generated. In image A, the uterus is grasped between the thumb and any finger, then the digits are rolled relative to one another until a firm clicking sensation is detected. The operators digits are not moved off the uterus at any time during this operation. Using an opened, pregnant uterus in image B, one can see how the edge of embryonic/fetal membrane (yellow arrows) is pinched gently between the operator's thumb and finger. As these digits are rolled relative to one another (decreasing the interposing angle between the thumb and finger) the membrane escapes the operator's grip (red and yellow arrows in image C) generating the clicking sensation.

Image size: 1024 x 768px.

In several studies, membrane slipping has been shown to be harmless to the conceptus.

As reviewed elsewhere in LORI, during early gestation, the allantois is not yet attached to the chorion. However, these two membranes gradually become inseparable and collectively, are known as the allantochorion or less commonly, the chorioallantois. Therefore in early gestation, it is either the allantois or chorion or allantochorion that is involved in membrane slipping; a fairly trivial point.

Although the production of pregnancy specific proteins, RNA segments and Interferon Tau have been developed for early pregnancy detection in cattle, ultrasonography is currently accepted as the gold standard for comparison of pregnancy diagnostic methods in cattle; it is both highly specific and sensitive after 28 days of gestation and especially valuable for the diagnosis of twins. However, membrane slipping continues to be a valuable diagnostic tool for routine pregnancy diagnosis. Membrane slipping is also a useful tool for the detection of early pregnancy in small ruminants and wild ruminants when laparotomy is performed for any reason. It cannot be used in new world camelids or equids because of the diffuse placental attachment in those animals.

Comment on the use of the word "fetal" in fetal membrane slip, still a widely accepted term in animal science:

It has been stated that the embryonic period in cattle extends from fertilization to the completion of differentiation at about 42 days (Committee on Reproductive Nomenclature, 1972. Recommendations for standardizing bovine reproductive terms. Cornell Vet 62: 216–237). However it is arguable that differentiation continues throughout pregnancy or even postnatal life and therefore, that the distinction between an embryo and fetus is arbitrary. Even in humans, where an embryo makes its sudden and completely artificial transition into fetal life at 8 weeks, this terminology is under review. Therefore, in this author's opinion, it is erroneous to use fetal membrane slip as a general diagnostic term in cattle, especially dairy cattle. This is because fetal membrane slip is most often used as a diagnostic tool before 42 days, when the conceptus is still formally classified as an embryo. This may be a trivial matter (and in the greater scheme of things, probably is) but like the term embryotomy it should probably be discarded in favor of a more global and accurate term. In that light, the author suggests that instead of the term fetal membrane slip, consideration be given instead, to using the term membrane slip throughout pregnancy.

Selected references:

Ioannidis, J. 2016. Circulating miRNA signatures of early pregnancy in cattle. BMC Genomics. 17: 184

Nation, D.P. et al. 2003. Accuracy of bovine pregnancy detection using transrectal ultrasonography at 28 to 35 days after insemination. Australian Vet. J. 81: 63–65

Preeti Rawat et al. 2016. Identification of potential protein biomarkers for early detection of pregnancy in cow urine using 2D DIGE and label free quantitation. Clin Proteomics. 13: 15.

Romano, J.E. 2006. Early pregnancy diagnosis by transrectal ultrasonography in dairy cattle.
Theriogenology. 66:1034–1041