Technique for artificial insemination; bovine.

Keywords:  bovine, vagina, insemination, A.I., AI, technique.

Routine artificial insemination (AI) in cattle is more complex than routine AI in mares.  The common method of AI in mares usually involves non-frozen semen. A gloved hand is simply inserted into the vagina then a finger into the cervix, enabling guidance of an insemination pipette into the uterus. In cattle, semen is almost invariably frozen and must be thawed carefully then inseminated by guiding an insemination rod through the cervix using transrectal manipulation. The bovine vagina, especially in heifers, will not permit the easy entry of a gloved hand and certainly, a finger cannot be passed through the cervix of a normal non-pregnant cow or heifer.

The intent of this entry is to illustrate the mechanics of handling an insemination rod and the act of insemination itself. The author is indebted to Select Sires US & Canada for allowing these images to be used in LORI. Select Sires holds the copyright to all images in the entry and should be contacted regarding their use outside of this image library.

After removing most of the feces from the rectum, the gloved hand is used to grasp the cervix transrectally. Then, using paper towel, the vulva lips are cleaned briefly, and the cow's tail is deflected to one side using the arm in her rectum. The vulva lips are parted (if a helper is not available, one of  the techniques described in the text box below can be used) and the AI gun is inserted into the vestibule at an upward angle. Obviously, care must be taken not to contaminate the vagina. See figure 1. It is important to grasp the cervix and push it cranially to eliminate vaginal folds that often impede passage of the AI gun.

Figure 1. An AI gun is inserted to the level of the external cervical os. In the author's opinion, the hand shown here should advance cranially to hold the entire cervical body. Holding the entire cervix in one's hand allows it to be manipulated effectively. At this point, the author also advances the AI gun so that it can be felt against the fifth digit (small finger) in the ventral part of the cervical fornix. With the entire cervix is within one's grasp, the external cervical os is lifted and fed over the tip of the AI gun. Image size:1000 x 680

Variations: One can hold the base of the loaded insemination between one's teeth; a valuable "third hand". The paper towel that has been wrapped around the AI gun to prevent semen cold shock, is removed. Some technicians fold this paper towel, kink it and place it in the ventral vulva commisure. This opens the vulva lips and serves as a surface upon which to slide the AI gun upward and into the vagina. In any case, the vulva lips should be wiped as clean as possible; generally not repeating this action without more paper towel, as that leads to more contamination than otherwise. The author uses a different approach, pushing down slightly on the cleaned perineal body using the elbow of the arm in the rectum. This causes the vulva lips to gape while the AI gun is inserted into the vagina.

  

Figure 2. An approach where the cranial vagina is narrowed over the external cervical os to facilitate entry of the AI gun into the external cervical os. As stated above, the author uses a different technique to locate an entry point for the AI gun. Although the external cervical os can also be located using an endoscope designed for this purpose, this does not necessarily facilitate passage of the AI gun through the cervix, often the most challenging part of AI in cattle. Image size:1000 x 680

Using either technique, after the AI gun has entered the cervical canal, the rings of the cervix are manipulated over the tip of the AI gun.  See figure 3. Note the emphasis on manipulating the cervix over the gun and not placing primary emphasis on moving the gun cranially, attempting to bypass the cervical "rings" (they are not true rings but a series of crescents). The AI gun should not be forced cranially in hopes of displacing the annular rings .

Figure 3: Manipulating the annular rings (folds) of the cervix over the AI gun as it is moved gently towards the uterine body. Image size: 800 x 554

Although it is not illustrated in this entry, it is essential to place a finger over the cranial aspect of the cervix to determine that the AI gun has emerged from the internal cervical os and lies within the uterine body. The plunger of the AI gun is then depressed, expelling semen into the uterus (see figure 4). One should ensure that the AI gun is not pulled caudally into the cervix while the plunger is depressed.

Figure 4. Depositing semen within the uterine body. Image size:1000 x 680

Clitoral stimulation in both cows and heifers immediately after insemination has been shown to increase pregnancy rates marginally in some studies but this effect (especially in heifers) is not consistent. 

Some studies show advantages to intra-cornual (in the uterine horns) insemination, others not. The situation remains unclear. For routine AI therefore, it is reasonable to suggest that semen should be deposited within the uterine body (the short section of the uterus that extends only 2 to 3 cm cranial to  the internal cervical os). On the other hand, there appear to be no detrimental effects when intra-cornual insemination is used. Therefore intra-cornual should be considered when sexed semen is used or when super-ovulation is practiced.  Passing an AI gun up the uterine horn requires different techniques to those shown here, akin to those used for embryo collection. 

Selected references:

Carvalho P.D. et al. 2013 Effects of deep-horn AI on fertilization and embryo production in superovulated cows and heifers. Theriogenology. 80:1074–1081

Ciro, M. et al . Comparison between deep intracornual artificial insemination (dIAI) and conventional artificial insemination (AI) using low concentration of spermatozoa in beef cattle. Braz. arch. biol. technol.[Internet]. 2012 June.cited 2019. 55: 371-374.

Lunstra, D.D. et al. 1983. Clitoral stimulation and the effect of age, breed, technician, and postpartum interval on pregnancy rate to artificial insemination in beef cattle. Theriogenology. 19:555-563

Momont, H. et al. 1989 Does intrauterine site of insemination in cattle really matter? Theriogenology 32:19-26

Segura, C.V.M. and Rodriguez, R.O.L. 1994. Effect of clitoral stimulation after artificial insemination on conception in Zebu-crossbred heifers in the tropics. Theriogenology 42:781-787

Acardiac fetuses

 Acardiac fetuses (Amorphus globosus monsters)

Keywords: amorphous, globosus, (AG),(AGM), acardiac fetus (AF), bovine, placenta, monsters, karyotype, cattle

The essential nature of these fetuses is that they do not have hearts. Therefore the term amorphous globosus (AG) is less specific than acardiac fetus (AFs). AFs are placental parasites originating from embryos that do not develop into normal fetuses. Although the term "monster" is commonly applied to these fetuses because of their grotesque appearance, that term is melodramatic and archaic. Indeed, the term "monster" has been largely discarded in human medicine. The term anidian monster has also been used to describe an AF i.e. The cytological sex of a bovine anidian (amorphous) twin monster. HO Dunn et al. Cytogenetic and Genome Research, 1967. This term is seldom used and its etymology remains a mystery.

Should one consider amorphous globusus monsters (AFs) as fetuses? Merriam-Webster defines a fetus as: "An unborn or unhatched vertebrate especially after attaining the basic structural plan of its kind". On perusing descriptions of AFs it appears that they probably contain endoderm, ectoderm and mesoderm although these basic embryonic layers do not develop normally. If these three layers are considered to be a "basic structural plan" (could that not also be the DNA in a single cell?) then AFs should be referred to as fetuses.

The image below shows an acardiac quadruplet born together with three normal triplets females also shown in another LORI entry.

Image size: 2816 x 2070 px

It would be convenient to assume that this AF was also a female because the triplet females were not androgenized (freemartins). However, the genital system of the acardiac fetus had not developed, therefore the possibility of it having an XY karyotype could not be discounted (see below).

Below, an AF and a radiograph of that fetus (inset). The radiograph shows a small center of mineralization/ossification. The umbilical cord has broken free of the host placenta.

Image size: 947 x 719 px

A radiograph of a bilobed acardiac fetus with a highly mineralized/ossified focus.

Image size: 1200 x 1039 px

Acardiac fetuses are most common in cattle, followed by the small ruminants, then horses and rarely, humans. The reason that they occur more frequently in cattle than other animals is probably because placental fusion and anastomosis of blood supply is very common in cattle, also explaining the high frequency of freemartins in these animals. Without a blood supply joined to that of a viable fetus (see below) an acardiac fetus can not survive. 

In humans some play is made of the fact that the viable fetus is the "pump" fetus that supplies the abnormal fetus with blood. However, this is of course the situation with any acardiac fetus because a fetus without a heart must rely on blood flow from its placenta, provided by at least one other "pump" fetus in the uterus. Therefore "pump" fetuses occur in animals as well.

Acardiac fetuses occur most frequently in the form of amorphous masses with hair coats and umbilical cords. In unusual cases, they may be recognizable as fetuses with legs, faces, muzzles teeth, vulva lips and tails. Rarely, two acardiac monsters may accompany a normal fetus (triplets). 

As mentioned, AFs do not have hearts. Internally, they may have blood vessels, irregularly shaped bones, foci of mineralization, cartilage and fibrous tissue. In humans, AFs are usually monozygous and therefore the same sex but in cattle, the karyotypic sexes of the viable twin and the acardiac twin can be the same or different.  In addition, the karyotype of the AF can be normal or abnormal. The precise genetic abnormalities leading to acardiac development are not known.

It is considered unlikely that a female co-twin to an acardiac monster can be a freemartin (because of the lack of gross male gonads in the amorphous co-twin). However, it is possible that male gonadal tissue is present if the amorphous globusus co-twin is a male. Therefore, it is wise to examine the viable  female conceptus for freemartinism.

Selected references

Anwar. M.T. et al. 2009. A rare case of globosus amorphus in a goat. Can Vet.: 854–856.

Blaicher. W. et al. 2000 Acardiac twin pregnancy: associated with trisomy 2: Case report. Human reproduction. 15:474-475

del Rio, N.S et al. 2006. Observed frequency of monozygotic twinning in Holstein dairy cattle. Theriogenology. 66:1292-1299

Kamimura, S. 993 A globosus amorphus from an in vitro fertilized embryo transferred to a japanese black cow. Theriogenology 40:853-858

Pearson, L.K.et al Theriogenology Question of the Month. J. Am.Vet.Med.Assn 238:1261-1263

Roberts, S.J. 1986. Veterinary obstetrics and genital diseases. Gestation period, pp 79-81. Published by the author, S.J. Roberts.

Weber J et al. 2017. Facets of Clinical Appearance and Aetiology in an Unusual Bovine Amorphus Globosus. Anat. Histol. Embryol. 46:502–506

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

Puberty. Bos indicus

Keywords: Afrikander, Bos indicus, bovine, breeds, puberty, nutrition,

These calves serve as an example for the discussion of puberty, occurring much earlier in Bos taurus var taurus breeds (English and European animal such as Herefords, Angus, Simmental etc) than in Bos taurus var indicus breeds such as Afrikanders, Texas Longhorns, Brahmans etc. Puberty is highly dependent on body weight and therefore, energy intake i.e. if an animal reaches 60% or more of its adult body weight sooner than a herd mate, it will reach puberty sooner.

In females it is often defined as the first time that ovulation and estrus occur together and in males, the first time that a fertile ejaculate is produced.