Introduction

Cattle fertility continues to fall worldwide, and the global human population is rising, accompanied by an ever-increasing need for sustainable protein production. There is therefore a requirement to amplify and exploit existing genetics, and improve cattle production efficiencies.

Background

Embryo transfer was first documented by Walter Heape who, in 1890, introduced two Angora rabbit fertilised ova into a previously mated Belgian hare doe rabbit that carried them to full term along with four of her own offspring (Heape, 1891). The technique was refined and utilised in many species, becoming a commercial breeding tool in cattle by the 1970s; conventional multiple ovulation and embryo transfer technology (MOET) has since then undergone serial improvements (Christie, 2001), such as non surgical recovery and transfer, cryopreservation, sexing, and enhanced regimes for superovulation, and been applied extensively to increase the reproductive rate of cows of high genetic merit. 

Since the birth of the first calf after transfer of an embryo produced by in-vitro fertilisation (IVF) was reported (Brackett et al., 1982), advances have been made in the development of relatively simple methods for producing bovine embryos in-vitro as reviewed by Thompson (1996).

The development of a technique known as ovum pick-up (OPU), which facilitated the recovery of preovulatory oocytes from live donors (Pieterse et al., 1988) was largely responsible for the expansion in commercial application of IVF, increasing the number of transfers of IVF embryos carried out worldwide to 41,000 during 2000 (Thibier, 2001).

Since 2000 there has been little change in the number of transfers of in-vivo produced embryos, with numbers having plateaued at around 550,000 per year, while the number of transfers of in-vitro produced embryos has steadily increased to over 630,000 in 2014 (Perry, 2015). This has been largely driven by a steady, steep increase in the number of transfers in South America (especially with bos indicus cattle which seem more suited to these techniques) until 2014 when this slowed. While between 2013 and 2014 there has been a sudden 91% increase in the numbers of OPU/IVP embryos in North America, yet there has been little change in the numbers transferred in Europe (Perry, 2015).

Advantages of OPU/IVP

The techniques of Ovum Pickup (OPU) and In-Vitro Embryo Production (IVP) have significant advantages over both traditional breeding programmes or the use of conventional multiple ovulation embryo transfer (MOET);
• The process is non-surgical and requires minimal treatment other than epidural anaesthesia minimising clinical risks and pharmacological interventions
• OPU collections can be performed more frequently, so more oocytes can be collected in a shorter time period
• Oocytes can be collected from both juvenile heifers and pregnant donors during the first trimester, extending the number of potential embryos, which can be produced
• The technique can be used on animals with a range of reproductive disorders which might not otherwise be able to continue breeding
• Less semen is used per fertilisation so multiple donors can be fertilised with a single straw – saving money and utilising limited semen stocks
• A wide range of bulls can be used, due to the frequency of collection, giving greater scope for genetic improvement.

IVP to amplify genetic gain

The Food and Agriculture Organization of the United Nations (UN FAO) predicts that by 2050, global demand for animal protein will rise by 85% from the level required in 2008, driven by population growth reaching 9 billion, and increasing affluence in developing countries with a concurrent switch from largely vegetable based diets to meat and dairy products. To achieve this as well as to reduce the impact livestock farming might have on climate change, greenhouse gas (GHG) emissions and the demand for fresh water, there is a plethora of existing and on-going research. It is imperative that there is a greater amplification and distribution of these outputs. Cattle breeders recognise that it is important to generate as many offspring as possible from genetically superior or important animals, and although the widespread use of artificial insemination has led to very significant improvements in the genetic merit of cattle, there is a need to amplify female genetic lines as well. Efficient OPU/IVP has a role to play in the future.

The potential of IVP

The rate of genetic selection for quantitative traits can be increased by using advanced breeding technologies such as multiple ovulation embryo transfer (MOET) or ovum pickup and in-vitro fertilisation OPU/IVF (Hansen and Block, 2004). This is achieved by improving the accuracy and intensity of selection, in conjunction with reducing the generation interval. Further technological developments are likely to further enhance the technique. With the bovine genome now mapped to over 3 billion base pairs, or 22,000 genes (Larkin, 2011) our current understanding of genotypes will further enhance our ability to select parents to use in IVP programmes, and even of individual embryos prior to transfer. 

In most dairy systems, farmers require replacement heifers for their herd of a breeding that they desire, so bull calves are unwanted and wasteful, unless they can be of a cross that will be suitable for fattening. The advances in reliability of sexed semen for conventional artificial insemination (AI) have led to an interest in this being used in IVP systems. Sexing of embryos by biopsy of the 7 day embryo using a micro-blade has been used successfully commercially (Lacaze et al., 2008), usually to select female embryos but is time consuming on farm, and there is wastage of the male embryos. Use of sexed semen means that 98% of all embryos produced are of the desired gender, and hence the number of embryos suitable for transfer is effectively doubled.

With declining pregnancy rates in dairy cattle (Royal et al., 2000, Dobson et al., 2007), any solutions to improve fertility are attractive to farmers, and IVP has been suggested as a means of bypassing, or at least limiting some of the known problems. Although Sartori et al. (2006) found that embryo transfer did not improve overall pregnancy rates compared with artificial insemination in lactating dairy cows there was an apparent benefit of ET when single ovulating follicles were small. Yet (Demetrio et al., 2007) concluded that the transfer of fresh embryos did increase the probability of conception of lactating Holstein cows and suggested it was because ET can bypass the negative effects of increased milk production and low progesterone on the early embryo. This effect was most evident in highproducing cows and is thought to be associated with the increased dry matter intakes associated with higher milk production resulting in lower circulating progesterone, possibly as a result of increased hepatic blood flow and therefore faster metabolic clearance (Vasconcelos et al., 2003). They also demonstrated that high body temperature measured on day 7 had a negative effect on conception rates and embryonic retention. This links to findings of a review paper (Rutledge, 2001) which suggested that a major pathway is in the effects of maternal heat stress on the early cleavage stage embryo. Thus higher pregnancy rates can be obtained with transfer of late cleavage stage embryos. 

The IVP technique is also useful in individual infertile cows, where the causes are failure of ovulation or fallopian transport or where the uterine environment will not support a pregnancy (such as low grade endometritis) or in situations of early embryonic death (Hansen, 2006). An essential part of the establishment of pregnancy is the production of interferon (IFNT) by the elongating blastocyst. This is the basis behind the technique of implanting ‘support’ or ‘cowstopper’ embryos one week after an insemination; part of their effect is to create an additional source of IFNT and therefore improve maternal recognition of pregnancy. IVP embryos can be produced relatively cheaply from abattoir ovaries to act as ‘support’ embryos – unpublished results (J.S. Mullan, personal communication) suggest that around 70% of calves born assisted by this method are the dam’s own, the others either being the implanted ‘support’ embryo or twins.

Crossbreeding has been widely used in the beef industry for decades and there has been a trend towards more crossbreeding in the dairy herd recently, particularly to avoid dystocia problems with Holstein heifers (Olson et al., 2009). At least 10% heterosis can be expected for total genetic merit, mainly due to increased longevity and improvement of functional traits. There is however some evidence of recombinant loss, and it is critical for long-term crossbreeding that genetic gain within the parental breeds is not reduced (Sorensen et al., 2008). So, IVP is likely to have a place not only in amplifying purebred genetics, but also in creating F1 embryos for crossbreeding programmes. 

IVP as a basis for other technologies

Nuclear cloning and transgenesis are possible, but are currently limited largely by societal concerns, which have swung from initial debate about the potential cloning of humans to that of using human embryos to produce stem cells for research (Wadman, 2007) – however these techniques will also benefit from improved IVP technologies, and are likely to become a breeding tool of the future (Campbell et al., 2007).

Intracytoplasmic sperm injection (ICSI) is a technique where a single sperm cell, with acrosome and sperm membrane intact is directly injected into a metaphase II oocyte, and then cultured in-vitro. Although this is a technique now widely used in human assisted reproduction, it yields relatively poor blastocyst numbers and pregnancy results in livestock. However it may be a technique to be used for genetic salvage, transgenic production, or to improve efficiencies in IVP systems especially when using sexed semen, which is less robust than conventional semen (GarciaRosello et al., 2009).

The diagnosis of genetic traits and/or diseases in IVP embryos or preimplantation genetic diagnosis (PGD) has been well established in humans for more than 20. Biopsy of bovine embryos using a laser and micromanipulator to extract between 1 and 10 cells is actively being developed in the UK. Whole genome amplification (WGA) then precedes interrogation with single nucleotide polymorphism arrays (SNP chips). Any ‘gaps’ in the SNP calls can be ‘filled in’ by comparison to the genomic DNA of the parents (a technique known as karyomapping). Thus, rather than targeting individual genes for sequence specific detection of traits or diseases, karyomapping uses linkage information to map the inheritance of chromosome specific segments upon which those loci are contained, thus it relies on association with multiple linked markers, rather than identification of causative alleles (Handyside et al., 2010a, Handyside et al., 2010b). Cattle breeders are increasingly making use of SNP chips to assess the genetic merit of animals and these have proven to be more efficient and cheaper than traditional progeny testing, while increasing selection pressure and greatly expediting the introduction of superior genetics into the breeding herd.

Traits targeted using SNP chips include somatic cell count, daughter pregnancy rate, productive life, stillbirth rate and calving difficulty, and this technology has also opened possibilities for increased power to select for lower heritability traits. The advantage of SNP chip use is its multiplicity – it is potentially applicable for any trait; the major disadvantage is the cost (monetary and environmental) of taking pregnancies to term before testing of offspring and subsequent introduction to the breeding programme.

While against a background of increasing dairy cattle disease incidence (NAHMS 2007), Immunity™ has been developed by Semex. Currently this involves testing sires for two different types of immune response; cell-mediated and antibody-mediated, with those top 10% of responders being designated as Immunity™ and with a predicted heritability of these traits being 30%. Studies by Thompson-Crispi (2014) have now identified genomic markers associated with these immune responses, and Chromosome 23 has been identified as carrying the genes for the Bovine Histocompatability Complex (BoLA) which is closely associated with regulating immunity in cattle. Most recently it has also been shown that resistance to bovine Tb has a genetic component, and these developments will in the future allow genomic selection for these very beneficial traits.

By genomically interrogating the embryo itself, there will be a reduction in the production and rearing of unwanted offspring, which in turn reduces ethical concerns and decreases the carbon footprint of cattle production. Conversely an artificial insemination (AI) stud bull is worth many thousands of pounds and young candidate sires can be marketed from 2 years old if the SNP chip indicates them to be of sufficient genetic merit. Commercial farmers will more readily be able to select genetics that suit their farm or ambitions, such as disease resistance or health traits.

IVP as an experimental tool

If parameters from within an IVP program such as oocyte collection rates, fertilisation rates, cleavage rates or blastocyst rates, could be correlated with subsequent pregnancy, then in the future these markers of fertility would facilitate smaller and faster studies being undertaken. It is also anticipated that specific genes in the cumulus cells may be markers of oocyte quality and this would give an even quicker assessment of intervention effects. If crossover trials of small groups of animals were to be possible, this would greatly enhance the statistical power of intervention studies.

Conclusion

OPU and IVP are ever more widely used in other parts of the world, and are now available in the UK and Europe. The embryo genomic techniques are at an early stage of development with initial markets being elite breeders, but these are certain to become mainstream breeding tools for all cattle in the future.

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