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.

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

Water

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

Salts

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

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.