Changes in the Fatty Acid Composition of Vegetable Oils Affect the Feeding Behavior, Feed Preference, and Thermoregulatory Responses of Sheep

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The introduction begins by suggesting that ruminant production may benefit from the use of fats and oils to improve efficiency, then moves to the effect of heat stress in semi-arid environments. However, the claim that increased fatty acid intake can relieve heat stress is not very convincing. Additional; reports of studies in this area might be useful, if there is good evidence that fat intake assists by reducing heat stress.

Then it is claimed that cashew nut shell liquid (CNSL) can maximise the benefits of feeding lipids, again with little or no convincing explanation of why this is the case. In lines 93-95 “positive effects” are claimed, but the effects appear to be ”no change”, so it is not clear why CNSL is beneficial.

The method of study ensured that the only difference between the diets was the source of fatty acids. This provided diets with the same energy value, and most nutrient measures were identical, with the only important difference being the fatty acid type, ranging from high saturated fatty acids, through high monounsaturated, to high polyunsaturated fatty acids. This was a good method to ensure that the only difference between the diets was the type of fatty acid.

However, the authors keep emphasising that the diets contained CNSL, implying that the effects of the fatty acids depend on the presence of CNSL. Therefore, it is difficult to know whether the results are applicable to sheep on other diets or only to sheep on diets containing CNSL, which would limit the value and relevance of this study.

Since the study puts some emphasis on feed efficiency and mentions palatability, it is surprising that the results for feed eaten per day are not clearly presented. Or feed not eaten per day could be given, which would be relevant to the particle distribution results. The sheep were weighed at the beginning of the trial, but there is no mention of weight at the end, to calculate weight gain on these diets. If the weight was recorded at the end, the weights and weight gain should be reported here.

The results are well presented, although there are so many possible treatment effects measured that it would be inevitable that some would reach p<0.05 by chance. Therefore, it is difficult to be certain that results such as boluses ruminated/day are meaningful at p = 0.044, since no other behavioural effects were significant. Similarly, cholesterol at p=0.05 may not be a real effect., with only two of the five treatments sufficiently different.

Without knowing how much feed was left uneaten each day it is difficult to assess the relevance of the particle size distribution. Are large lumps left because the sheep failed to chew on the feed? Are many small particles left because the sheep repeatedly scraped at the pellets in the feed container, trying to get more? Or is the pellet size related to the type of fat that is holding the pellets together? Possibly fats with higher melting points could provide a more solid lump than the fats with low viscosity. A report of feed eaten per day and variation in feed intake would be useful in understanding these results.

Overall, the study appears to be a valid method of comparison of the effect of different fats in a diet containing CNSL, but whether it has wider relevance to those not using CNSL is not clear. A greater attention to setting out the relevance of fats and CNSL in the Introduction might improve the relevance to a wider audience. Results for weight gain and % of the diet eaten each day should be reported if these values are known.

Comments on the Quality of English Language

The article is generally well written, with only occasional incorrect word usage, e.g. "manly factors" and "Extrato etereo" in Table 3.

Author Response

Dear Reviewer #1

We would like to thank the associate editor and reviewers for the suggestions made. We appreciate the attention, time and reviewers' contributions to the improvement of this version of the manuscript. We have modified the manuscript according to the suggestions and below we present point-by-point answers to the questions. All corrections were addressed, as shown below and in the attached file. Answers to the questions are provided below. All the manuscript changes have been highlighted in red font. 

Sincerely yours,

Dr. Leilson Bezerra

Point by point answer to reviewers:

The introduction begins by suggesting that ruminant production may benefit from the use of fats and oils to improve efficiency, then moves to the effect of heat stress in semi-arid environments. However, the claim that increased fatty acid intake can relieve heat stress is not very convincing. Additional; reports of studies in this area might be useful, if there is good evidence that fat intake assists by reducing heat stress.

Response = Thank you for this clarification. The relationship between increased fatty acid intake and the fact that the increase in the energy density of the diet can explain heat stress. Fat is a concentrated energy source, and energy production is linked to the length of the carbon chain of each fatty acid, as well as the presence of double bonds in unsaturated fatty acids. When sheep ingest fat, it is metabolized and generates energy, part of which is released as heat. Animals that eat diets with higher energy levels result in reduced feed intake and fermentation heat, consequently losing less energy and improving feed conversion of the animals, and this is even more important in tropical environments where temperatures are quite high. Thus, adding fat to the diets of growing and finishing sheep generally improves feed efficiency, increasing weight gain and influencing carcass measurements. The Introduction section has improved this, and we have added the References below.

https://doi.org/10.1016/j.aninu.2020.05.008

https://doi.org/10.3390/ani9070400

https://doi.org/10.1016/j.anifeedsci.2017.02.005 https://doi.org/10.1093/jn/124.suppl_8.1377S.

Then it is claimed that cashew nut shell liquid (CNSL) can maximise the benefits of feeding lipids, again with little or no convincing explanation of why this is the case. In lines 93-95 “positive effects” are claimed, but the effects appear to be ”no change”, so it is not clear why CNSL is beneficial.

Response = The CNSL is not an oil similar to other vegetable oils. It is rich in phenolic compounds, such as anacardic acid, cardanol, and cardol, and these compounds have antioxidant, antimicrobial, and anti-inflammatory properties. Furthermore, in their free form, these compounds can be toxic, but at the same time, they promote much more biohydrogenation than other vegetable oils. So, " In vitro studies conducted by Watanabe et al. (2010) demonstrated that the application of CNSL reduced methane production by 70% and increased propionate production by 44%. This could also contribute to thermal comfort, as metabolic production is a factor in thermal stress. Shinkai et al. (2012) observed a rise in rumen propionate production and a 38% reduction in methane emissions in cows fed a diet of hay and concentrate (60:40 ratio) with the inclusion of 4 g of CNSL per 100 kg of body weight. " This was added in the text.

L281: The method of study ensured that the only difference between the diets was the source of fatty acids. This provided diets with the same energy value, and most nutrient measures were identical, with the only important difference being the fatty acid type, ranging from high saturated fatty acids, through high monounsaturated, to high polyunsaturated fatty acids. This was a good method to ensure that the only difference between the diets was the type of fatty acid.

Response = Thank you for recognizing our efforts.

However, the authors keep emphasizing that the diets contained CNSL, implying that the effects of the fatty acids depend on the presence of CNSL. Therefore, it is difficult to know whether the results are applicable to sheep on other diets or only to sheep on diets containing CNSL, which would limit the value and relevance of this study.

Response =Thanks. Since the effects of CNSL are known and in moderate quantities, such as the one we used, the fermentative behavior will not be so different from that in other diets that may or may not use fats. This is because we ensured that the amount of ethereal extract was not extrapolated.

Without knowing how much feed was left uneaten each day it is difficult to assess the relevance of the particle size distribution.

Response =Thanks. We offered an average of 3.1 kg of natural matter, and the animals left an average of 0.45 kg, approximately 15% of leftovers (Table 1), which varied according to each animal. Daily weighing of the offered feed and leftovers allowed for daily adjustments, and weighing the animals every two weeks allowed for weekly adjustments of the offer. Below is a table with the average by treatment of offer and leftovers so that the Reviewer can better visualize.

Table. Offer of diets, and consumption in kg and relationship between leftovers and supply (%).

Are large lumps left because the sheep failed to chew on the feed?

Response =Thanks. The diet did not contain large pieces; the bulk was made of sorghum silage processed to contain particles of an average size of 3 cm (this was added in text). The animals left larger particles because they could select and eat more of the bran concentrate. This demonstrates that they did not reject the combination of CNSL with other vegetable oils. This can be observed by evaluating the distribution of particles in the diets (Table 2) because when we compare it with Table 3 in the article, we can see that there was indeed a selection by the animals since, for example, 16% of the larger particles were offered, through the 19 mm sieve. This percentage increased during intake through the sieves 12 and 24 hours after the offer, with an average of 19% and 26%, respectively. This demonstrates that, even though the diet was well homogenized at the time of the offer, the animals could select the smaller particles from the larger ones.

Are many small particles left because the sheep repeatedly scraped at the pellets in the feed container, trying to get more? Or is the pellet size related to the type of fat that is holding the pellets together? Possibly fats with higher melting points could provide a more solid lump than the fats with low viscosity. A report of feed eaten per day and variation in feed intake would be useful in understanding these results.

Response =Thanks. The feed was not pelleted but rather in the form of bran. The animals did not leave many small particles; on the contrary, they consumed more small particles than large ones. To prepare the concentrate (CNSL and oils soybeans, corn, and mineral mixture), the CNSL was mixed with part of the corn; the same happened with the oil, and then the rest of the corn, soybeans, and mineral mixture was incorporated into the mixture and homogenized for 10 minutes in a Y-type mixer. All the oils had the same consistency, resulting in concentrates with the same physical appearance, changing only in chemical composition.

Since the study puts some emphasis on feed efficiency and mentions palatability, it is surprising that the results for feed eaten per day are not clearly presented. Or feed not eaten per day could be given, which would be relevant to the particle distribution results. The sheep were weighed at the beginning of the trial, but there is no mention of weight at the end, to calculate weight gain on these diets. If the weight was recorded at the end, the weights and weight gain should be reported here.

Response = Thanks. We agreed with the reviewer and applied different statistical analyzes as contrasts, but it was still not enough to point out the significance. Applying a less rigorous test could be an alternative, but this has not been done.

Overall, the study appears to be a valid method of comparison of the effect of different fats in a diet containing CNSL, but whether it has wider relevance to those not using CNSL is not clear. A greater attention to setting out the relevance of fats and CNSL in the Introduction might improve the relevance to a wider audience. Results for weight gain and % of the diet eaten each day should be reported if these values are known..

Response = Thanks. We agreed with the reviewer and applied different statistical analyzes as contrasts, but it was still not enough to point out the significance. Applying a less rigorous test could be an alternative, but this has not been done.

Overall, the study appears to be a valid method of comparison of the effect of different fats in a diet containing CNSL, but whether it has wider relevance to those not using CNSL is not clear. A greater attention to setting out the relevance of fats and CNSL in the Introduction might improve the relevance to a wider audience. Results for weight gain and % of the diet eaten each day should be reported if these values are known.

Response = Thanks. Thank you for all your consideration to improve the paper. We have improved the introduction section to make the manuscript more transparent and more objective to the public. We have inserted the animals' weight data and the % of the diet eaten each day.

The article is generally well written, with only occasional incorrect word usage, e.g. "manly factors" and "Extrato etereo" in Table 3.

Response = Thanks. We apologize for these minor errors. We sent the manuscript to a native English Editor for correction.

Sincerely yours,

Dr. Leilson Bezerra

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, the author provide a new insight on vegetable oils on sheep. It is intersting. However, the desigh and result provided in this manuscript was not clear. The writting need improved. 

Table 1~4: Provided ADF, GE and OM. Why not set a group without oils? 

The author weight the sheep, however, i am very double the data of growth performance?

Table 2: "-" means not detect?

Table 4:  Letter need superscript. Ues the abbre in the item.

Figure 1:  SEM.

 Line 167 uL

Suggest provide the rumen fermentation parameters.

Comments on the Quality of English Language

Need improve!

Author Response

Dear Reviewer #2

We would like to thank the associate editor and reviewers for the suggestions made. We appreciate the attention, time and reviewers' contributions to the improvement of this version of the manuscript. We have modified the manuscript according to the suggestions and below we present point-by-point answers to the questions. All corrections were addressed, as shown below and in the attached file. Answers to the questions are provided below. All the manuscript changes have been highlighted in red. 

Point by point answer to reviewers:

In this manuscript, the author provide a new insight on vegetable oils on sheep. It is intersting. However, the desigh and result provided in this manuscript was not clear. The writting need improved.

Response = We appreciate the attention, time and reviewers' contributions to the improvement of this version of the manuscript. We apologize for these idiome errors. We sent the manuscript to a native English Editor for correction. We have modified the manuscript according to the suggestions and below we present point-by-point answers to the questions. All corrections were addressed, as shown below and in the attached file. Answers to the questions are provided below. All the manuscript changes have been highlighted in red font.

Table 1~4: Provided ADF, GE and OM. The author weight the sheep, however, i am very double the data of growth performance? Why not set a group without oils?

Response = We have provided ADF, OM, ME and weight in tables. It is known that vegetable oils interfere in ruminal fermentation. Thus, the central hypothesis of the work is that depending on the oil's fatty acid profile, ruminal biohydrogenation will be more or less extensive. Consequently, the fermentation products will alter the animal blood's thermal comfort and physiological and biochemical variables. Therefore, a group without the addition of oils would not make sense. 

Table 2: "-" means not detect?

Response = Yes. This was corrected. Variables were not detected from the chromatography peak.

Table 4:  Letter need superscript. Ues the abbre in the item.

Response = Thanks. We have put tetters were superscript.  And used abbreviations for variables.

L322: The authors suggest that the low WI observed during BW loss could be attributed to greater metabolic water production. However, dry matter intake is much lower as well.

Response =Yes. This is by design while creating the experimental model. To achieve maintenance while feeding cattle or to achieve body weight loss, animals had to be feed-restricted, however water was always ad libitum. Basically, animals decide how much water they would drink while feed restricted.

Figure 1:  SEM.

Response = This was added line 236.

Line 167 uL

Response =Thanks. This was corrected “ul” to “uL”

Suggest provide the rumen fermentation parameters.

Response =Thanks. We sincerely apologize but data on ruminal fermentation parameters were not collected.

Sincerely yours,

Dr. Leilson Bezerra

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The changes primarily in the Introduction are acceptable. Results for amount eaten and growth rate have been added, as requested.

Author Response

We would like to thank the reviewer for the suggestions made. We appreciate the attention, time and reviewers' contributions to the improvement of this version of the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Table 1 and 3：Unify NDF or aNDF.

Table 4： define the abbre one by one.

Author Response

We would like to thank the Reviewer 2 for the suggestions made. We appreciate the attention, time and reviewers' contributions to the improvement of this version of the manuscript.

We have corrected:

Table 1 and 3: We have used aNDF in all text.

Table 4： We have defined the abbreviations individually, directly in the table or footnote.