Bovine advanced reproductive technology

This article explores and provides an overview of advanced reproductive technologies (ART) through the eyes of a large animal practitioner.

The author is a member of a large animal practice that used to provide an embryo transfer (ET) service, but does this service need to be rekindled? The author uses his own training programme and experiences from a trip to western Canada shadowing a leading ET practitioner for a day to consider a new set up.

Rapid advances have been made in the field of ART in all species, from the metamorphosis of the first artificial insemination in a bitch performed by Lazzaro Spallanzani (Spallanzani, 1780) to the first ET performed in a rabbit by Walter Heape (Heape, 1890). In the human field, the major breakthrough occurred in 1978 through in vitro fertilisation (IVF) with the birth of Louise Brown by Patrick Steptoe and Robert Edwards (Steptoe and Edwards, 1978). In the area of cattle, the first calf born from multiple ovulation and ET (MOET) was in 1950, with the first IVF calf born in 1982.

At present, no organisation represents those involved in ART in the UK. To this end, the major protagonists in the industry conducted a survey looking at the perceived future of ART and changes within the field. The author was interested in these and other statistics from the area of ART to answer his question.

The survey was facilitated by the Association of Embryo Technology in Europe (AETE), Vetoquinol and Eggtech. There were 90 respondents to the online survey, of these 65 were practising ET commercially.

A similar survey was undertaken at the AETE annual conference in Barcelona in 2016, with the chart below summarising the findings. This year, a more extensive survey was carried out.

An optimism appears to exist in the representatives for the future provision of ART services. At the 33rd annual meeting of the AETE in Bath the author attended a practitioner day, which was a primarily workshop-based day with presentations in ovum pick-up (OPU), embryo freezing, flushing and embryology. It proved to be a good overview of the field where practitioners could mix with specialists in the field.

Commercial activity in Europe

As part of the proceedings an annual record of commercial activity in Europe was presented (Mikkola, 2017). In 2016, a total of 20,783 in vivo embryo collections were made in Europe, resulting in the production of 128,877 viable embryos, with an average of 6.2 viable embryos per collection. The UK centres performed 959 collections, resulting in 4,786 viable embryos.

The greatest activity occurred in France: 6,260 collections with 34,896 viable embryos. Interestingly, other countries with the most activity included Belgium, Italy, the Netherlands and Russia. In vitro embryo production activity in 2016 throughout Europe was made up of 10,651 sessions, resulting in 94,407 oocytes and 18,879 embryos. A rapid increase has been seen in transvaginal OPU in the UK (Dawson, 2017a; 2017b).

From these technologies, and the alternative technique from collecting oocytes to produce embryos from the abattoir, a total of 116,403 in vivo embryo transfers were made in 2016, with the UK contributing to 1,309 transfers. Although the numbers of bovine embryos collected and transferred by MOET remains static, a huge increase was made in IVP.

Undoubtedly, the increase in OPU-IVP is caused by the advantages, specifically the ability to collect more oocytes and, therefore, produce embryos due to collections being undertaken as frequently as every week in a younger population of animals, including prepubutal animals. Advancement in the culture and fertilisation has also contributed to the increase. However, it does raise questions – and did at the conference – of the ethics of such technology, particularly in young animals where the OPU technique may be compromised due to their small size and difficulty in manipulation rectally.

UK projects

In the UK, through Innovate UK, the Technology Strategy Board, a collaborative project led by Paragon, XLVets, Cogent and The University of Nottingham, resulted in a company called Activf-ET. This established a network of OPU regionally based XLVet collection teams and a network of regionally based XLVet IVP transfer teams to provide a national service for UK farmers.

Huge inroads have been made in areas of preimplantation genetic diagnosis (PGD). A major advance is the ability to carry out genomic tests, which is the equivalent of a genetic fingerprint. Certain nucleotide sequences can be chosen to specific production and health traits – single nucleoid polymorphisms (SNPs).

SNPs are used in progeny testing, so only animals implanted and born with a correct genotype, resulting in less wastage and improvement of animal welfare if positive health traits/disease resistance are used in the selection. Sex can be determined by removal of a small proportion of cells from the blastocysts, done in the field by micro-manipulation by use of a blade. The sex is determined by amplification using PCR.

The use of blastocoel fluid compared to embryonic cells for sex determination has been investigated (Herrera et al, 2017). No significant differences were evident in post-warming survival rates, depending on the method of diagnosis used. Hatching rates and amplification rates for PCR were significantly higher in embryos that had blastocoel fluid collected.

Methods to retrieve embryos

MOET relies on the adoption of super-ovulation protocols. It relies on multiple injections. Modification of the protocols have been proposed by using slow-release follicle-stimulating hormone (FSH; Lindeberg et al, 2017), necessitating the use of only two injections.

Despite the number of embryos yielded not differing between the traditional protocol and the slow-release FSH protocol, the latter yielded a higher percentage of viable embryos and lower numbers of unfertilised embryos. Popov and Kosovsky (2017) mixed one of two polymers, poly-vinyl alcohol (PVA) and polyethylene glycol (PEG), to the FSH injections that were injected once as part of the protocol. These substances are thought to prolong the release of the FSH. A total of 86.4% of the PEG group responded to treatment compared to 74.5% of the PVA group, with a greater number of ovulations and collected embryos from this group. It appears PEG will be an aid to optimise super-ovulation responses.

Many different methods can be used to retrieve embryos. The method may be determined by personal presence or statuary rules, the most common is the requirement to use a closed system for those embryos destined for the export market. Other methods include the open system, which relies on sequential flushes with a syringe, and a variety of three-way flush systems, differing in sizes of the stylette – depending on the size and age of the donor.

The embryos are identified and sorted by a grading system of one to four, which relates to their likelihood of successful pregnancy and ability to be successfully frozen, and a stage classification of one to nine that relates to the embryo’s developmental stage in days. It is essential to implant the embryo into a uterine lumen at the same stage of the cycle to ensure the uterine milieu and embryo are best matched.

Eight embryos were implanted into synchronised recipients. Prior to implantation, all the possible recipients had their reproductive tracts rectally palpated. The size of the ovary and presence of a corpus luteum (CL) was noted. This information was also related to the strength and time of the reference heat. A strong reference heat seven days previous to the day of transfer was the optimum for selection of a recipient, combined with a large, palpable CL. Through extensive investigation no difference in outcome appeared, as measured by the number of pregnancies, in whether the embryo was transferred into the ipsilateral or the contralateral horn to the CL (McDonald, personal communication).

So many variables occur in the success of ET related to donor, embryo and recipient factors. The laboratory conditions are part of the attention to detail to achieve a successful pregnancy and the role of nutrition of the donor is paramount. From a cellular perspective, there is thought to be an affect on oviduct physiology. Bovine oviduct epithelial cells and zygotes were co-cultured, and non-esterified fatty acids (NEFAs) negatively affected embryonic development (Jordens et al, 2017).

Another group of researchers matured oocytes in physiological or elevated NEFA conditions by the increase of palmitic acid, the principle NEFA that is found to be elevated in the follicular fluid during negative energy balance. Elevated levels of palmitic acid at in vitro maturation had a significant negative effect on embryo elongation and interferon tau – a chemical messenger involved in the maternal recognition of pregnancy – was significantly reduced. This metabolic stress at the time of oocyte maturation has long-lasting effects on embryonic viability and likelihood of pregnancy.

Markers of oocyte and embryo viability are continually being investigated. A measure of cellular energy metabolism and mitochondrial function is oxygen consumption. A bio-analyser, which is practical and fast, was used to directly measure oxygen consumption rate – such analysis had no detrimental effect on the sequential development stage of the oocyte to fertilisation (Muller and Sturmey, 2017).

Ultrasonic findings of structures on the ovarian and their ability to predict outcomes may be of practical use to ET practitioners. The presence (nonechodense cavity; CLcav) or lack of cavity (compat CL; CLcom) in the CL was used as an indicator to the likelihood of pregnancy in heifers following ET into the ipsilateral horn. Pregnancy rate, as measured two months after ET, was 51.1% (CLcav) versus 34.7% (CLcom).

Interestingly, pregnancy rates were higher in those embryos placed in the left horn in the CLcav compared to the right horn (41.8% versus 59.5%). The findings were in the same direction with a CLcom presence – 30.4% versus 37.3%. If P4 concentrations were found to be greater than 10.88ng/ml, it was associated with a CL with a cavity (Jaskowski et al, 2017).

Frozen storage

Embryos not immediately transferred into recipients are stored in liquid nitrogen. Optimum cell viability is achieved by cooling slowly enough to allow water to move out of the cells before crystallisation, but not so slow as to prolong the cells to high external salt concentrations. Cryoprotectants, most commonly 1.5 moles per litre ethylene glycol with or without the addition of sucrose, are used.

The embryos are loaded into straws to ensure they stay in the middle. This is done by the use of short columns of air. Loaded embryos are placed in a freezer at −6°C, where a procedure called “seeding” is performed – the controlled initiation of ice formation. From this point, they are cooled at a rate of 0.5°C a minute to −32°C to −35°C, when they are stored in liquid nitrogen.

Looking around the animal kingdom, biological molecules are known to exist that are isolated from organisms inhabiting sub-freezing conditions. One group of molecules are the bacterial exopolysaccharides. They trigger ice nucleation, or inhibit ice nucleation and growth. Combined with vitrification (an alternative method of embryo preservation), the authors in one study concluded supplementation did not induce significant changes with the benefit seemingly being greater in prepubutal heifers than adult bovine oocytes (Arcarons et al, 2017).

Yellow straws are used for the freezing of embryos. Several loading protocols can be used. The straws are stored in a cane that is fully recorded in line with ET legislations.

Conclusion

ART in cattle represents an exciting field for the large animal practitioner to offer an enhanced service to what they do in routine fertility herd management. It is a field that is in rapid flux, most notably due to the increase in the use of OPU-IVP. The author sees a demand within his locality to be filled.

We will know in the near future whether a British Embryo Transfer Society will be formed to represent those professionals in the field of ET, offering a portal for continuing professional development and offer support to those embarking on a career in this exciting field.

Acknowledgements

Thanks from Brian Graham of Eggtech for his assistance in co-ordinating the sourcing of information that formed the basis of this article. Thanks also to Peter May of BCF, John Dawson of Embryonics and David Black for his tabulation of the International Embryo Technology Society survey results. Also, a thank you goes to Gordon McDonald of Emtech Genetics, Langley, Canada, and Pol Butte Holsteins, Coaldale, Canada.