C. Wells1, R. Killingsworth1,2
1EmGenisys, Houston TX 77002
2Shamrock Veterinary Hospital, Shamrock TX 79079
Introduction
Embryo transfer (ET), including in vivo derived (IVD) and in vitro produced (IVP), is a routine breeding strategy in cattle operations as it enables the perpetuation of individuals with high genetic merit, improves herd performance, decreases calving interval, and increases fertility in cows under heat stress and repeat breeder cows (Hasler, 2014; Putney et al., 1989, Ambrose et al., 1999; Drost et al., 1999). ET can also be used to help producers control the sex ratio of their herd, which is often referred to as the “most economical genetic trait” as heifer calves are valued at a premium in a dairy herd due to their ability to produce milk. Additionally, ET is an important tool to enable “beef-on-dairy” strategies to add profitability to the dairy industry as dairy cows giving birth to beef calves keep the milking herd lactating while producing offspring which can be sold into the beef marketing chain. Due to these benefits, ET is becoming an increasingly common breeding strategy for the cattle industry, with some companies reporting a 20% increase year-over-year in number of embryos created and transferred (Genus, 2019).
Unfortunately, success rates of ET are still low and limits the return on investment. Research from controlled studies report pregnancy rates of 70-80% from embryo transfer, but practitioners and producers in the industry encounter lower rates of these procedures on the farm or ranch (Youngs, 2011). The Canadian Embryo Transfer Association (CETA) reports pregnancy success of conventional fresh ET <57.3% successful with further reduced outcomes with IVF embryos (30%), which is comparable to pregnancy rates achieved in the United States (CETA, 2022). While causes of failed pregnancy are multi-factorial and can stem from embryonic, maternal, environmental stressors, or technician competence it is estimated that 20% of transferred embryos are likely non-viable at time of transfer and will never result in pregnancy (Prien et al., 2015, Vanroose et al., 2000; Diskin and Morris, 2008; Alfieri et al., 2019).
A contributing factor to this problem is the reliance on a subjective grading system dependent on a technician’s ability to grade embryos based on morphological characteristics. While practical and economical, the morphological analysis fails to account for many factors which contribute to an embryo’s health and viability including genetic defects, metabolic activity, acute stress, mitotic activity, and response to environmental factors. Therefore, emerging technologies which are non-invasive and non-subjective to evaluate embryo health can enable ET practitioners to select the healthiest embryos for transfer and subsequently improve pregnancy outcomes of IVF/ET in cattle.
The objective of this study was two-fold. 1.) To determine if embryo morphokinetic activity can be evaluated in short observatory periods and 2.) To determine if embryo morphokinetic activity can be used as an indicator of embryo health and viability.
Methods
In a pilot study, 94 (n=94) in vivo derived (IVD) embryos were flushed by a licensed veterinarian from beef cattle in the Texas panhandle region. <6 embryos were arranged in a line left to right, top to bottom in the field of view of the image capture. A 35 second video of each embryo was recorded under standard embryo culture with a Nikon CoolPix camera mounted in a trinocular stereo zoom scope. Once recorded, videos were filtered with the video motion magnification system platform Lambda Vue, which takes a standard video sequence as input, and applies special decomposition, followed by temporal filtering to the frames. The resulting signal is then amplified to reveal hidden information (Wu et al., 2012). Video motion magnification parameters included Reiz motion at 300x amplification with low cutoff set to 0.2 and high cutoff set to 0.7.
Embryo competency was determined by pregnancy outcomes as determined by rectal palpation, ultrasound, blood test or calving. To study the embryos morphokinetic changes over time, ImageJ software (national institute of Health) was used to record measurements of embryos area, diameter, and perivitelline space. Measurements were collected at 5s time intervals from 0-30s.
Changes in the embryo’s area, diameter and perivitelline space were recorded. Comparisons were made between embryos in each group (embryos videoed together) to determine if morphokinetic changes were unique and independent to each embryo (rather than artifacts inducing noise into the video data). Then, comparisons of the embryo’s diameter, area and perivitelline space measurements were made between embryos which established pregnancies and embryos which did not establish pregnancies.
Results
Raw, unfiltered videos portrayed each embryo as a static organism. However, once filtered with Video Motion Magnification, movement in both the inner cell mass and zona pellucida were visually evident. Protrusions, bulges, depressions, pulses, and changes in embryo shape were observed.
To determine if individual embryo activity could be measurable, measurements of embryos in a single video file were plotted on a line graph (Figure 3). Embryo mass diameter demonstrated low correlation, suggesting embryos in a single video file demonstrated unique, individual growth trends (Figure 3).
All embryos which met the International Embryo Transfer Society (IETS) morphological grading scale for Grade 1 or Grade 2 requirements were recorded and transferred as singletons into eligible recipient females. In the bovine model, data suggests video motion magnification software is effective at demonstrating changes in embryo morphokinetics in real-time and videos of embryos amplified with video motion magnification software demonstrate unique morphokinetic changes which are independent of the morphokinetic changes of other embryos in the video. Pregnancy results from this study suggest embryos with moderate morphokinetic activity establish pregnancies at a greater rate than embryos with a high or low level of morphokinetic activity (Figure 4).
It is generally accepted by domain experts that not all high-quality embryos will result in pregnancy, due to the number of factors which can cause a pregnancy to fail, including maternal, environmental, stress and nutritional factors. Therefore, one can assume not every embryo which did not establish a pregnancy was incompetent at time of transfer. Functionally, one can expect only 20% of grade 1 and 2 embryos are incompetent at time of transfer. Data from this study suggests embryos outside of 2 standard deviations of the mean can represent these incompetent embryos, thus suggesting this technology may be effective at identifying these 20% of low-quality embryos prior to transfer.
Additionally, it was found that the range of embryo movement within the subzonal space can be an important indicator of embryo competency (Figure 5).
These findings can build metrics to predict the likelihood of an individual embryo establishing a pregnancy (Figure 5). These outcomes allow veterinarians and producers to customize their breeding programs according to the success goals of their program and tolerance level (Figure 5).
Conclusion
This field study demonstrated embryo morphokinetic changes can be measured in short observatory periods. Furthermore, results suggest embryos with a high degree of change and embryos with a low degree of change in a 30 second duration do not establish pregnancies as frequently as embryos with a moderate degree of change. This can be used to build mathematical models correlating to an embryo’s developmental potential, competency, and viability to provide a more comprehensive evaluation of an embryo’s health, spanning beyond a morphological analysis.
While the biological nature of the morphokinetic activity observed in these embryos is not fully elucidated, researchers hypothesize analysis of embryo video data provides a non-invasive assessment of embryo metabolic activity as the findings of these studies are comparable to data reported in metabolic studies showing embryos with moderate metabolic activity are more viable because they do not have to waste energy attempting to survive, a theory coined as the “Quiet Embryo Hypothesis” (Leese, 2002). A real-time assessment of embryo metabolic activity as inferred from morphokinetic activity from video data can enable ET practitioners to identify stressed embryos prior to transfer. Reducing the transfer of low quality and non-viable embryos can improve pregnancy outcomes of IVF/ET up to 20% and maximize the potential of IVF/ET to achieve genetic gains.
Future studies will collect more data from IVD and IVP embryos from beef and dairy cattle to further understand the relationship between embryo morphokinetic activity and developmental potential. Additionally, utilization of computer vision and machine learning techniques can automate the video evaluation and improve performance for use in robust laboratory or field conditions.
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