In Vitro Fertilization

Chapter 81
In Vitro Fertilization

John F. Hasler1 and Jennifer P. Barfield2

1 Bioniche Animal Health, Inc., Laporte, Colorado, USA

2 Department of Biomedical Sciences, Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado, USA


In Latin, in vitro means “in glass,” and in vitro fertilization (IVF) is frequently used as a general term for the process of generating embryos outside the body, which also includes in vitro maturation (IVM) and in vitro culture (IVC). These procedures are conducted in the sequence IVM–IVF–IVC to produce embryos exclusively in vitro (IVP).

In 1959, the rabbit was the first mammalian species in which live offspring were produced by IVF,1 followed by laboratory mice in 1968.2 Because the mouse was not a good model for cattle, the first successful IVF with cattle was in 1977 when sperm were capacitated in the oviduct or uterus of a cow or rabbit3 and the first live calf born in 1981 when a 4-cell embryo was transferred into the oviduct of a recipient cow.4 The first calves produced entirely from IVM, IVF, and IVC were born in 1987.5 Since that time, research on in vitro procedures has grown dramatically. In addition, in vitro procedures are now being used commercially in conjunction with embryo transfer in a number of countries. In this chapter we intend to describe the most current techniques for oocyte collection, IVP of embryos, and the utilization of IVP embryos in the cattle industry. Some of the applications of IVF technology in cattle are listed in Table 81.1.

Table 81.1 Applications of IVF technology.

Commercial applications
Offspring from infertile cattle
Offspring from pregnant cattle
Offspring from young heifers prior to breeding age
Salvage of genetics from terminally ill/injured cattle
Efficient use of sexed semen
Use of resorted semen (frozen-thawed and sexed after thawing)
Use of multiple sires in a short period of time
Utilization of slaughterhouse-derived oocytes for production of research and/or inexpensive embryos

Research applications
Improvements of IVF technology
Improvement of IVC for cloning and transgenic procedures

Collection of oocytes

Originally, in vitro procedures in cattle were conducted primarily for research purposes and utilized oocytes collected from superovulated females. When collected from preovulatory follicles or oviducts 20–24 hours after onset of estrus, oocytes have already undergone maturation and are ready for IVF and IVC. Oocytes collected from slaughterhouse-procured ovaries require IVM, so research utilizing these oocytes lagged until IVM techniques were improved. Today, ovaries from cattle slaughterhouses are used extensively as a source of oocytes.

Oocyte collection from living cattle

The primary method of collecting oocytes from live cattle is aspiration of ovaries manipulated per rectum and guided by a vaginally inserted ultrasound probe and needle. Prior to the development of this technique, oocytes were obtained surgically through a flank incision6 or laparoscopic procedures via the paralumbar fossa,7 though these approaches were expensive, inefficient, and risked the formation of adhesions with subsequent loss of fertility. The introduction of real-time transrectal ultrasonic imaging (for review, see ref. 8) led to the development of techniques for the repeated collection of oocytes from bovine females. Ultrasound-guided collection of oocytes via the paralumbar fossa was described in 1987 by Callesen et al.9 and the first repeatable efficient technique involving transvaginal ultrasound-guided aspiration was developed in 1988.10 This technique has become widely known as ovum pick-up (OPU) or transvaginal aspiration. An ultrasound transducer and an attached needle guide of the type used in cattle are shown in Figure 81.1. A diagram of how the transducer is inserted into the vaginal fornix so that a needle can be guided through the vaginal wall and into the ovary is also shown.


Figure 81.1 (a) Ultrasound transducer with oocyte aspiration needle protruding from needle guide. (b) Diagram showing position of the ultrasound transducer pressed against vaginal fornix, with ovary manually manipulated per rectum and held up against the vaginal wall.

Reproduced with permission from Hasler.11

The number of oocytes collected from a cow during a single session of OPU depends on a variety of factors. On the mechanical side, the vacuum pressure used to remove the oocyte from the follicle, the gauge of the needle,12 and the length of the bevel on the needle13 all impact the number of intact cumulus–oocyte complexes collected. Frequency of collection is another factor. OPU can be performed once or twice a week on the same cow without the use of exogenous hormones. Studies have demonstrated no advantage of interval length for the number of oocytes retrieved per collection when 3, 4, or 7 days were compared.14–16 The ultimate goal is to generate embryos that can be transferred to recipients and from that perspective there is evidence that a 3- or 4-day interval (8 oocytes per OPU) is better than a 7-day interval (5.5 oocytes per OPU).17

The number of oocytes collected per OPU can be increased by pretreatment of the donor with gonadotropins.18,19 In a very comprehensive comparison of different OPU intervals with and without follicle-stimulating hormone (FSH), Chaubal et al.20 reported that the most productive protocol in terms of oocyte and embryo production involved dominant follicle removal, FSH treatment 36 hours later, and OPU 48 hours after FSH. This treatment was alternated weekly with simple once-weekly OPU. The OPU weekly session involving FSH averaged 10.6 oocytes and 2.4 blastocysts, while the alternating weekly OPU with no FSH averaged 4.6 oocytes and 0.9 blastocysts. The interaction of factors, including the frequency of OPU and the inclusion or absence of FSH stimulation, has been recently reviewed.21

Breed of cattle is also an important factor. Low mean numbers of usable oocytes, ranging from 4.1 to 5.3, were reported by Hasler et al.22 and Looney et al.23 from combined populations of dairy and beef breeds, including older females with a variety of fertility problems. The use of FSH with a coasting period is now successfully employed in several commercial North American bovine IVP businesses and has resulted in a mean of approximately 20 oocytes collected from Bos taurus cattle once every 2 weeks (data courtesy of Trans Ova Genetics). In South America (primarily Brazil), 17–25 oocytes are routinely collected with no FSH priming from Nelore cattle, an indigenous breed of Brahman origin known to develop higher numbers of follicles in each follicular wave than Bos indicus females (data courtesy of In Vitro Brazil).

Age of the donor can also impact the number of oocytes recovered per OPU. Evidence in Holsteins suggests that a significantly higher number of oocytes can be collected in cows 6–9 years old than in cows aged 14–18 years.16 However, these were primarily cattle that were undergoing OPU/IVP due to previously existing fertility problems. Recent unpublished data from a large commercial in vitro program involving reproductively healthy donors indicated little variation in recovered oocyte numbers to age 13 and a steady decline from age 14 to 18 years (data courtesy of Trans Ova Genetics). Interestingly, the developmental potential of oocytes was similar from all donor age categories in this program.

There is considerable variation among donors in terms of oocyte production over time. For example, an 8-year-old Holstein donor, previously diagnosed as infertile, produced a total of 176 embryos from 167 collections by 23 different sires over a period of 167 weeks.16 With repeated OPU collections, the mean number of oocytes decreased; however, the developmental potential of oocytes produced by OPU did not change. This clearly shows the potential for production of large numbers of embryos by OPU–IVP methods and the advantageous opportunity to use different sires weekly or even biweekly.

Oocyte collection from excised ovaries

In addition to bovine oocyte retrieval by OPU, there are several situations in which oocytes are removed from excised ovaries. When cows develop terminal diseases or become crippled, ovaries can be removed via a flank laparotomy or through a vaginal incision and the oocytes recovered by follicular aspiration or slicing. Stringfellow et al.24 reported collecting an average of 46 oocytes from 18 culled dairy cows by slicing the ovarian cortex. In a highly successful commercial program, Green and McGuirk25 reported producing an average of 46 oocytes and nine embryos per donor in more than 100 cases of chronically ill, injured, and senile cows.

Oocyte collection from slaughterhouse-procured ovaries

A large source of oocytes is ovaries from cows processed in slaughterhouses. All follicles between 3 and 8 mm in diameter are aspirated and the collected oocytes used to generate IVP embryos. In a mass-production system using ovaries from the slaughterhouse, Lu and Polge26 reported producing more than 200 000 in vitro-derived blastocysts from approximately 700 000 oocytes in 2 years. Although this program is no longer in operation, it demonstrated the potential for production of IVP embryos from cattle after slaughter. Reports indicate that as many as 100 and 200 oocytes can sometimes be harvested from one pair of Bos taurus ovaries.26 In Japan, ovaries from individual Kobi cows are procured at the time of slaughter and sent to an IVP laboratory for processing. Later in the same day, carcass values of the individually identified cattle determine which oocytes are retained and processed through the entire IVP procedure. In addition to their commercial value, IVP embryos are produced in universities and laboratories around the world as a valuable source of material for bovine reproduction experiments.27


Once oocytes are collected, they are cultured in a series of media that support IVM, IVF, and in vitro development. In general, there are at least six different media utilized for the production of in vitro embryos. Most of these media are not commercially available and must be produced in the laboratory, though some commercial media systems exist. All these media have common components, which are briefly reviewed here, followed by specifics about media used during specific stages of development.


Pure water is the single most abundant component of all media used for all types of embryo procedures. In fact, water quality might be considered the single most important component of embryo media. What constitutes pure water and how best to produce it has been thoroughly reviewed.28


A number of salts, such as sodium chloride and potassium phosphate, are used in the preparation of virtually all media that are utilized for IVP. These components, and in fact all chemicals used in media preparation, should be reagent quality and whenever possible should be certified as having been “mouse embryo tested.”

Energy sources

Glucose, fructose and/or pyruvate are often included in media as energy sources. It is well established that optimal glucose levels for bovine embryos must be low, around 0.5 mmol/L, during early embryonic development. Once the late morula stage is reached, however, higher glucose levels are desirable, and most embryo transfer media contain approximately 2.0 mmol/L glucose.


Macromolecules include serum, bovine serum albumin (BSA), hyaluronic acid, and synthetic molecules such as polyvinyl alcohol. The most important reason to include a macromolecule in IVP media is the surfactant properties of these molecules, which reduces the chances that embryos will float or stick to equipment, pipette tips, straws, and Petri dishes. More specifically, serum contains a wide range of components that are beneficial for embryo development, such as proteins, growth factors, vitamins, minerals, and energy substrates, and it is well established that serum promotes blastocyst development.29 Serum and BSA also provide a number of physical properties, including chelation, colloidal osmotic regulation, and pH regulation. Unfortunately, the inclusion of serum during IVC has been associated with a high degree of abnormal pregnancies and offspring, often referred to as large offspring syndrome.30–33

Polyvinyl alcohol is primarily used as a surfactant in flushing and holding media that are formulated to include no components of animal origin. These media have been used on hundreds of thousands of bovine donors with highly satisfactory results, based on extensive repeated sales and verbal expressions of satisfaction from a large number of bovine and equine embryo transfer practitioners (unpublished results courtesy of Bioniche Animal Health, Inc.).

Hyaluronic acid (HA) is a glycosaminoglycan that is present in follicular, oviductal, and uterine fluids.34 Embryos have been shown to have surface receptors for HA, and it is involved in the regulation of gene expression, cell proliferation, and cell differentiation. Addition of HA to bovine embryo culture systems improves development to the blastocyst stage, increases hatching rates, and improves cryotolerance.35–38 The traditional source of HA has been cock’s combs, but can be produced biosynthetically in a Streptococcus equi fermentation that does not contain any products of animal origin.

Amino acids

The addition of all 20 essential and nonessential amino acids to embryo culture systems is almost universal. The role of certain individual amino acids and also that of all 20 combined in the development of embryos and enhancing their viability was reviewed by Gardner.39 When serum is absent from culture media, the inclusion of amino acids substitutes for some of the benefits that serum provides.


Antioxidants reduce the formation of damaging free radicals in media. Glutathione, BSA, ascorbic acid, and catalase are frequently used as antioxidants in embryo media.


Chelators are chemicals that bind heavy metal ions. In a perfectly pure medium, there would be no need for chelators, but there is always a chance that traces of heavy metals can enter the formulation via water or reagent salts. Transferrin, BSA, and ethylenediaminetetraacetic acid (EDTA) all function as chelators.


Osmolytes are organic compounds that play a role in maintaining volume of cells and organelles within cells as well as fluid balance. When cells swell due to external osmotic pressure, membrane channels open and allow efflux of osmolytes, which carry water with them, restoring normal cell volume. Glycine is an osmolyte, and the smallest of the so-called nonessential amino acids that are usually added to culture media and to some formulations of holding and cryopreservation media. The physiological basis for the osmolyte properties of glycine in embryo culture was described in detail by Steeves et al.40

pH and buffers

The intracellular pH (pHi) of in vitro-derived bovine embryos is 7.2 as measured with a pH-sensitive probe.41 This study also demonstrated that bovine embryos in culture recovered after being subjected to an acid pH more much readily than from a basic pH. In fact, pHi was decreased only to approximately 7.8 when embryos were subjected to a pH above 8. In a classic study, Kane42 showed that the highest percentage of rabbit blastocysts hatched during IVC when pH was in the range 7.2–7.4, with a precipitous decline when pH was below 7.0 or above 7.8. Most bovine IVP media are in the 7.3–7.4 range except for fertilization media which are generally 7.5–7.6.

Phosphate-buffered saline (PBS), which has been widely used for in vivo-derived embryo recoveries, contains phosphate as a buffer. Although phosphate is not a particularly effective buffer, the inclusion of either serum or BSA adds additional buffering capacity to PBS. IVP media, on the other hand, are normally maintained in an incubator with a 5% CO2 atmosphere. The inclusion of bicarbonate in these media provides a stable pH due to the equilibrium that is established between the bicarbonate and the CO2. However, when media based on this system are removed from the incubator, the pH rapidly decreases and will reach an unacceptable level of acidity within a few minutes in air. As a consequence, the media used for rinsing ova/zygotes between IVP steps and for holding them in air prior to transfer or cryopreservation contain MOPES or HEPES zwitterionic buffers.


The optimum osmolarity for maintaining bovine embryos during IVP procedures is approximately 275 mmol/L, which is also true of other species such as the rabbit, rat, and pig.43 On a practical basis, bovine and probably equine embryos are not adversely affected by exposure to osmolarities ranging between 250 and 300 mmol/L for relatively short periods. Research has suggested that a total osmolarity between 250 and 270 mmol/L appeared to be optimal when bovine in vitro-derived embryos were cultured in KSOM.44

An important consideration when adjusting the osmolarity of media is the issue of what molecules are added or reduced in order to raise or lower osmolarity. The most abundant salt in all media utilized in IVF procedures is NaCl, but it had been shown to be detrimental to bovine embryo development at concentrations above 95 mmol/L.44 Thus the benefit of having osmolarity below 300 mmol/L in most studies is likely due to lowered Na+ as opposed to lowered osmolarity per se (oviduct fluid is >300 mmol/L).


Much of the success of embryo IVP depends on the quality of the starting material, the oocyte. Photomicrographs of the four basic categories of freshly collected oocytes are shown in Figure 81.2. Oocytes in these four categories have greatly different potentials for maturation and development into embryos, ranging from type 1 with the best potential to type 4 with almost zero potential.16 Table 81.2 demonstrates the potential for these oocytes to be fertilized, develop to blastocysts, and result in pregnancies.


Figure 81.2 Types of bovine oocytes collected from cattle ovaries prior to IVM. Brief descriptions: I, more than four layers of cumulus; II, one to three layers of cumulus; III, nude oocyte with no cumulus; IV, expanded cumulus.

Reproduced with permission from Hasler.11

Table 81.2 Potential for fertilization and embryonic development in different types of immature bovine oocytes.11

Source: reproduced with permission from Hasler J. Embryo transfer and in vitro fertilization. In: Schatten H, Constantinescu GM (eds) Comparative Reproductive Biology. Ames, IA: Blackwell Publishing, 2007, pp. 171–211.

Oocyte type Cumulus No. oocytes Percent cleaved Percent blastocysts Percent hatched
I >4 layers 571 65a 29a 19a
II 1–3 layers 228 40b  8b  6b
III Nude 289 38b <1c <1c
IV Expanded 151 38b  7b  5b

Values within a column with different superscripts (a, b or c) differ significantly (P < 0.025).

An oocyte that is considered competent for IVP is referred to as “matured.” Maturation involves a series of events that begin in fetal life with the initiation of meiosis. At birth oocytes are arrested at the diplotene stage while continuing to grow and they do not become meiotically active again until puberty, when they are exposed to preovulatory surges of the gonadotropins luteinizing hormone (LH) and FSH, which induce changes in gene expression as well as in morphological and physiological features of the theca, granulosa, cumulus, and oocyte compartments of the ovarian follicle. A subset of oocytes is continually recruited to proceed with meiosis during each estrous cycle, only to arrest at metaphase II, the stage at which they are ovulated. The changes that occur between the diplotene and metaphase II stages of arrest are considered maturation.45

IVM of immature oocytes occurs by a different mechanism from that of in vivo-matured oocytes. Removal of an oocyte from the inhibitory follicular environment followed by favorable culture conditions results in spontaneous maturation without the physiological series of events that occur in vivo, though some morphological changes can be observed (e.g., expansion of cumulus cells).46 In vitro conditions simulate some of the physiological changes with the addition of hormones, including LH, FSH, and estradiol-17β, which are typical of the preovulatory follicle, and growth hormones or epidermal growth factor.47

The way in which oocytes are collected and their maturation status dictate how they must be handled after collection. Immature oocytes can be collected from unstimulated cows undergoing OPU every 3–7 days, irrespective of the stage of their estrous cycle; however, it is also possible to time aspiration after ovarian stimulation and injection of LH or gonadotropin-releasing hormone (GnRH) so that one collects mature oocytes. These in vivo-matured oocytes do not require further maturation in vitro though some hours in maturation medium prior to fertilization may be beneficial. Oocytes aspirated from ovaries collected from cows processed in slaughterhouses are primarily from follicles 3–8 mm in diameter and the resulting cohorts of oocytes vary in their maturational status. Even so, the majority of these oocytes can achieve maturation during a 24-hour period of IVM.


Once IVM is complete, oocytes are ready to be fertilized. This involves the coincubation of oocytes with spermatozoa and is generally done in a 4-well dish, but also can be done in a microdrop under oil. Most laboratories allow for 18 hours of coincubation, even though the majority of fertilization events will be complete by 12 hours of coincubation.

On ejaculation, spermatozoa are motile but not capable of fertilizing an oocyte. The changes that a spermatozoon must go through before it is capable of fertilizing an oocyte are collectively called capacitation. Even though this process was discovered in 1951 by Chang48 and Austin,49

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Aug 24, 2017 | Posted by in GENERAL | Comments Off on In Vitro Fertilization

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