Embryo Morphokinetic Activity Evident in Short Videos of In Vitro Bovine Embryos

Round 1

Reviewer 1 Report

In this study, the authors evaluated the bovine embryo real time morphokinetic activity by 30s video recordings with a SMZ-1000 Stereo zoom microscope and TE-300 Nikon inverted microscope. They found that embryos with lower morphokinetic activity demonstrated higher hatching rates and developmental outcomes, suggesting measurement of embryo morphokinetic activity is a noninvasive and non-subjective method to evaluate embryo competency prior to transfer. It seems to be useful for improving the reproductive efficiency and profitability of IVF/ET of dairy cattle. However, I have some concerns about the results provided. The authors should address these issues.

Here are several issues that need to be addressed.

Major points

1.      In Abstract section, the author indicated that the morphokinetic activity of Day 6 morlulars were recorded based on 30s video, which is different form the description in Materials and Methods (Day 7.5, morulas and early blastocysts, Line 72-76). Similarly, the number of embryos recorded by SMZ-1000 Stereo zoom microscope and TE-300 Nikon inverted microscope is inconsistent in Abstract (103 embryos, Line 21) and Materials and Methods (88 embryos, Line 71). Please check the numbers carefully and make descriptions more clearly.

2.      I propose to simplify the Abstract since too much background are introduced.

3.      As linking bovine embryo morphokinetic activity to embryo development and viability is one of the focuses of this research, I suggest to introduce more about it to support the result of this paper (Line 58-63). In line with this, more discussion is needed in line 201-210, rather than listing a bunch of references.

4.      Line 149-150 Please provide more description about how embryo morphokinetic activities change over time in Figure 6.

5.      In Figure 8 Are the data expressed as mean±SD/SEM? If so, you’d better show the SD/SEM in the panel.

6.      L231-237 There seems to be no direct evidence to support this view. I propose to provide the relavent images or figures which show the morphokinetic activity characteristics between viable and inviable embryos.

7.      The Conclusion section needs to be simplified. For instance, “Reducing the transfer of…” (Line 258-260) is more appropriate to be shown in Introduction or Discussion sections instead of Conclusion section.

8.      The Reference list is imcomplete. For example, references “Henchion et al., 2017; Westhoek et al., 2017” (Line 34) , “Capper, 2007” (Line 40), “AETA, 2019” (Line 53), “Wells and Killingsworth, 2022” (Line 61, 63, 201), “Leese 1999” (Line 249) are missing in the list.

Minor points

1.      Line 68 The meaning of this sentence is confusing, please check it.

2.      Line 73 Provide some information or relavent links of International Embryo Transfer Society (IETS) standards.

3.      Figures 2, 3C, 4, 5, 6 Please indicate the magnification of these images in the caption, respectively.

4.      Figure 9 Lable the panel with statistical symbol since P<0.05 (Line 231).

Author Response

Dear Reviewer,

Thank you so much for your thoughtful review of our paper. It is our goal to generate quality research and publications and your insights are greatly appreciated.

 

In regard to your major points:

 

1. I apologize for the confusion. We began with 103 embryos but only 88 met the inclusion criteria of being a IETS Quality Grade 1 or 2. I have changed the abstract and the narrative to be consistent with sample size n=88 to be consistent.
2. I have modified the abstract to be more succinct with less background information. Additionally, I have looked up several other papers and abstracts published by this journal, Dairy, to make this abstract more cohesive.
   
   
3. I have added literature to support the use of morphokinetics in time lapse imaging systems to evaluate embryo health. The new lines 84-117 add information to support this study. In the discussion, lines 317-327 were also provided to add more support for morphokinetics research.
   
   
4. Figure 6 is an attempt to visually show the changes in the embryos which were included in this study. In the raw video (not pictured), a human could not detect any change at all. However, with the image subtraction, changes are perceptible. As someone who has studied embryos throughout my career, this was fascinating but it looks best in video format. For obvious reasons, I cannot put a video into a published paper, so I am challenged to put these results in a print format. Despite the fact that I can “see” changes in these videos, the changes are not obvious between viable and inviable embryos. Therefore, we need a computer to interpret the results and automatically calculate the differences between the changes in the viable and inviable embryos. I have added information to explain this both in the results section (lines 236-242) and discussion (331-341)
   
   
5. I have added error bars to figure 8.
   
   
6. I have attempted to add information in the discussion to support viable embryos have less change and a less variability (range) in change over time. This is definitely the strongest point I want to make in this paper and it is critical that I do.
   
   I have spent many hours trying to figure out how to portray this data impactfully. Prior to making the box and whisker plot, I had individual embryos graphed out frame by frame on a line graph. This showed the patter of their changes over time which was interesting (such as in Figure 7) but did not reflect the trends found in the entire group of viable or inviable embryos. I wanted to consolidate this data into a single, more concise statistic. I have added verbiage to explain the findings in this figure.
   
   
7. I have simplified the conclusion
   
   
8. I have double checked the references. Thank you so much for pointing out these flaws. I have added new references, as I added more peer reviewed literature to both the introduction and discussion section. I omitted the Westhoek citation because, while I was able to find the source embedded in the Henchion et al 2017 paper, the Westhoek is “no longer found”.
   
   

Minor Points

 

1. “380 bovine embryos (n=380) were produced from post-mortem harvest of oocytes (Simplot Animal Science; Kuna, ID)”.
   
   Simplot is a company in Idaho which is connected to a USDA inspected slaughterhouse. They have an animal science lab connected to the slaughterhouse which takes the ovaries of the slaughtered animals and dissects the oocytes from the ovaries. Because the animal was dead at the time which the oocytes were dissected from the ovaries, we called this “post mortem harvest of oocytes”. We purchased the oocytes from Simplot and had them sent to Texas A&M for the study.
   
   I have changed this sentence to “380 bovine embryos (n=380) were produced from post-mortem harvest of oocytes, a process which oocytes are dissected from the ovaries of slaughtered cattle (Simplot Animal Science; Kuna, ID)”.
   
   
2. I have added information about the IETS Developmental Stage and Quality Grading system.
   
   
3. I included the magnification of the images
   
   
4. I have labeled the statistical symbol.
   
   

Again, I want to thank you for your in-depth review of my paper. These comments will undoubtedly add value to this paper and scientific dissemination.  

Reviewer 2 Report

Dear authors,

When I first read the title I had great expectations about this research. Unfortunately, it was just my expectations. 

 

Although the introduction talks about the need of increasing animal protein production, and the genetic selection and brings some information about ET/IVF stats, little is cited about embryo evaluation or how could embryo evaluation could enhance ET/IVF results.

 

In the M&M section the authors describe when the embryos were evaluated and why, but they do not explain clearly how they evaluated objectively (in a non-subjective way) these embryos. The statistical analysis also seems to be inadequate. Once the experimental unity is the embryo, the analysis should be done with time-repeated measurements or something like a contingency analysis.

 

In the Results section, we can find more about the methods used. Lines 128 through line172 of Results are part of the methods, where the authors try to explain how the embryos were evaluated. Even so, the authors did not show any number of how the embryos were evaluated in a non-subjective way. The authors say about embryo activity and a difference in pixel number, but no number is shown to demonstrate that. The results found are that 36% of the evaluated embryos were considered viable which agrees with the ET/IVF results cited before. But, again, the author did not show any novelty.

 

In the discussion section, the authors just try to justify the technic and bring more about the method used. There is no discussion about their results and little is said about how they used it to evaluate the embryos or embryo activity and the comparison with other embryo evaluation methods.

 

This manuscript has great potential and so does the technic the authors tried to demonstrate. On the other hand, the authors did not show the results of embryo evaluation or the evaluation method in a non-subjective method as they promised. 

 

One of the references which are cited is not in the reference list: Wells and Killingsworth, 2022.

Author Response

Dear Reviewer,

 

Thank you so much for your thoughtful review of our paper. It is our goal to generate quality research and publications and your insights are greatly appreciated.

I am disappointed that we failed to meet the expectations which you had for this paper. However, I appreciate your enthusiasm for the title and am striving to write a quality paper which is worthy of your enthusiasm. I believe that your comments, as well as comments from the other reviews, is helping to meet these expectations.

In the introduction, I have added a paragraph to explain the methods used by nearly 100% of the industry to evaluate embryo health. These methods are a simple morphological evaluation of the microscope by the technician or examiner.

 

On the M&M section, I think you and I are on the same page except I did failed to communicate the non-subjective analysis appropriately. It is non-subjective because we use quantitative methods (counting pixel changes over time) to generate our results, rather than subjective methods like judging the embryos appearance. I have added more information for how the image subtraction allows us to count the pixel changes, as well as the mathematical methods we used to analyze the data and generate results. These changes can be found thorough out the paper to add clarity regarding the analysis.  

 

On Figure 6, I agree that it does not convey the analysis appropriately. I wanted to provide a visual image so readers can understand how the data looks after the videos are processed with graphic image processing and image subtraction. However, there is nothing that a human can interpret from this data other than the embryos do look a little different whereas they look identical had the methods not been used. The quantitative part of the analysis comes in when the computer actually counts the pixels which change.  I attempted to make this more clear.

 

I have expanded the discussion section to further discuss the results. I appreciate the comment in which you suggested to add information about evaluating embryos and comparing the method with other embryo evaluation techniques.  

 

We have double checked our references section.

 

Again, I want to thank you for your review and comments. We believe this manuscript has great potential and are working hard to develop research which can benefit many producers and veterinarians. We appreciate your comments and have tried to address them to strengthen the scientific merit of this manuscript.

Reviewer 3 Report

The manuscript describes the steps to follow to obtain the morphokinetic activity of embryos. The steps to be followed to obtain the short videos in in vitro embryos are clearly outlined. In addition to analyzing the embryo, all possible noises and artifacts that may interfere with the analysis are studied.

I propose to review the following paragraphs:

In line 10, the authors state "Dairy producers routinely utilize embryo transfer (ET) and in vitro fertilization (IVF). Please revise this statement, my area is top in dairy production and ET is minimal and occasional. With the phrase they have used they are assuming that the use of ET is widespread in many areas.

In line 42, “Genetic selection and advancement of dairy cattle can serve as a useful strategy to meeting food security, environmental and economic goals” If you state that genetic selection is going to be the solution to feed the world's population. Specify more how it will be achieved, will we continue selecting by production as it has been done so far or will new selection parameters be taken into account, please, make this paragraph more specific.

In line 45, “Producing genetically favorable animals” Please specify which animals are to be produced.

In line 211, “Lapse imaging systems are cost prohibitive are not practical for use in dairy” This sentence should be avoided, because making an essay that cannot be applied in practice is uninteresting. Therefore I suggest you rewrite it.

Author Response

Dear Reviewer,

 

Thank you so much for your thoughtful review of our paper. It is our goal to generate quality research and publications and your insights are greatly appreciated.

 

I agree with you about line 10. When I first said routinely, I meant that it is no longer an experimental procedure and it is often times used. I have modified the sentence to “Embryo transfer (ET) and in vitro fertilization (IVF) are increasing in use by dairy producers as a mean to breed their animals as these assisted reproductive techniques can optimize the genetics of the dairy breed or to enable “beef on dairy” programs to increase the profitability of the dairy”.

 

Thank you for challenging me to emphasize the importance of Genetic Selection. This is at the crux of why ET and IVF should be used in the first place. I have expanded the introduction through the new lines of 39-54 to cite literature about the advancements and achievements of Genetic Selection historically, as how it can improve animal health in the future.

 

“Producing genetically favorable animals”. As you and I can probably agree, producers definition of “genetically favorable” is subjective and dependent on the goals of the individual dairy. Some producers like purebred animals, others like crosses, etc. I thought a lot about how to described this and ultimately decided to scrap the term genetically favorable all together and replaced it with “Embryo transfer (ET) and in vitro fertilization (IVF) can maximize the impact of genetic selection because these breeding methods enable superior animals to have more genetic offspring in a single year than can be achieved in nature. ET and IVF reduces the generation interval required to generate genetic progression to quickly obtain demonstrable advantages of genetic selection.” I feel this is broader and more applicable to everyone.

 

“Time lapse imaging systems are cost prohibitive and are not practical for use in dairy” – this sentence you feel is uninteresting. I am struggling with how to best address this and aim to do so with the best of our ability.

 

From a personal perspective which should not be included in this manuscript as it is my personal opinion, I believe that the pregnancy rates of ET and IVF are quite shameful. They have not improved in the past 40 years, despite all of the advancements in assisted reproductive technologies. I am very active in the Clinical Human IVF industry as well as the livestock industry. Their pregnancy rates are actually lower than what we can accomplish in livestock, but they have so many technologies that the veterinarians and dairy producers do not have access to. Therefore, we really have to lean on the human IVF side of things to draw conclusions and hypothesis for why and how our technology can work.

 

The technology that we have developed to analyze embryo real time morphokinetic activity is novel. This year alone, we have had 4 patents issue. The problem with being novel, is there is not a precedent, and we have a lot more research to do to really explain why and how the technology works. The only precedent we have is time lapse imaging. Time lapse imaging is very different than the research described in this paper as it clearly allows you to study embryo cleavage. Our technology shows morphokinetic activity, but the 30 seconds does not allow us to observe a full cellular division cycle (which may be future research). Therefore, we have to figure out what we are seeing.

 

I made many references to time lapse imaging as it is really the foundation for embryo morphokinetics. However, it is not used for cattle embryology because it is not practical or affordable. If we can shorted the observation time of embryos required to generate objective information of embryo health, we have developed something both practical and affordable which can benefit the dairy industry. I want this paper to be interesting but I also want the paper to be factual. I have omitted the sentence you suggested and tried to replace it with factual, yet interesting statements.

 

Thank you again for the review. I appreciate your comments and time commitment to helping me strengthen the merits of this paper.

 

 

Round 2

Reviewer 1 Report

The revised manuscript is suitable for publication after modify the text editing.