Incidence, Reproductive Outcome, and Economic Impact of Reciprocal Translocations in the Domestic Pig

Round 1

Reviewer 1 Report

The manuscript of Lewis et al. titled by “Incidence, Reproductive Outcome, and Economic Impact of Reciprocal Translocations in the Domestic Pig” describes application of the reciprocal translocation (RT) screening program, instead of just karyotyping, using multiple FISH based methods for pig breeders. I am not familiar to application of the basic biological methods to the applied fields such as agriculture and animal husbandry. So, my questions are given from a view of basic Biology.

 

1. I was surprised by the high number of RT (and different types) incidences in the pig populations. Does it mean that they have been accumulated during many generations because undetected in the past? or were they produced during one or a few generations?
2. What about inversions or other rearrangements?Do they affect litter size or viability of pig without apparent abnormalities?
3. If not, why do you think RT frequently is found in pig populations?
4. What about RT on sex chromosomes? Does it happen and how affects to pig growing?

Author Response

We sincerely thank the reviewer for all the comments on this paper, which we have addressed as discussed below.

 

Point 1: I was surprised by the high number of RT (and different types) incidences in the pig populations. Does it mean that they have been accumulated during many generations because undetected in the past? or were they produced during one or a few generations?

Response 1: A few studies have found a significant prevalence of translocations in pigs (several papers from Ducos et al. and O’Connor et al., 2021). As seen in the present and previous works, RTs can affect a variety of different chromosomes. Interestingly, here most of the RTs were on chromosomes 1+2 and 9+18, but we know this occurred due to the selective screening of an affected boar and its offspring/relatives. So in essence, the RTs here discussed were mostly spread across just 2 generations (parent and offspring).

 

Point 2: What about inversions or other rearrangements? Do they affect litter size or viability of pig without apparent abnormalities?

Response 2: We do believe that any chromosomal rearrangement could lead to infertility issues affecting e.g. the litter size. In our experience, however, RTs are the prevalent kind of chromosomal rearrangement in pigs.

 

Point 3: If not, why do you think RT frequently is found in pig populations?

Response 3: This is an interesting question. In pigs, the effect of RTs is readily detected as a reduction in litter sizes. In other animals, such as cattle, the result will be more subtle (pregnancy or no pregnancy, where lack of pregnancy can likely be attributed to any number of other factors) and therefore difficult to detect. We propose that RTs are common in other species as well but underreported. RTs are, however, well described in pigs since this species farrows litters and significant economic interest has led to many screening programmes being in place today.

 

Point 4: What about RT on sex chromosomes? Does it happen and how affects to pig growing?

Response 4: We were able to check for any RT that would involve chromosome X and another autosomal chromosome. Unfortunately, we were not able to screen chromosome Y. Until now, we have not found any RTs involving chromosome X, but we believe this would affect fertility in the same way as RTs in autosomal chromosomes do.

Reviewer 2 Report

The manuscript Incidence, Reproductive Outcome, and Economic Impact of Reciprocal Translocations in the Domestic Pig by Lewis et al. provides new, improved data on the frequency of reciprocal translocations in pigs revealed by FISH using subtelomeric probes. This method is more sensitive than classical karyotyping and requires less training than classical karyotyping. In addition, the authors calculate the loss of pig breeders due to RTs. The article is well written, straightforward, contains valuable data, and therefore deserves to be published in DNA. 

I have the following comments and questions: 

1) I must admit that I was confused by the naming of best, average, and worst-case scenarios. I understand that the worst-case scenario has the highest commercial losses, but it is because the authors take into account all the other parameters (such as litter size and survival after weaning) better than other scenarios. I have no suggestion on how to improve this, I am just describing a reader's feeling. Perhaps it would be less confusing to show only the Average scenario (which the authors say is "based on the authors' understanding of the current pig market, portray a realistic average scenario"). Also, if the average scenario reflects the current pig market, I wonder where the data for the other two scenarios came from. Please specify in more detail the source of the data for these scenarios. 
Similarly, in Table 1, much of the data is from interviews. Please indicate how many interviews the information came from.

2) Please explain the abbreviation GGP in Table 1. 

3) In Table 2, the number of t(1;2) is much higher than the number of other RTs. Are these two chromosomes more prone to rearrangement? Please comment. 

4) Figure 1: I suggest showing not only the current image here, but also the latter picture from the same slide, but with the probe for chromosome 18. Now, it is a little confusing, since the legend describes the reciprocal translocation, but the reciprocity cannot be seen without the latter probe. 

5) Since the primary motivation of this work is to save breeders money, there should be some information about how much it costs to test for RTs. I assume that such a test is not yet commercially available, but can the authors give a qualified estimate of the price of the test? This would also emphasize the discrepancy between losses due to RTs and the cost of the test. I assume such information would be of utmost interest to readers.

Author Response

We sincerely thank the reviewer for all the comments on this paper, which we have addressed as discussed below.

 

Point 1: I must admit that I was confused by the naming of best, average, and worst-case scenarios. I understand that the worst-case scenario has the highest commercial losses, but it is because the authors take into account all the other parameters (such as litter size and survival after weaning) better than other scenarios. I have no suggestion on how to improve this, I am just describing a reader's feeling. Perhaps it would be less confusing to show only the Average scenario (which the authors say is "based on the authors' understanding of the current pig market, portray a realistic average scenario"). Also, if the average scenario reflects the current pig market, I wonder where the data for the other two scenarios came from. Please specify in more detail the source of the data for these scenarios. Similarly, in Table 1, much of the data is from interviews. Please indicate how many interviews the information came from.

Response 1: We conducted a total of 5 interviews across different major breeding companies. This information is now in the manuscript. We have also removed any mention of both worst- and best- case scenario analysis to improve clarity.

 

Point 2: Please explain the abbreviation GGP in Table 1.

Response 2: The definition of GGP was added to Table 1.

 

Point 3: In Table 2, the number of t(1;2) is much higher than the number of other RTs. Are these two chromosomes more prone to rearrangement? Please comment.

Response 3: The number displayed in table 2 is the total number of events. In this case specifically, we extensively screened the offspring of a boar carrying a t(1;2), resulting in a high number of RT detections for this specific error. To improve clarity, we have added a column to Table 2 reporting how many cases were de novo, as opposed to cases discovered in known relatives of an affected individual. We have also reviewed several figures across the manuscript as this change has prompted us to discover a de novo error that was previously missed.

 

Point 4: Figure 1: I suggest showing not only the current image here, but also the latter picture from the same slide, but with the probe for chromosome 18. Now, it is a little confusing, since the legend describes the reciprocal translocation, but the reciprocity cannot be seen without the latter probe.

Response 4: We agree it could be confusing. New images were added with the RT clearly shown on both chromosomes 9 and 18 using probes specific for each of them.

 

Point 5: Since the primary motivation of this work is to save breeders money, there should be some information about how much it costs to test for RTs. I assume that such a test is not yet commercially available, but can the authors give a qualified estimate of the price of the test? This would also emphasize the discrepancy between losses due to RTs and the cost of the test. I assume such information would be of utmost interest to readers.

Response 5: We agree with the reviewer on this point but we found it difficult to provide a reliable estimate for the cost of screening. This is likely to vary considerably between different centres, according to the methodology used, and depending on the number of samples screened. Instead, we tried to provide guidelines for breeders in section 3.3, where we made it clear that it would be economically beneficial for them to perform the screening as long as its cost is £328 per animal or less for karyotyping and up to £629 for FISH analysis.

 

Reviewer 3 Report

Line 27: Considering removing the full citation, and representing it with a number [1], as was done for each of the other citations.

Line 29: Consider adding a comma between “10 years” and “in line”.

Lines 47-48: An important distinction could be made that the 1:200 estimate is more indicative of a population subject to cytogenetic screening overtime, thus only de-novo cases are represented, while the 1:30 figure is more representative of a population not previously subject to screening, in which there is a breadth of repetitive cases (For example in Sánchez-Sánchez et al., 2019, they found 7 carriers of a rcp(1;6) and 11 carriers of a inv(4) in Spain), and calculated total prevalence rather than de-novo prevalence.  

Lines 68-72: The beginning of this sentence reads a little awkwardly, here is a suggested edit: “A potential solution is a novel device developed by our laboratory group and discussed in O’Connor et al (2017) (or “and colleagues”), where we reported the development of a new screening protocol using fluorescence in situ hybridisation (FISH) based on multiple hybridization of sub-telomeric probes [7,15].”

Lines 204-207: You indicate that the higher prevalence of rearrangements may be due to the use of FISH allowing the detection of cryptic rearrangements, however this is very little direct evidence that the higher prevalence of rearrangements found relative to the established de-novo rate is directly due to the use of FISH as a detection mechanism. For example, the prevalence of cryptic rearrangements amongst the 21 de-novo rearrangements which is suggested to explain the difference in prevalence is not reported. In addition you performed karyotyping on a subset of animals, however do not report the prevalence of de-novo rearrangements identified via karyotyping, and the prevalence identified via FISH? Was karyotyping performed on any of the samples where a RT was identified through FISH? I believe an argument could be made that you provide no evidence that any of the rearrangements identified through FISH would not also be identified through karyotyping which is important to the thesis. Providing this information would allow a better gauge of how many additional rearrangements may have been found that would not have been were traditional karyotyping used, and has implications for your conclusions regarding the effectiveness of FISH relative to karyotyping. Consider also that other factors such as sample size, and the length of time the herds have been subject to karyotyping may influence the prevalence of rearrangements, alongside the use of FISH as a detection mechanism. Overall I would be cautious making statements indicating that FISH will identify nearly 2x the RTs as karyotyping without sufficient backing evidence.

Line 17-18: From the abstract: “FISH based screening detected more errors than karyotyping” Consider whether you actually demonstrated this through your results (see above).

Line 252-253: This raises an interesting question of have the herds screened in this study previously been subject to cytogenetic screening (either through karyotyping or FISH). This sentence suggests that the herds screened had previously been subjected to cytogenetic screening, however no statement to this effect is provided in the methods section (although the high prevalence of duplicated rearrangements suggests that those herds had not been previously subjected to cytogenetic control). If these herds had previously been subject to systematic cytogenetic screening, this could be discussed and would strengthen the argument that the use of FISH identifies a greater proportion of RTs, alongside additional data indicating the proportion of RTs identified through karyotyping vs FISH (see above)

Line 198-200: You may wish to consider discussing the methodology by which you came to these economic impact figures in more depth. These numbers are heavily influenced by the number of matings, however that is not directly displayed in this section. You may wish to demonstrate the impact of a RT carrier on a single litter to provide a contextual number, then demonstrate how that number when scaled to reflect the average number of matings inflates to tens of thousands of pounds.

Line 230: Consider a change to “Incur significant losses in the pig industry.”

Lines 232-236: Consider providing a source that discusses the environmental impact of pigs, and how optimization of litter sizes may mitigate negative environmental affects. This is brought up in the introduction and discussion however no literature is cited. 

Lines 257-262: As above, consider adding additional evidence to support the conclusion that FISH is a superior detection method to karyotyping.

Author Response

We sincerely thank the reviewer for all the comments on this paper, which we have addressed as discussed below.

 

Point 1: Line 27: Considering removing the full citation, and representing it with a number [1], as was done for each of the other citations.

Response 1: We corrected the citation to [1].

 

Point 2: Line 29: Consider adding a comma between “10 years” and “in line”.

Response 2: Thank you for the correction. The comma was added.

 

Point 3: Lines 47-48: An important distinction could be made that the 1:200 estimate is more indicative of a population subject to cytogenetic screening overtime, thus only de-novo cases are represented, while the 1:30 figure is more representative of a population not previously subject to screening, in which there is a breadth of repetitive cases (For example in Sánchez-Sánchez et al., 2019, they found 7 carriers of a rcp(1;6) and 11 carriers of a inv(4) in Spain), and calculated total prevalence rather than de-novo prevalence.

Response 3: We believe the reviewer has pointed out an important distinction. We agree the current text could be misleading, and we have therefore updated this section to provide a more adequate evaluation of the two studies. The more realistic 1:200 figure was used for comparison elsewhere in the manuscript.

 

Point 4: Lines 68-72: The beginning of this sentence reads a little awkwardly, here is a suggested edit: “A potential solution is a novel device developed by our laboratory group and discussed in O’Connor et al (2017) (or “and colleagues”), where we reported the development of a new screening protocol using fluorescence in situ hybridisation (FISH) based on multiple hybridization of sub-telomeric probes [7,15].”

Response 4: Thank you for the suggestion. The sentence is now edited.

 

Point 5: Lines 204-207: You indicate that the higher prevalence of rearrangements may be due to the use of FISH allowing the detection of cryptic rearrangements, however this is very little direct evidence that the higher prevalence of rearrangements found relative to the established de-novo rate is directly due to the use of FISH as a detection mechanism. For example, the prevalence of cryptic rearrangements amongst the 21 de-novo rearrangements which is suggested to explain the difference in prevalence is not reported. In addition you performed karyotyping on a subset of animals, however do not report the prevalence of de-novo rearrangements identified via karyotyping, and the prevalence identified via FISH? Was karyotyping performed on any of the samples where a RT was identified through FISH? I believe an argument could be made that you provide no evidence that any of the rearrangements identified through FISH would not also be identified through karyotyping which is important to the thesis. Providing this information would allow a better gauge of how many additional rearrangements may have been found that would not have been were traditional karyotyping used, and has implications for your conclusions regarding the effectiveness of FISH relative to karyotyping. Consider also that other factors such as sample size, and the length of time the herds have been subject to karyotyping may influence the prevalence of rearrangements, alongside the use of FISH as a detection mechanism. Overall I would be cautious making statements indicating that FISH will identify nearly 2x the RTs as karyotyping without sufficient backing evidence.

Response 5: We thank the reviewer for raising this central point. This very kind of analysis formed a significant part of a previous paper from this group (O’Connor et al. 2021), which is referenced in the present work as well [7]. As such, we did not repeat the investigation here. We have reviewed the present manuscript to explicitly report this and to provide a more conservative comparison between Karyotyping and FISH analysis.

 

Point 6: Line 17-18: From the abstract: “FISH based screening detected more errors than karyotyping” Consider whether you actually demonstrated this through your results (see above).

Response 6: Line 17-18 was removed from the abstract.

 

Point 7: Line 252-253: This raises an interesting question of have the herds screened in this study previously been subject to cytogenetic screening (either through karyotyping or FISH). This sentence suggests that the herds screened had previously been subjected to cytogenetic screening, however no statement to this effect is provided in the methods section (although the high prevalence of duplicated rearrangements suggests that those herds had not been previously subjected to cytogenetic control). If these herds had previously been subject to systematic cytogenetic screening, this could be discussed and would strengthen the argument that the use of FISH identifies a greater proportion of RTs, alongside additional data indicating the proportion of RTs identified through karyotyping vs FISH (see above)

Response 7: We thank the reviewer for their interesting observations. The database contains information from several herds from across Europe, which are managed by different companies. Some of these companies have their own regular cytogenetic screening programmes and have used our services in addition or in place of theirs. Other herds would have been screened by ourselves exclusively. It is unfortunately quite difficult to dissect this information and complete an assessment in the way the reviewer suggests.

 

Point 8: Line 198-200: You may wish to consider discussing the methodology by which you came to these economic impact figures in more depth. These numbers are heavily influenced by the number of matings, however that is not directly displayed in this section. You may wish to demonstrate the impact of a RT carrier on a single litter to provide a contextual number, then demonstrate how that number when scaled to reflect the average number of matings inflates to tens of thousands of pounds.

Response 8: As the reviewer correctly explains, the number of matings of an affected boar is a key factor in each calculation. The full analysis with regards to how the number of matings was estimated, is presented in supplementary table S1. This has been better clarified in the text.

 

Point 9: Line 230: Consider a change to “Incur significant losses in the pig industry.”

Response 9: The sentence has been improved as suggested.

 

Point 10: Lines 232-236: Consider providing a source that discusses the environmental impact of pigs, and how optimization of litter sizes may mitigate negative environmental affects. This is brought up in the introduction and discussion however no literature is cited.

Response 10: A new reference [26] has been included.

 

Point 11: Lines 257-262: As above, consider adding additional evidence to support the conclusion that FISH is a superior detection method to karyotyping.

Response 11: This section has been edited in line with previous comments (point 5).

 

Reviewer 4 Report

Nice work, very important for practical purposes. Unfortunately, I do not fully agree with the conclusions. According to what I believe, the FISH method certainly allows a rapid diagnostic approach to the problem, but the karyotype, still used all over the world even in human diagnostics, is the basis for an accurate screening of all chromosomal anomalities. Therefore, I believe that the term "abandonment of karyotypic practices" is inappropriate, since the use of the karyotype, in a synergistic way, must and can be used in diagnostics with the FISH technique. So, I suggest, in my opinion, to review this last part.

Author Response

We sincerely thank the reviewer for their comment on this paper, which we have addressed as discussed below.

 

Point 1: Nice work, very important for practical purposes. Unfortunately, I do not fully agree with the conclusions. According to what I believe, the FISH method certainly allows a rapid diagnostic approach to the problem, but the karyotype, still used all over the world even in human diagnostics, is the basis for an accurate screening of all chromosomal abnormalities. Therefore, I believe that the term "abandonment of karyotypic practices" is inappropriate, since the use of the karyotype, in a synergistic way, must and can be used in diagnostics with the FISH technique. So, I suggest, in my opinion, to review this last part.

Response 1: We agree that an expertly performed karyotype analysis is still a powerful tool to diagnose not just RTs but many kinds of chromosomal errors including, for example, inversions. We have therefore reviewed the conclusions section to provide a more balanced argument.