2-Hydroxy-4-(Methylthio)-Mutanoate Supplementation Affects Production, Milk Fatty Acid Profile, and Blood Metabolites of High-Producing Holstein Cows

Round 1

Reviewer 1 Report

The manuscript (dairy-2690596) investigates the impacts of 2-hydroxy-4-(methylthio)-butanoate (HMTBa) supplementation in high-producing Holstein cow diets on their growth performance, milk production and composition, milk fatty acid profile and blood metabolites.

The content of this manuscript totally falls within the scope of the special issue: Recent Advances in Dairy Cattle Feeding and Nutrition of Dairy. To the best of my knowledge this paper has not been published elsewhere. The authors have investigated an interesting research field. They have done a huge and complicated experiment with 158 high-producing multiparous cows in a commercial scale. The manuscript was well-prepared, following the journal’s instruction for authors. After carefully reviewing the manuscript, I feel that it will be suitable for publication in this journal with a minor revision. I have several comments for the authors to consider as follows:

Please use the full form of the words at the first time instead of its abbreviation and use full word forms at the beginning of sentences such as DIM, UV and AST.

Why did the author only choose 1 HMTBa supplementing level (0.12%MD)? Please provide a convincing reason and explanation along with citations in the introduction section.

Clarify which yield range is high-producing.

Normally, the adaptation period of feeding trials are at least 2-3 weeks, why the pre-trial period in this study was only 3 days, please explain.

The statement: “In conclusion, HMTBa supplementation appears to influence the ruminal metabolism of high-producing Holstein cows fed a diet with moderate fiber inclusion” is too general, please specify.

Author Response

Reviewer 1

Please use the full form of the words at the first time instead of its abbreviation and use full word forms at the beginning of sentences such as DIM, UV and AST.

Au: Thank you for your comment. We applied these suggestions throughout the text.

Why did the author only choose 1 HMTBa supplementing level (0.12%DM)? Please provide a convincing reason and explanation along with citations in the introduction section.

Au: We sought to adjust the dose with similar situations found in the literature. The supplementation dose was set at 0.12% of DM (35 g) of MFP to provide 11.76 g of metabolizable methionine for high-producing Holstein cows consuming 29.3 kg DM/d of feed. With supplementation of 35 g of MTBa we sought to increase methionine availability in the ruminal environment. We also sought to increase the flux of metabolizable methionine to achieve an adjustment in the ratio of Lysine:Methione (2.97:1) of the metabolizable protein. However, we emphasize that it would be very interesting to test more supplementation levels, but it would be very difficult to implement in a commercial farm three or more experimental groups. In the study of Baldin et al. (2018, 2022), treatments were an unsupplemented control (CON) or HMTBa (0.1% of diet DM, targeting 25 g/d at 25 kg DMI). The HMTBa was provided in a corn carrier (10% HMTBa) and mixed in the TMR. An equal amount of the same ground corn carrier was added to the control treatment. For Lee et al. (2021), HMTBa was implemented at a level of 0.11% DM (28g/d) for 700 kg-cows that had an intake of 24 kg DM and 35 kg/d milk yield. For Jenkins et al. (2020), the diets were top-dressed with 25 g of HMTBA with 225 g of ground corn as a carrier to target HMTBA inclusion at 0.1% of diet DMI in cows with 22 DMI and 625 kg BW.

References

Baldin M, Garcia D, Zanton GI, Hao F, Patterson AD and Harvatine KJ 2022. Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on milk fat, rumen environment and biohydrogenation, and rumen protozoa in lactating cows fed diets with increased risk for milk fat depression. Journal of Dairy Science 105, 7446–7461.

Baldin M, Zanton GI and Harvatine KJ 2018. Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on risk of biohydrogenation-induced milk fat depression. Journal of Dairy Science 101, 376–385.

Jenkins CJR, Fernando SC, Anderson CL, Aluthge ND, Castillo-Lopez E, Zanton GI and Kononoff PJ 2020. The effects of 2-hydroxy-4-methylthio-butanoic acid supplementation on the rumen microbial population and duodenal flow of microbial nitrogen. Journal of Dairy Science 103, 10161–10174.

Lee C, Copelin JE, Park T, Mitchell KE, Firkins JL, Socha MT and Luchini D 2021. Effects of diet fermentability and supplementation of 2-hydroxy-4-(methylthio)-butanoic acid and isoacids on milk fat depression: 2. Ruminal fermentation, fatty acid, and bacterial community structure. Journal of Dairy Science 104, 1604–1619.

Clarify which yield range is high-producing.

Au: We classify milk production levels into three groups: high-producing cows averaged ≥ 40 kg/d, and low-producing cows averaged ≤ 30 kg/d. Cows with moderate-milk production have an average production in the range of 31 to 39 kg/d. A similar classification was used by Baldin et al. (2018) where: milk production level: high-producing cows averaged 44.1 ± 4.5 kg/d and low-producing cows averaged 31.4 ± 4.3 kg/d at the end of the pretrial period.

References

Baldin M, Zanton GI and Harvatine KJ 2018. Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on risk of biohydrogenation-induced milk fat depression. Journal of Dairy Science 101, 376–385.

Normally, the adaptation period of feeding trials are at least 2-3 weeks, why the pre-trial period in this study was only 3 days, please explain.

Au: Thanks for the opportunity to clarify this point. In fact, the collection of data (BW and BCS determination, milk yield for blocking, and milk samples collection which were used as a covariable in the statistical model) took 3 days (9 milkings) only. But the adaptation period was actually 18 days, because the milk yield and the milk components’ yields and contents were determined on days 19-20-21 of each supplemental period (two periods of 21 days each). So, in fact, we adopted exactly the adaptation period that you suggested above.

The statement: “In conclusion, HMTBa supplementation appears to influence the ruminal metabolism of high-producing Holstein cows fed a diet with moderate fiber inclusion” is too general, please specify.

Au: Under similar conditions in this study, it appears that HMTBa supplementation may impact the rumen metabolism of high-producing Holstein cows fed a diet with moderate fiber inclusion. 

Author Response File: Author Response.pdf

Reviewer 2 Report

Author Response

Reviewer 2

There were no suggestions. We do appreciate your comments about our manuscript.

Reviewer 3 Report

Review Manuscript ID: dairy-2690596

Brief summary: The manuscript entitled "2-hydroxy-4-(methylthio)-butanoate Supplementation Affects Production, Milk Fatty Acid Profile, and Blood Metabolites of High-Producing Holstein Cows” investigate on to evaluate the effects of supplementing the diet of high yielding Holstein cows with 2-hydroxy-4-(methylthio)-butanoate (HMTBa) on milk production and composition, milk fatty acid profile, blood metabolites and body parameters. The cows were divided into two treatment groups: cows in the control treatment received 100 g/cow/day of maize meal, while cows supplemented with HMTBa received 35 g HMTBa + 65 g/cow/day of maize meal. Supplementation with HMTBa did not alter milk yield, but improved milk fat content, showing an increase in the concentration of medium chain fatty acids. Blood levels of serum urea and aspartate aminotransferase were lower in cows supplemented with HMTBa, but their body condition score increased. The authors conclude by providing further information on the use of a methionine analogue as a feeding strategy to optimise milk quality in high yielding Holstein cows.

The manuscript is in line with the theme of the journal, but in its current form it requires minor revision.

Below I report my considerations line by line:

General consideration: when authors refer to blood urea nitrogen and milk urea nitrogen, it is more appropriate to rewrite as Blood Urea Nitrogen BUN and Milk Urea Nitrogen MUN, in order not to confuse the reader.

Lines 75-70: I suggest that authors include a reference.

Line 86: HMTBa cows (n = 79; 130 ± 78 DIM) had a milk production of 47.70 ± 6.95 kg/d (mean ± SD) and the control cows (n = 79; 138 ± 131 DIM) had a milk production of 47.76 ± 6.65 kg/d.  My simple observation: DIM variability, was higher in the control group than in the HMTBa group.

Lines 126-127: During each collection period, nine consecutive milkings were followed, with an 8-hour interval. Three milkings for days were only used in the last trial period? should be better explained and the number of daily milkings should be put in context with the whole trial period.

Lines 151-155.  Concentrations of milk fat, total protein, lactose, total solids, casein, milk urea nitrogen, and somatic cell count (SCC) were determined in the milk laboratory from the Holstein Breeders Association of Paraná State (APCBRH) using mid-infrared spectrometry methodology (Nexgen, Bentley Instruments®). Fat, total protein, lactose, total solids, and casein values were also expressed as absolute values (in kg/d), multiplying their average contents by the daily milk production.

To date, I do not believe that somatic cells can be detected by FTIR methodology. There is a need to better explain the different instruments used and how they work, such as 'flow cytometry' for SCC.

Line 223: SCS = log2 (SCC/100) + 3 [26]. Insert the formula with reference also in M&M (line 185)

Line 180: 2.4 Statistical Analysis. Insert in paragraph: Results are presented as lsm ...

Line 327: “The adjusted means for AST suggest an effect between treatments”. Is “adjusted means” a correct term? Rewrite. 

Line 342: “5. Conclusions”. I suggest that the authors rewrite the conclusions more completely.

Check reference n. 40 “Kaneko, J.J.; Harvey, J.W.; Bruss, M.L. Clinical Biochemistry of Domestic Animals, Sixth Edition; 2008;

The manuscript is in need of a minor English language revision.

Author Response

Reviewer 3

General consideration: when authors refer to blood urea nitrogen and milk urea nitrogen, it is more appropriate to rewrite as Blood Urea Nitrogen BUN and Milk Urea Nitrogen MUN, in order not to confuse the reader.

Au: Ok, it was done.

Lines 75-70: I suggest that authors include a reference.

Au: Reference included in text.

Baldin M, Garcia D, Zanton GI, Hao F, Patterson AD and Harvatine KJ 2022. Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on milk fat, rumen environment and biohydrogenation, and rumen protozoa in lactating cows fed diets with increased risk for milk fat depression. Journal of Dairy Science 105, 7446–7461.

Line 86: HMTBa cows (n = 79; 130 ± 78 DIM) had a milk production of 47.70 ± 6.95 kg/d (mean ± SD) and the control cows (n = 79; 138 ± 131 DIM) had a milk production of 47.76 ± 6.65 kg/d. My simple observation: DIM variability, was higher in the control group than in the HMTBa group.

Au: You are right. It would be that the random draw ended up with a greater variability on DIM in the control group.

Lines 126-127: During each collection period, nine consecutive milkings were followed, with an 8-hour interval. Three milkings for days were only used in the last trial period? should be better explained and the number of daily milkings should be put in context with the whole trial period.

Au: Milk samples collection for milk composition analysis were collected during the last 3 d of treatment periods (19^th, 20^th and 21^st d), with 21-day intervals. Nine consecutive milkings were followed, with an 8-hour interval for each collection period (pre-trial period, first, and second supplementation periods). So, in other words, each experimental cow had 27 milk samples collected and analyzed.

Lines 151-155. Concentrations of milk fat, total protein, lactose, total solids, casein, milk urea nitrogen, and somatic cell count (SCC) were determined in the milk laboratory from the Holstein Breeders Association of Paraná State (APCBRH) using mid-infrared spectrometry methodology (Nexgen, Bentley Instruments^®). Fat, total protein, lactose, total solids, and casein values were also expressed as absolute values (in kg/d), multiplying their average contents by the daily milk production. To date, I do not believe that somatic cells can be detected by FTIR methodology. There is a need to better explain the different instruments used and how they work, such as 'flow cytometry' for SCC.

Au: Milk analyses were performed using FTIR (concentrations of milk fat, total protein, lactose, total solids, casein, milk urea nitrogen), and Flow Cytometry (for somatic cell count) methods. State-of-the-art equipment was used, calibrated with international reference samples. The Laboratory also participates in interlaboratory and proficiency tests, to guarantee the accuracy of the results. The Laboratory began its activities in 1991 (APCBRH, UFPR and McGill University-Canada agreement), receiving its first accreditation through SDA Ordinance 46 on 05/25/1992, it is part of the Brazilian Network of Milk Quality Control Laboratories – RBQL since the creation of RBQL in 2002, being accredited by CGAL (General Coordination of Laboratory Support) of the Ministry of Agriculture since 2008. The Laboratory meets the demands of official programs for monitoring the quality and improving the quality of milk, inspection services of products of animal origin, in addition to carrying out Milk Control and Quality Control Management (GCQ) analyses.

Line 223: SCS = log2 (SCC/100) + 3 [26]. Insert the formula with reference also in M&M (line 185)

Au: Ok, the formula has been included in the text.

Line 180: 2.4 Statistical Analysis. Insert in paragraph: Results are presented as lsm ...

Au:  Results are presented as Least Squares Means and multiple mean comparisons were per-formed using Tukey test. For BW, BCS, milk fatty acid profile, and blood metabolites data, which did not involve repeated measures, the model included the effects of treatment (Control vs. HMTBa), and the random effects of block and cow within treatment. Statistical significance was declared at P ≤ 0.05, while trend was declared at 0.05 < P ≤ 0.10.

Line 327: “The adjusted means for AST suggest an effect between treatments”. Is “adjusted means” a correct term? Rewrite.

Au:  The averages for aspartate aminotransferase suggest an effect between treatments, however these values (105.6 vs. 89.4 U/L) remained within the range of 78 to 132 U/L, which is an indicative of a normal condition.

Line 342: “5. Conclusions”. I suggest that the authors rewrite the conclusions more completely.

Au: Ok, it was completed in the manuscript with small adjustments.

Cows supplemented with HTMBa over a 42-d period showed higher milk components and increased concentrations of odd- and branched-chain milk fatty acids. These changes were accompanied by changes in body condition score and reduced blood urea nitrogen concentrations. Therefore, it seems that the HMTBa supplementation at 0.12% of DM inclusion can have an impact on the de novo FA synthesis in the mammary gland, as well as the plasmatic FA uptake. Under similar conditions in this study, it appears that HMTBa supplementation may impact the rumen metabolism of high-producing Holstein cows fed a diet with moderate fiber inclusion.

Check reference n. 40 “Kaneko, J.J.; Harvey, J.W.; Bruss, M.L. Clinical Biochemistry of Domestic Animals, Sixth Edition; 2008;

Au:  Reference was checked and included in the manuscript with small adjustments.

Kaneko, J.J.; Harvey, J.W.; Bruss, M.L. Clinical Biochemistry of Domestic Animals, Sixth Edition; 2008, ISBN 9780123704917, https://doi.org/10.1016/B978-0-12-370491-7.00031-3