Combining Embryo Transfer and Artificial Insemination to Achieve Twinning in Beef Cattle, and Effects of Different Twin Calf-Raising Methods on Neonatal Behavior and Growth

Round 1

Reviewer 1 Report

Summary

The present study was designed to improve the current methods and technologies in producing twin calves in a commercial beef cattle operation to increase the efficiency within the herd. Frozen-thawed embryos were transferred into the contralateral uterine horn 7 days after AI. The goal was to create two calves for one cow, produce one calf from AI and one from ET. Ultrasound was conducted between gestational day 45 and 53. From those 63 cows that received both AI+ET, 27 cows were pregnant with twins (42.9%) and 12 were pregnant with singletons (19%). Pregnancy loss was 20.5% from GD 53 to term for the AI+ET group. Overall the study was successful in demonstrating that the AI+ET protocol is capable of producing twins and these twins result in similar weights of singleton calves by d280. 

General Comments

1.      This whole paper should be ONE experiment. Experiment 1 contributes very little novel information to the scientific community and therefore should be removed as its own experiment and included within the materials and methods as but not its own experiment. 

2.      This entire experiment is nothing novel as it does not provide any new information to the industry, just aids in additional information regarding this specific topic (specifically adding information to the previous studies cited by the authors). 

3.      Line 127-128, the cows that did not show heat (<50% patch score), were they bred or removed from study?

4.      Please include a reference on why scoring a CL on a 1-3 basis has been shown to be beneficial and significant. 

5.      What was concluded with the late gestation progesterone that was mentioned in the bottom of the introduction? If this was collected, the results should be included. 

6.      How many (n=?) calves were in each TS and TT groups and how were these groups truly assigned? Were they assigned prior to calving or after? Were they randomly assigned or were the TT calves only left with dams that could support 2 calves?

7.      Table 1 needs clarification. What is the relevance of using groups and total for pregnancy risk? These are not 2 separate groups and data is confusing. 

8.      Please state if these were in vivo or in vitro produced embryos.

a.      Similar grade of embryos?

b.      Same dam/sire?

9.      Was “pregnancy loss” determined as “pregnant to open”, “twins to singleton”, or both?

10.  Why did the authors separate out the first and second born calves? Consider averaging them for a more clear understanding of TT to TS. Based on your results there was no significance between first and second born (table 4). 

11.  In the conclusion, it is important to address and make a small note of the concerns with twinning (dystocia, retained placentas, longer postpartum intervals , more management practices). 

12.  Some of the details included in the second paragraph of the materials & methods of experiment 1 were not necessary. It has much to do with experiment 2 and therefore, should be moved. 

13.  In experiment 1, you did not mention the number of observers for the behavioral data collected at parturition. There could be an inter-observer variability. How did you account for it?

Specific Changes

Line 43: Delete the second “when” in the sentence.

Line 61-63: Insufficient reference. Add sufficient references.

Line 71: Here it is stated conception rate and calving rate but later throughout the manuscript it is referred to pregnancy risk and calving risk. Remain consistent. 

Line 82: Change “wight” to “weight”

Line 106: Change “ever” to “every”

Line 166-169: provide reference for the score scale used at parturition.

Line 177: “progesterone variables” did not mention anything about blood samples being collected from cows. 

Line 197: define “latency to stand” and “latency to first suckle.”or provide the ethogram.

Line 310 and Table 4 does not agree with line 420-431 in regard to what calves were heavier at days 200 and 280.

Author Response

Reviewer 1

Summary

The present study was designed to improve the current methods and technologies in producing twin calves in a commercial beef cattle operation to increase the efficiency within the herd. Frozen-thawed embryos were transferred into the contralateral uterine horn 7 days after AI. The goal was to create two calves for one cow, produce one calf from AI and one from ET. Ultrasound was conducted between gestational day 45 and 53. From those 63 cows that received both AI+ET, 27 cows were pregnant with twins (42.9%) and 12 were pregnant with singletons (19%). Pregnancy loss was 20.5% from GD 53 to term for the AI+ET group. Overall the study was successful in demonstrating that the AI+ET protocol is capable of producing twins and these twins result in similar weights of singleton calves by d280. 

AU: On behalf of all authors, we would like to thank the reviewers for the comments and suggestions to improve our manuscript entitled "Combining Embryo Transfer and Artificial Insemination to Achieve Twinning in Beef Cattle, and Effects of Different Twin Calf-Raising Methods on Neonatal Behavior and Growth" and submitted to your journal for consideration. We carefully reviewed and answer all the comments and made revisions when applicable.

General Comments

1. This whole paper should be ONE experiment. Experiment 1 contributes very little novel information to the scientific community and therefore should be removed as its own experiment and included within the materials and methods as but not its own experiment. 

AU: sections that separated experiment 1 from 2 were removed and reworded (highlighted)

2. This entire experiment is nothing novel as it does not provide any new information to the industry, just aids in additional information regarding this specific topic (specifically adding information to the previous studies cited by the authors). 

AU: The authors of this paper respectfully disagree. Perhaps you are thinking of the Dairy cattle industry. Dairy producers are not likely willing to producing twins in their industry due to extensive studies reporting decrease in milk yield and complications of twin pregnancies in dairy cows. In fact, they may be more interested in current trends of producing beef/dairy offspring to add to the current market's deficit and increase profit of surplus calves. On the other hand, the U.S. beef industry utilizes very little reproductive technologies compared to the dairy industry, mainly because beef cow-calf operations are extensive and vary greatly in animal husbandry methods. The only other research group that we know of that used similar technology was from Tani et al. (2010). Tani et al. used Japanese dairy cows that have a different genetic from the cows utilized in our study. To the best of authors knowledge this is the first time AI+ET is performed to produce twins in beef cows in the US, using the contralateral method. In addition, Tani et al. (2010) outcomes were very different than ours with only 2 singletons and one set of twins. This particular project also demonstrates that twinning is possible in beef production, when many cow-calf operators would not consider the technology because twinning requires more intensive methods, such as bringing the dams up to a barn and observing dams and calves during the perinatal phase. While this may sound like a standard practice in the dairy industry, this is not a normal practice in most beef cow-calf operations. The target audience for this work is researchers and producers who are interested in increasing beef cattle efficiency, not dairy cattle. The animal husbandry and animal welfare methods applied to this work will be a good foundation to improve beef cattle efficiency. Currently, this work may be of interest, especially as beef cattle production has decreased and the cost and demand for beef has increased. This work is important to publish in a scientific journal so that there are credible resources for beef producers to refer.  

3. Line 127-128, the cows that did not show heat (<50% patch score), were they bred or removed from study?

AU: In beef cattle when reproductive technologies are used, the patches and GnRH method is commonly implemented. That aids in the cost of using an GnRH to all cows, instead only cows with inactivated patches will receive a GnRH injection to ensure ovulation. For this study, all cows were bred and kept enrolled in the study. The GnRH injection is used in synchronization protocols to ensure ovulation and that is what our rational to keep those cows enrolled even if they did not present estrous behavior. We added a line to clarify.

Line 143: Nevertheless, all cows were kept enrolled in the study for the next phase: artificial insemination.

4. Please include a reference on why scoring a CL on a 1-3 basis has been shown to be beneficial and significant. 

AU: There are varied results regarding scoring CL for embryo transfer, therefore, we included Alkan et al., (2020) and Spell et al., (2001) in this manuscript. However, CL scoring is the standard practice adopted by the veterinarian who performed the embryo transfer. This veterinarian is a cross-country genetics specialist who is an expert in cattle reproduction physiology. If you would like to reach out to this veterinarian for your own pursuits, he is a coauthor on this manuscript (Dr. Clay Breiner). The authors do recognize that this wonderful resource is a limitation for others to reproduce these methods, therefore we are as transparent   with the procedures performed as possible. Further explanation was added to the manuscript:

Lines 157-161: Regardless of conflicted results about the correlation between CL scores and pregnancy success, progesterone concentrations required for pregnancy maintenance have been associated with CL quality [22–24]. Scoring the CL is standard practice used by the fertility specialist veterinarian.

5. What was concluded with the late gestation progesterone that was mentioned in the bottom of the introduction? If this was collected, the results should be included. 

AU:  Thank you for pointing this out. The progesterone data were non-significant and did not end up adding any information to the final manuscript. This manuscript was originally pulled from the first author’s dissertation, which the committee wanted to see that data included in the dissertation. We removed references to progesterone data from the manuscript.

6. How many (n=?) calves were in each TS and TT groups and how were these groups truly assigned? Were they assigned prior to calving or after? Were they randomly assigned or were the TT calves only left with dams that could support 2 calves?

AU: Calf numbers for each group were added to the materials and methods section (TT=10; TS=18; S=11). Calves were not randomly assigned to groups TS and TT they were selected by the commercial herd manager to mimic common practices of the industry (external validity). Explanation was added:

Line 115-117: Calves were grafted to cows that lost their own claves at parturition (stillborn) or their calves died in the immediate post-partum period.

7. Table 1 needs clarification. What is the relevance of using groups and total for pregnancy risk? These are not 2 separate groups and data is confusing. 

AU: The authors feel that the information about pregnancy risk is very important for target audiences to understand the scope of inference for this research. Pregnancy risk is an indicator of fecundity within a herd, and other herds from other ranches (or research operations) will need this information. One of the groups only received Artificial insemination and the other received Artificial insemination and embryo transfer. This is to show descriptively what happen to all cows that were initially enrolled in the study.

8. Please state if these were in vivo or in vitro produced embryos.

AU: in vivo was added to line 164 for clarity.

1. Similar grade of embryos?

AU: Yes, all embryos were grade 1 or 2, mostly grade 1. Added to line 164.

1. Same dam/sire?

AU: No, we had 7 cow donors and 7 sires.

9. Was “pregnancy loss” determined as “pregnant to open”, “twins to singleton”, or both?

AU: Pregnancy loss was determined as pregnant to open and it was added to the materials and methods.

10. Why did the authors separate out the first and second born calves? Consider averaging them for a more clear understanding of TT to TS. Based on your results there was no significance between first and second born (table 4). 

AU: In other species, second born twins had a higher probability of complications at birth and higher perinatal mortality. To the best of the authors’ knowledge, this is the first-time twin beef calf performance is reported for first and second born twin calves. Even though there is not significant, the rates for this data are reported and now a reference point can be used for other researchers.

11. In the conclusion, it is important to address and make a small note of the concerns with twinning (dystocia, retained placentas, longer postpartum intervals, more management practices). 

AU: We have added a note to the conclusion based on previous points made during the discussion.

Lines 44-448: It is important to note that many factors need to be considered when implementing twinning in commercial herds, including time commitment of intensively management and increase in parturition assistance for cows birthing twins. For researchers who wish to use some of these technologies, the authors highly recommend that the more intensive animal husbandry methods be used (i.e. up close pens and continuous observation) for successful births.

12. Some of the details included in the second paragraph of the materials & methods of experiment 1 were not necessary. It has much to do with experiment 2 and therefore, should be moved. 

AU: The authors agree and have merged the two experiments and moved sections of the materials and methods accordingly.

13. In experiment 1, you did not mention the number of observers for the behavioral data collected at parturition. There could be an inter-observer variability. How did you account for it?

AU: All behaviors recorded at birth were recorded by a single observer (main author) who is an expert in applied ethology with over 10 years of experience. For nursing behaviors, it is mentioned that two trained observers were used. We have added the inter-observer reliability calculated using ICC.

Specific Changes

Line 43: Delete the second “when” in the sentence.

AU: It was deleted.

Line 61-63: Insufficient reference. Add sufficient references.

AU: More references were added.

Line 71: Here it is stated conception rate and calving rate but later throughout the manuscript it is referred to pregnancy risk and calving risk. Remain consistent. 

AU: It was updated to maintain consistency

Line 82: Change “wight” to “weight”

AU: Fixed

Line 106: Change “ever” to “every”

AU: Fixed

Line 166-169: provide reference for the score scale used at parturition.

AU: Reference added

Line 177: “progesterone variables” did not mention anything about blood samples being collected from cows. 

AU: Removed and explained above.

Line 197: define “latency to stand” and “latency to first suckle.”or provide the ethogram.

AU: Definition was added.

Line 310 and Table 4 does not agree with line 420-431 in regard to what calves were heavier at days 200 and 280.

AU: Reworded for clarity, thank you for catching that.

Lines 419-421: In the current study, twin-twin and twin-single calves were not different at 200d nor at 280 d of age.

Reviewer 2 Report

Dear Authors,

Nice work! Once if you have such a good paper please do something with the Figure. In its' current form it is not fitting into the journal.

My short recommendations are here:

L28:accuracy is twice in the sentence 

L34:light in bodyweight

L132: Artificial insemination and ovary diagnostics

L156-158: please specify if pregnancy diagnosis carried the examination of the embryonic heartbeat and the examination of the corpora lutea. Not a problem if not, but if yes, pls let the text contain it.

L256-257: AI and ET description is not necessary

L264-269: regarding to the twin pregnancy Se and Sp, I don't really see the data where these numbers are coming from. I think rewording these lines is necessary.

L296: I have contradictory feelings with the figure. On one hand I welcome that finally somebody creates good quality pictures with good quality content, on the other hand, even with serious magnification the true fetal presentations are not visible nor the colours of the calves. Please once if you have this figure that I highly recommend let it undergo some professional editing. 

L307, L309: lighter in bodyweight

English language quality is appropiate

Author Response

Reviewer 2

Dear Authors,

Nice work! Once if you have such a good paper please do something with the Figure. In its' current form it is not fitting into the journal.

AU: On behalf of all authors, we would like to thank the reviewers for the comments and suggestions to improve our manuscript entitled "Combining Embryo Transfer and Artificial Insemination to Achieve Twinning in Beef Cattle, and Effects of Different Twin Calf-Raising Methods on Neonatal Behavior and Growth" and submitted to your journal for consideration. We carefully reviewed and answer all the comments and made revisions when applicable.

My short recommendations are here:

L28:accuracy is twice in the sentence 

AU: We fixed this sentence. Thank you!

L34:light in bodyweight

AU: We fixed this sentence.

L132: Artificial insemination and ovary diagnostics

AU: We have changed the sections as recommended by reviewer 1.

L156-158: please specify if pregnancy diagnosis carried the examination of the embryonic heartbeat and the examination of the corpora lutea. Not a problem if not, but if yes, pls let the text contain it.

AU: We have not examined embryonic heartbeat and CL for pregnancy diagnosis. Pregnancy was determine when one or more embryos were visible during US scan, as stated in lines 174-175.

L256-257: AI and ET description is not necessary

AU: Thank you for the suggestion. We will maintain the description to abide with the journal guidance in order for the tables to stand alone.

L264-269: regarding to the twin pregnancy Se and Sp, I don't really see the data where these numbers are coming from. I think rewording these lines is necessary.

AU: Sensitivity and specificity were calculated using FREQ and NLMIXED from SAS as state in the statistical analysis portion. It was calculated based on ultrasound and twin or singleton delivery at time of calving. We have added more descriptive data to make it clear (see below)

Lines: 267-270: “When the ultrasound was used between 40 to 53 gestational days, detected 18 cows pregnant with twin calves of the 24 calving twins; and all 11 cows pregnant with singleton calves. The sensitivity to detect twin pregnancies was 75% (18/24), while the specificity was 100% (11/11) (Table 2).”

L296: I have contradictory feelings with the figure. On one hand I welcome that finally somebody creates good quality pictures with good quality content, on the other hand, even with serious magnification the true fetal presentations are not visible nor the colours of the calves. Please once if you have this figure that I highly recommend let it undergo some professional editing. 

AU: Thank you for the suggestion. We have removed the figure and if possible, will added as supplementary material.

L307, L309: lighter in bodyweight

AU: We fixed this sentence.

Reviewer 3 Report

There study was well designed, and the results would be very useful to improve the fertility of beef industry. One suggestion was provieded, authors should provied the calulation of cost and benefit by doing AT + ET, which would be important for readers.

Author Response

Reviewer 3

AU: On behalf of all authors, we would like to thank the reviewers for the comments and suggestions to improve our manuscript entitled "Combining Embryo Transfer and Artificial Insemination to Achieve Twinning in Beef Cattle, and Effects of Different Twin Calf-Raising Methods on Neonatal Behavior and Growth" and submitted to your journal for consideration. We carefully reviewed and answer all the comments and made revisions when applicable.

There study was well designed, and the results would be very useful to improve the fertility of beef industry. One suggestion was provieded, authors should provied the calulation of cost and benefit by doing AT + ET, which would be important for readers.

AU: The authors agree that cost will be an important factor for readers and producers; however, it was not the main objective of this study. Further studies will address the cost of this biotechnology by assessing this technology through the economic standpoint accounting for price of technology and relating to profit.

Reviewer 4 Report

The manuscript is clear, with an excellent experimental design and brings new results to the literature. However, I believe that it could be improved by better exploring why using artificial insemination combined with embryo transfer instead of just using the transfer of two embryos, with each side of both uterine horns having one of them inovulated. It is known that, in general, female recipients of embryos have low or lesser genetic merit compared to donors. Therefore, it would be important to make it clear why the recipient should have a pregnancy together with an embryo with the donor's genetics.

Furthermore, it would be interesting to discuss whether there were statistical differences between the calves with the donor's genetics and those of the recipient where the AI technique was used together with ET and which were born single.

Certainly the conclusion can be improved and although one of the statements were that although twins are born lighter compared to singles, there would be a greater amount of kg/cow production, this may be a mistaken view of the scenario. This is because there was no mention of the return to cyclicality of females who became pregnant. If the days without cycling were much longer or the durability of these females in serving as breeders, this could reduce the kg/cow produced. Therefore, I suggest that this statement is also related to birth (kg/cow/pregnancy).

Author Response

Reviewer 4

AU: On behalf of all authors, we would like to thank the reviewers for the comments and suggestions to improve our manuscript entitled "Combining Embryo Transfer and Artificial Insemination to Achieve Twinning in Beef Cattle, and Effects of Different Twin Calf-Raising Methods on Neonatal Behavior and Growth" and submitted to your journal for consideration. We carefully reviewed and answer all the comments and made revisions when applicable.

The manuscript is clear, with an excellent experimental design and brings new results to the literature. However, I believe that it could be improved by better exploring why using artificial insemination combined with embryo transfer instead of just using the transfer of two embryos, with each side of both uterine horns having one of them inovulated. It is known that, in general, female recipients of embryos have low or lesser genetic merit compared to donors. Therefore, it would be important to make it clear why the recipient should have a pregnancy together with an embryo with the donor's genetics.

Furthermore, it would be interesting to discuss whether there were statistical differences between the calves with the donor's genetics and those of the recipient where the AI technique was used together with ET and which were born single.

AU: The authors agree with the statement that recipients of embryos usually have less genetic merit and therefore are used in ET. In this study the main goal was to improve pregnancy risk in beef cattle and therefore we used this innovative method. We believe that the use of this technology could be apply to commercial herds, in hopes that embryos could be generated within our commercial herds and not only from cows with higher generic merit. The use of commercial herd cows to produce embryos could aid the economically to reduce price of ET; however, it was not the main goal of this project to address the economic impact of this technology. Further studies will evaluate the economical aspect of using AI and ET combined, addressing cost of embryos from commercial cows.
We were unable to genetically test the calves in this study, which would be a necessary tool to be able to calculate statistical differences.

Certainly the conclusion can be improved and although one of the statements were that although twins are born lighter compared to singles, there would be a greater amount of kg/cow production, this may be a mistaken view of the scenario. This is because there was no mention of the return to cyclicality of females who became pregnant. If the days without cycling were much longer or the durability of these females in serving as breeders, this could reduce the kg/cow produced. Therefore, I suggest that this statement is also related to birth (kg/cow/pregnancy).

AU: We have addressed the kg/cow/pregnancy. We were unable to follow cows for another cycle to investigate cyclicality of them. We hope to find new funding to follow the cows for longer and address gaps of knowledge not addressed in this initial study.

Round 2

Reviewer 1 Report

The authors have addressed a significant amount of comments associated with the review that make it more acceptable for publication. A few responses to comments from the authors to reviewer requests:

General Comments, question #2: The response to this comment leaves this review concerned that the authors failed to read some of the citations that they provided in their manuscript. While I believe this manuscript provides additional data to support twinning in beef cattle, the experiment was not novel. The authors cited Dahlen et al., (2012), which provided two methods of twinning in beef cattle and also cited other research associated with beef cattle. Outside of placing an embryo in the contralateral horn of the CL, the authors do not provide anything that is new or novel. Calf performance of the offspring adds additional contributions to the significant amount of data previously provided through other groups that have conducted twinning work in beef cattle (see Citation #19 - Echternkamp et al., 2007). 

General Comments, question #4: If the authors read the Spell et al., (2001) manuscript they would have noted that Cross Country Genetics was involved in that study that concluded that the scoring (1 to 3) system did not support pregnancy rates.  The conclusion of that study were: "There were no significant differences in mean CL diameter, luteal volume or plasma progesterone concentration among recipients that did or did not become pregnant after embryo transfer. We conclude that suitability of a potential embryo transfer recipient is determined by observed estrus and a palpable corpus luteum, regardless of size or quality."

Certainly this does not mean that there is a concern or a reflection on the practices of embryo transfer by Cross Country Genetics who aim to provide the best services to their customers. We are aware of the scoring system and they are respected as leaders, but the science does not support the scoring system. Therefore, this citation does not support the statement!

General Comments, question #5: Authors should not consider eliminating 'non-significant' results from manuscripts.  Non-significant data is also important in publications to ensure that researchers do not replicate the work.

Overall the authors sufficiently addressed many of the requests form this reviewer.  However, there is a significant concern that the authors claim the work to be novel for twinning in beef, in spite of the significant amount of work already published in beef cattle cattle associated with beef cattle. It is a little disheartening that the authors cite a significant amount of research conducted for decades, on tens of thousands of beef cattle at the USDA-MARC and USDA-Ft Keogh (i.e., Echternkamp and Bellows). I recommend significant language correction to ensure that the work already published is given the credit it deserves to demonstrate that the authors are awrae that much of this work has already been done.

Author Response

The authors have addressed a significant amount of comments associated with the review that make it more acceptable for publication. A few responses to comments from the authors to reviewer requests:

AU: The authors would like to take the reviewer for the second round of revisions. All your comments were considered and addressed to improve our manuscript. We would like to thank for challenging our statements as a way to significantly improve our work.

General Comments, question #2: The response to this comment leaves this review concerned that the authors failed to read some of the citations that they provided in their manuscript. While I believe this manuscript provides additional data to support twinning in beef cattle, the experiment was not novel. The authors cited Dahlen et al., (2012), which provided two methods of twinning in beef cattle and also cited other research associated with beef cattle. Outside of placing an embryo in the contralateral horn of the CL, the authors do not provide anything that is new or novel. Calf performance of the offspring adds additional contributions to the significant amount of data previously provided through other groups that have conducted twinning work in beef cattle (see Citation #19 - Echternkamp et al., 2007). 

AU: Addressing both this comment and the overall final comment: We made significant improvement to our introduction to better describe how the extensive and important previous work has been used to design our study. Our goal was to reopen the discussion of twinning in beef cattle. We recognize all the amount of work that has been done in this topic before, including genetic selection, hormonal superovulation, embryo transfer, artificial insemination and embryo transfer combined. We made sure to correct our language and demonstrate that the aim of our project was to use the contralateral embryo transfer technique with basis on previous research by applying to a U.S. commercial cow calf herd. It was never the goal of the authors to minimize the importance of the previous work, in fact our work was only possible based on previous research. We understand the constrains and challenges that comes with producing twins in cattle and we are thankful that because of previous research we can keep moving forward to improve cattle efficiency.

Line 56: Biotechnologies applied to production of twins in cattle were extensively studied and reported in literature.

Line 60: Genetic selection was done in a long-term program by selecting dams with multiple ovulations, and testing sires progeny increased twinning frequency [6,9,16]. This program was ground breaking, and due to its increase in ovulation the frequency of fraternal twins and triplets birth reached an annual rate of 60%[15]

Line 66: When genetic selection is considered unilateral multiple ovulations (same ovary), or bilateral ovulations (both ovaries) can occur.  Despite the biotechnology chosen to produce twins (e.g. genetic selection, bilateral embryo transfer) researchers reported an increased survival rate, gestation length, and birth weight when twins were gestated in bilateral uterine horns [12,20,21]

Line 74: The reasoning for using the contralateral technique is not clear in their report; however, improved fetal and performance outcomes of embryos gestated in different uterine horns could be involved.

Line 79: In views of extensive previous research done in cattle twinning, and the need to reopen this discussion aiming to improve efficiency of U.S. beef cattle, we developed a twinning trial in a commercial U.S. cow-calf operation.

Line 84: Furthermore, once twins are born in commercial cow-calf operations it is common that one of the calves gets grafted to another cow in hopes to improve their growth rate and maintain that cow in the herd. Therefore, our secondary hypothesis is that twin calves grafted will present an improved growth rate compared to twin calves that were both kept with their dam. To the best of the authors knowledge, little research data is available comparing behaviors between these two groups of calves. 

General Comments, question #4: If the authors read the Spell et al., (2001) manuscript they would have noted that Cross Country Genetics was involved in that study that concluded that the scoring (1 to 3) system did not support pregnancy rates.  The conclusion of that study were: "There were no significant differences in mean CL diameter, luteal volume or plasma progesterone concentration among recipients that did or did not become pregnant after embryo transfer. We conclude that suitability of a potential embryo transfer recipient is determined by observed estrus and a palpable corpus luteum, regardless of size or quality."

AU: We have corrected this statement to better explain why we collected that information. It was not our main goal to test between CL scoring, but we thought it was important to be transparent of our exact technique.

Line 172: Even though, using the CL scores to determine recipients present conflicted results in the literature,  the suitability of recipients is mainly determined by estrus and palpable CL [24–26]. Nevertheless, we recorded the CL scores as it was standard practice used by the fertility specialist veterinarian to perform or not the embryo transfer.

Certainly this does not mean that there is a concern or a reflection on the practices of embryo transfer by Cross Country Genetics who aim to provide the best services to their customers. We are aware of the scoring system and they are respected as leaders, but the science does not support the scoring system. Therefore, this citation does not support the statement!

General Comments, question #5: Authors should not consider eliminating 'non-significant' results from manuscripts.  Non-significant data is also important in publications to ensure that researchers do not replicate the work.

AU: We understand your concern and agree with your statement. Our samples were collected at a fixed time, unfortunately our parturition due days differed, and we did not have enough samples to account for that difference in our model. In addition, we added 3 cows with natural twins that were not collected.

Overall the authors sufficiently addressed many of the requests form this reviewer.  However, there is a significant concern that the authors claim the work to be novel for twinning in beef, in spite of the significant amount of work already published in beef cattle cattle associated with beef cattle. It is a little disheartening that the authors cite a significant amount of research conducted for decades, on tens of thousands of beef cattle at the USDA-MARC and USDA-Ft Keogh (i.e., Echternkamp and Bellows). I recommend significant language correction to ensure that the work already published is given the credit it deserves to demonstrate that the authors are awrae that much of this work has already been done.

AU: We have addressed this concern with comments for questions #2. When we mentioned that little was done in beef cattle, it was referent to the biotechnology we have used (ET+AI) and not all the work done in beef cattle twinning. We are aware and we consider extremely important all the work previously done by USDA-MARC and USDA-Ft Keogh (i.e., Echternkamp and Bellows). Without that work many questions about twining and viability of twin calves would remain answered. Once again, the authors would like to thank you for your comprehensively review of our manuscript. It is clear to us the amount of improvement done from the first draft to the last and this was only possible with the help of you and other reviewers. We hope our manuscript reopens the discussion for twinning in selected beef cattle operations.

Line 459: The present study follows extensive cattle twinning work done in previous research and reopens the discussion of a possible efficiency improvement during the cow-calf phase in U.S. beef cattle operations.