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.

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.

Two and three wave estrous cycles

Key words: bovine, estrous, estrus, cycle, follicle, wave, Ovsynch, prostaglandin

A diagram modified from several sources of two and three wave estrous cycles. One cow has two waves of follicle growth with the follicle in the second wave eventually ovulating. The other has three waves, with the last wave providing the follicle that ovulates. The length of the estrous cycle is one or two days longer in cows that have three follicle waves instead of two. A minority of cows have four follicle-wave estrous cycles.

This physiology is important in everyday dairy practice when prostaglandins are used to shorten the inter-estrous period because follicles are more likely to large and ready to ovulate if cows have more follicle waves during the estrous cycle. There is usually only one follicle per wave but once a follicle has grown to its maximum size it begins to regress, it cannot be utilized. Therefore, many follicles and their oocytes are wasted. The physiology of two or three follicle waves is also important during “Ov-sych” treatment to synchronize ovulation in cattle.

Bovine uterine involution

Key words: bovine, involution, uterus, cycling, estrous, cycles, postpartum, palpation

The uterus of a cow, two weeks after calving, opened to show the state of involution. At this stage of involution, the areas of the endometrium have already involuted (this occurs by about 10 days after calving) but the caruncles themselves have not even sloughed their epithelium completely. It is only by 12 to 14 days that (at about the stage of the uterus in this image) that the sloughing process reaches the capillary beds, releasing red blood cells into the lumen. It also at this time, that neutrophils enter the lumen, attracted by chemotaxis to the contaminated lochia. Therefore lochia that are somewhat purulent and blood tinged at this time are normal.

The contents of the uterus are normally contaminated (not infected) by bacteria up until 25 to 30 days postpartum. The bacterial swab in the inset has been added as a reminder to this fact. Uterine cultures taken before this time are of limited value.

Image size: 1229 x 1162px

During transrectal palpation, it is only possible to grasp the cranial border of the uterus by this stage of involution i.e. 10 to 14 days after calving; much later than in mares. The size of an average human hand is given to scale to allow one to appreciate that challenge. Depending on management and health of the animal it has returned to its pre-pregnancy size by about 35 days after calving; in some reports as early as 25 days; in others, as late as 50 days. It is easily retracted at that time. After calving, the uterus of a cow is always larger than that of a heifer.

Although the corpus luteum of pregnancy is visible for several weeks after parturition (see the inset at right above) it has ceased to function at the time of calving.

Uterine involution is, for all practical purposes, disconnected from ovarian function in cattle. It is also disconnected from estrous behavior. In dairy cows (where human interference prevents calves from suckling) the first ovulations after calving often occur before 20 days postpartum but those ovulations are seldom accompanied by signs of estrus. Over the next two to three ovulations (which occur at increasing intervals between one another) estrous display becomes more obvious. Interestingly, the process of uterine involution is slower than both the resumption of normal ovarian activity and estrous display.

Although the epithelial regeneration over the caruncles is largely complete by 30 to 35 days postpartum, global involution of the uterus continues over the next 10 to 15 days. Certainly, the uterus is completely involuted by 70 to 75 days, when the first inseminations start occurring after calving in most dairy cattle i.e. the "voluntary waiting period". In beef animals, where suckling occurs, and breed and energy intake can also have profound negative effects on postpartum anestrous intervals, uterine involution is complete long before re-breeding occurs and is seldom a limiting factor in postpartum fertility.

Note of interest: Although the complex physiology of postpartum anestrus (nutrition, breed, presence of milk in the udder, calf contact etc) is still not completely understood, one of the cornerstones of anestrus appears to be a lack of LH secretion. Strangely, FSH secretion is a not a limiting factor in the resumption of cyclicity, well developed follicle populations being plentiful well before the first ovulations occur. A major negative effect on LH secretion is that of endogenous opioids (endorphins). This is not only true for cattle in the postpartum period but for mammals in general, including humans. LH secretion increases almost immediately after morphine antagonists such as naloxone and naltrexone are administered.

The bovine estrous cycle

Keywords: estrus, estrous, progesterone, prostaglandin, estradiol, LH, FSH, bovine, cow

Note: The use of estrus as an adjective is less common than estrous (1.23 million vs 840 thousand Google hits respectively). Nevertheless, estrus is often used as an adjective e.g estrus cycle, estrus behavior, estrus synchronization etc. Indeed, it has become so common as to be regarded as both a noun and an adjective. However, the author adopts the more common term estrous as an adjective in this entry. In the British commonwealth, the terms oestrus and oestrous are used for the noun and adjective respectively.

The image shown here is useful for explaining the bovine estrous cycle*. It shows the rise of estradiol 17 beta that brings the cow into heat and causes a surge of luteinizing hormone (LH). Ovulation usually occurs about 16 to 24 hours after the onset of the LH surge i.e. when the cow is no longer showing signs of estrus.

After ovulation has occurred, a corpus luteum forms from the structure that was previously the follicle, producing progesterone (the yellow profile)  for approximately 19 days. If the cow is not pregnant, at about 15 to 16 days after ovulation, the endometrium releases prostaglandin F2 alpha (PG) which causes luteolysis i.e. the demise on the corpus luteum. Immediately after ovulation has occurred a new wave of follicle growth begins because inhibin production by the previous large follicle, is no longer present. This results in an elevation of follicle stimulating hormone (FSH).

A dominant follicle is selected in each follicle wave, suppressing its cohorts. If that follicle does not ovulate, it undergoes regression and a new follicle wave begins. There are usually three such follicle waves during each estrous cycle but in young animals, there may be only two waves, resulting in an interovulatory interval that is a few days shorter than a conventional estrous cycle of  21 or 22 days.

Standing estrus last for about 8 to 12 hours in most dairy cows but may be much shorter (and difficult to detect) in high producing cows. Very short estrous periods are typical of zebu-type cattle as well.

*Readers are strongly encouraged to consult an excellent resource on the bovine estrus cycle developed by Dr Roberto Palomares, Kip Carter and colleagues at The College of Veterinary Medicine, University of Georgia. The presentation is available for Mac or PC use.

The Mac OS version has full animation. Lacking some animations, the PC (epub) version however remains valuable for all students of reproductive physiology. On most PC computers, the ePub version can be viewed without downloading ePub readers.

Kamar heatmount detector

Keywords:  Kamar, bovine, management, cow, estrous, cycle, detection

The Kamar device is applied to the rump of a cow with adhesive. When the cow is in standing estrus and allows another cow to mount her, a chamber containing dye is crushed, turning the device red throughout its length. Although valuable, the device is prone to false positives in crowded barns when cows are moving in or out of the building; in such cases mounting may be less specific than otherwise.