Assisted reproductive technologies used in domestic species

Chapter 27
Assisted reproductive technologies used in domestic species

Assisted reproductive technology (ART) is the term used to describe treatments and procedures in the field of reproductive biology used to establish pregnancy by artificial or partially artificial means. This usually involves the hormonal manipulation of reproductive cycles as well as the utilisation of gametes or embryos, including artificial insemination, induction of ovulation, in vitro fertilisation, gamete and embryo cryopreservation and similar procedures designed to enhance pregnancy in treated animals.

The goals of ARTs vary depending on the species or groups of animals to which they are applied. In humans, ARTs are used almost exclusively to overcome issues of either male or female infertility or, in some cases, for pre‐implantation diagnosis of genetic disorders. In rodents such as mice, they are used as a valuable research model to facilitate better understanding of the underlying regulation of normal and abnormal developmental processes; for example, the production of ‘knockout’ mice has provided an extremely informative research tool in the study of gene function. Initially, the focus of ARTs in domestic species was concerned with genetic improvement or production aimed at allowing the livestock industry to respond to continually increasing demands for improved productivity and quality (for example, producing more offspring from a valuable animal than would be possible through normal mating), as well as the conservation of rare or endangered breeds or species. In recent years, however, the use of certain techniques, such as somatic cell nuclear transfer (SCNT) and genome editing has opened up applications in the biomedical model area.

Several generations of ARTs have been developed for domestic animals. These include artificial insemination (AI), multiple ovulation embryo transfer (MOET), in vitro embryo production (IVF) and related techniques including transvaginal oocyte recovery (often referred to as ‘ovum pick‐up’ or OPU), cloning and transgenesis. The main impetus for the development of these technologies arose from their potential application in the refinement of breeding strategies for improved production and health in animal husbandry. More recently, biomedical applications of these technologies, in particular, somatic cell nuclear transfer and stem cell culture, have been pursued in domestic mammals as possible models applicable to human clinical conditions or with potential application in therapeutic drug development (Table 27.1).

Table 27.1 Major developments in assisted reproductive technologies used in domestic animals from the eighteenth century onwards.

Year Event
1784 First successful AI – dog
1890 First successful embryo transfer – rabbit
1900s First AI in cattle and sheep
1930 First bovine embryo recovered from the reproductive tract
1950 Prima – first calf born following embryo transfer
1951 Spermatozoa capacitation first described
1951 Frosty – first calf born after AI with frozen semen
1950s AI in cattle becomes widely established
1959 First successful IVF in the rabbit
1969 Fertilisation of human oocytes in vitro
1970s Non‐surgical embryo transfer developed in cattle
1972 First successful embryo transfer in the horse
1973 Frosty II – first calf born after transfer of a frozen embryo
1977 Fertilisation of bovine oocytes in vitro
1978 Louise Brown – first human baby born following IVF
1981 Virgil – first calf born after IVF
1985 First transgenic livestock – by pronuclear injection
1986 Cloning in sheep by nuclear transfer using embryonic cells
1987 First cloned cattle using embryonic cells
1988 First twin calves after IVM/IVF/IVC
1989 Development of ‘ovum pick‐up’ in cattle
1989 Sex‐sorting of spermatozoa by flow cytometry
1996 Dolly – first mammal, a sheep, produced by SCNT using adult donor cells
1997 Polly – first transgenic sheep produced by SCNT – donor cell a foetal fibroblast transfected with gene coding for human blood clotting factor IX
2002 CC – first cloned cat
2003 Idaho Gem, a mule, born – first member of the horse family to be cloned
2003 Prometea born – first member of the horse family to be cloned from an adult cell
2005 Snuppy – first cloned dog born
2015 First pups born after IVF

As there are major differences in reproductive physiology between mammalian species, it is not surprising that reproductive technologies can be highly efficient in some animal groups, including most farm animals and laboratory rodents, but are very inefficient when used with carnivores, which often have species‐specific reproductive features. Among domestic species, the application of ARTs has been extensively employed in cattle, which reflects their economic importance in food production. Accordingly, in this chapter, emphasis will be placed on the use of ARTs in cattle followed by a discussion of their application in other species.

The impact of reproductive technologies on animal breeding

The rate of genetic improvement in most breeding programmes is controlled by four main factors: (1) the selection intensity, a measure of how selective breeders are in the choice of animal; the fewer animals selected based on their superior performance for a particular trait, the faster the rate of genetic progress; (2) the accuracy with which the genetic merit of an individual animal can be predicted; the greater the accuracy, the greater the potential improvement; (3) the genetic variation in the particular trait in question; the greater the variation for a given trait, the greater the scope breeders have to select animals which are far from the mean level of performance for the trait; and (4) the generation interval, a measure of how long it takes the selected animal(s) to contribute their superior genes to the next generation. In domestic species, this tends to be quite a long period, up to three years. Coupled with these four parameters are the selection differential (the difference in performance in a particular trait between the selected animals and the overall group from which they are selected) and the heritability of the trait (the proportion of the selection differential which, on average, is passed on to the offspring).

Several reproductive biotechnologies can influence one or more of these parameters and, thereby, enhance the rate of genetic improvement (Fig 27.1). Advantages of MOET, for example, include higher female selection intensity and increased selection accuracy. However, variability in an individual animal’s response to superovulation and the low average number of transferable embryos recovered are still limiting factors. In vitro embryo production can potentially affect generation interval through its use on prepubertal donors. Indeed, with the recent advent of genomic selection, it is now possible to select embryos based on a biopsy taken a few days after fertilisation.

Radial diagram of technologies used in genetic improvement: artificial insemination, transgenesis, nuclear transfer, sperm sexing, embryo sexing, in vitro fertilization, and multiple ovulation.

Figure 27.1 Reproductive technologies used to enhance genetic improvement.

Assisted reproductive technologies in cattle

Artificial insemination

Among the available reproductive technologies (Fig 27.2), artificial insemination (AI) has made the single biggest impact on animal production and genetic improvement. Artificial insemination allows high selection intensity on the male side, permitting superior males to sire in excess of 100,000 offspring in their lifetime. At each ejaculation, a bull deposits up to 10 billion spermatozoa in the cranial vagina of the cow. All of these spermatozoa can potentially compete to fertilise the single oocyte that is typically ovulated and only one spermatozoon is required for fertilisation. Indeed, penetration by more than one spermatozoon results in polyspermy, leading to an abnormal chromosome number and embryo mortality. In reality, however, the vast majority of spermatozoa never reach the site of fertilisation in the uterine tube. Thus, there is substantial wastage associated with natural service. In contrast, typical semen doses used in conventional bovine AI, where semen is placed directly into the uterus, contain between 10 and 20 million spermatozoa when frozen‐thawed semen is used. Furthermore, when fresh or sex‐sorted spermatozoa are employed, concentrations as low as one to three million spermatozoa are used. Depending on volume and spermatozoa concentration, a single ejaculate can potentially be used to inseminate more than 1000 cows.

Schematic flow diagram of the methods used in generating multiple offspring from a valuable dam, with MOET to AI, slaughter and ovum pick-up to IVF, and cloning to NT.

Figure 27.2 Methods employed for the generation of multiple offspring from a valuable dam. MOET: multiple ovulation embryo transfer; AI: artificial insemination; IVF: in vitro fertilisation; NT: nuclear transfer.

Advantages of AI include:

  • allowing the widespread use of the semen of outstanding sires;
  • storage of semen for future use, even after the death of the sire;
  • accelerating the introduction of improved genetic characteristics;
  • flexibility in the choice of sire compared to natural service (for example, on the basis of breed, calving ease, production traits and other desirable characteristics);
  • optimising the cost effectiveness – bulls are expensive to buy and rear, without the assurance of high fertility;
  • minimising the risk of disease transmission compared to natural mating;
  • reducing the risk of injury to humans and animals;
  • facilitating the use of oestrous synchronisation and sex‐sorted semen.

Multiple ovulation embryo transfer (MOET)

High genetic merit sires produce billions of fertile gametes at each ejaculation and can sire thousands of offspring during, or even after, their lifetime. However, the contribution of a genetically superior cow to a breeding programme is limited by the fact that cows are monovulatory (that is, only one oocyte is ovulated during each oestrous cycle) and gestation lasts nine months following which a period of uterine involution is required before another pregnancy can be initiated. In addition, these animals have a relatively short productive lifespan of approximately five years.

Induction of multiple ovulations (MO) in a donor, often referred to as ‘superovulation’, coupled with AI, embryo recovery and embryo transfer (ET) to surrogate recipients, provides an opportunity to increase the impact of superior females on a breeding programme, comparable to the impact of AI on the role of the male, but on a much smaller scale.

As the name suggests, MOET involves several steps including (1) synchronisation of the oestrous cycles of the donor and recipient females, (2) induction of superovulation in the donor, (3) AI of the donor, (4) recovery of embryos from the donor, and (5) transfer of embryos to recipients or cryopreservation of the embryos for future transfer (Fig 27.3).

Schematic flow diagram of the overview of multiple ovulation and embryo transfer in cattle, from donor of high genetic merit and crossbred recipients undergoing estrus synchronization.

Figure 27.3 Overview of multiple ovulation and embryo transfer in cattle. Following superovulation, the high genetic merit donor is inseminated with semen from a high genetic merit sire. Approximately seven days later, embryos are recovered by non‐surgical uterine flushing. Good quality embryos are transferred to synchronised recipients. Meanwhile, the valuable donor can be resynchronised and superovulated to produce more embryos, or she can be returned to natural breeding.

Sir Walter Heape is credited with carrying out the first embryo transfer in 1890 using rabbits. The first successful embryo transfer in cattle was recorded in 1951. Since that time, this procedure has played an important role in cattle breeding worldwide. Historically, embryo recovery and transfer involved the use of surgical procedures. However, the development of simple non‐surgical recovery and transfer procedures in the 1970s ensured more widespread accessibility of the technology. Data collated annually by the International Embryo Technology Society ( indicate that approximately one million cattle embryos are transferred worldwide each year.

Superovulation protocols have become more refined over the past 50 years. The use of commercial pituitary extracts and prostaglandins in the 1970s, and partially purified pituitary extracts and progesterone‐releasing devices in the 1980s and 1990s, have facilitated the development of many of the protocols used today. Furthermore, knowledge of follicular wave dynamics through the use of real‐time ultrasonography and the development of strategies for control of follicular waves have provided new practical approaches to superovulation. Despite the fact that much research has focused on methods to maximise the number of ovulations in the donor female, the total yield of transferable embryos has not changed markedly over the last 40 years (typically about five to seven transferable embryos are recovered per superovulated donor). Variability in the donor’s response to superovulation continues to be one of the main limitations of embryo transfer in cattle.

Many of the benefits or potential applications of AI which also apply to ET include:

  • multiplication of genetically superior females;
  • long‐distance/international transport of embryos without the necessity of acclimatisation and health issues associated with live animal transportation;
  • long‐term storage of embryos;
  • increased twinning rates in cattle, either by the transfer of multiple embryos or the transfer of a single embryo to the uterine horn contralateral to the corpus luteum of a previously inseminated recipient;
  • providing twins for research, thereby reducing the numbers of animals required;
  • overcoming the negative effects of heat stress, particularly in lactating dairy cows, where the quality of the oocyte can be compromised;
  • facilitating the control of the sex of a calf (by sexing the embryo prior to transfer or by predetermining the sex of the embryo using sex‐sorted spermatozoa);
  • conservation of endangered species;
  • maximising the number of calves per semen dose when used with superovulated donors;
  • minimising the risk of transmission of infectious agents compared with natural service.

In vitro fertilisation (IVF)

The ability to produce embryos in vitro (in vitro embryo production, IVP) has been possible for more than three decades in domestic mammals, particularly cattle. The first human conceived following IVF, Louise Brown, was born in 1978. The first calf produced by IVF was born in 1981. In both instances, the procedure involved the removal of a matured ovulated oocyte from the uterine tube, incubation with spermatozoa in vitro and subsequent replacement in the female reproductive tract.

The in vitro production of bovine embryos is a three‐step process involving oocyte maturation (in vitro maturation, IVM), oocyte fertilisation (IVF) and embryo culture (IVC) (Fig 27.4). Despite these three distinct steps, the term ‘IVF’ is often used generically to refer to the entire process. Immature oocytes are typically collected either from the ovaries of slaughtered heifers and cows at an abattoir or from the ovaries of live animals using transvaginal oocyte recovery procedures (see below). Good quality cumulus‐oocyte‐complexes are selected, based on their morphological appearance, and are matured in an incubator for 24 hours, typically in an appropriate culture medium supplemented with gonadotrophins and/or growth factors. The first morphological sign that maturation has occurred is the expansion of cumulus cells surrounding the oocyte. Removal of the cumulus cells and more detailed examination of the oocyte itself reveals the first polar body extruded into the perivitelline space, indicating attainment of metaphase II, the stage at which the oocyte is normally ovulated in vivo and at which fertilisation occurs. For IVF, oocytes are incubated in vitro with fresh or, more typically, frozen‐thawed spermatozoa. Prior to addition, motile spermatozoa are isolated, typically using density gradient centrifugation. Gametes are co‐incubated for up to 24 hours after which the presumptive zygotes are washed and cultured for seven days in an appropriate medium to the blastocyst stage, when they are transferred to a recipient or cryopreserved for subsequent transfer.

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Sep 27, 2017 | Posted by in GENERAL | Comments Off on Assisted reproductive technologies used in domestic species

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