Ruminal Solubility and Bioavailability of Inorganic Trace Mineral Sources and Effects on Fermentation Activity Measured in Vitro
Round 1
Reviewer 1 Report
The present study aimed to evaluate the effects of supplementation of inorganic sources of Mn, Zn and Cu on in vitro rumen fermentation, solubility and bioavaliability. The work is well performed and very well written and the subject is interesting for the field of trace elements supplementation of ruminant animals. However, authors should justify the levels used for each trace element and why they only used copper sulfate.
Specific comments on Tables:
Table 2. What do you mean by “trace-mineral content of rumen juice”? It seems that the trace minerals presented in Table refer to the diet of donor cows. Please clarify.
Table 3. SEM should have one more decimal place than the mean. Please correct.
Author Response
Response to Reviewer 1 Comments
Thank you for the positive feedback regarding this work and for pointing out some improvement points.
Point 1: “authors should justify the levels used for each trace element and why they only used copper sulfate”
Response 1: The high mineral dosages applied in this study were based on previous in vitro studies, assessing the effects of high trace-element supplementation levels on fermentation and solubility (Arelovich et al. 2000; Eryavuz and Dehority 2009; Petrič et al. 2021). Furtheremore, we aimed high dosages of trace minerals (12x or 10x above recommended dosages for ruminant supplementation) in order to highlight some of the negative effects of trace-minerals on rumen fermentation, that is demonstrated is some other in vitro studies with high levels of additional minerals.
Concerning the use of only one source of inorganic Cu, as in this study we focused only on inorganic sources of trace-minerals, the most communly used inorganic sources (by complete feed and premix producers) were: for Mn and Zn the -sulfate and -oxide forms; and for Cu only the -sulfate form (as Cu oxide is considered unavailable for ruminant animals; Spears 2003).
Specific comments on Tables:
Table 2: Ideed, the trace-mineral content of the diet is presented in this table, and not of the rumen juice mineral content. This mention was an error from the first draft of the paper, and corrected in this revised version.
Table 3: SEM corrected as indicated.
Kind regards,
Reviewer 2 Report
Many congratulations for the excellent work!
I only suggest the revision of the italics type for in vitro expression. For instance, in lines 11,36 and 72, italics is missing.
Author Response
Thank you for the positive feedback.
Yes, I will make sure to use italics every time in vitro is mentionned in the text.
Best regards.
Reviewer 3 Report
General comments
The manuscript submitted examined the effect of trace minerals, including Mn, Zn, and Cu, on rumen fermentation in vitro. The subject falls within the aims and scopes of the journal and may provide some insight into the subject area of rumen fermentation. However, there are some concerns in relation to Materials and Methods which should be addressed properly prior to publication. Please see below for specific comments.
Major comments
Line 88 and elsewhere, Materials and Methods: Rumen fermentation characteristics in vitro, although it is debatable, have been a standard procedure for years to examine any changes in a small bottle over time. Fermentation interval varies depending on the purpose of the experiment, but 70 hr is not a regular fermentation interval found occasionally in the literature. Please provide a decent explanation of why 70 hr incubation period was chosen for this study.
Line 90: For Mn and Zn treatment, 4 replications was employed. However, for Cu, 6 replications were employed. Is there any specific reason for this? An appropriate explanation is needed in the text.
Line 135: Line 135: Appropriate permission to use rumen-cannulated animals should be described in the text.
Lines 176`178: In the Materials and Methods, the addition of trace minerals during in vitro rumen fermentation at 24 or 48 h was performed without an appropriate explanation. The authors must explain in the text why such experimental treatments were established in the Materials and Methods section. Another point to highlight was that without appropriate substrates supplying energy and protein to the incubation bottle, only trace minerals were supplemented, which requires proper explanation.
Line 189-190: The nylon bags containing undegraded substrate were frozen at -18°C for 24 h after being removed from the flask and briefly rinsed with cold water. Is there any specific reason to freeze the nylon bags before washing and oven-dried for 48 h at 60°C? If nylon bags were frozen, then the pore of the nylon bag is highly likely to be damaged during the freezing process, and the rinsing procedure after thawing may overestimate the true digestibility of the substrate. Please explain the reason in the text.
Specific comments
Lines 175, 176, and 180: (the whole text) The acronym for “hour(s)” were presented in mixed form (i.e., hr, hours, hrs with or without a proper space). They should be revised in uniform.
Line 388: The typo “precise” should be corrected.
Line 394: The terminology “rumen juice” delivers its meaning but is less appropriate than “rumen fluid.” Please revise it.
Tables 3, 4, 5, 6, 7, and 8: The “SEM” stands for “standard error of the mean.” Please revise them.
Author Response
Thank you for the feedback regarding the relevance of this study in the rumen fermentation subject area; and for highlighting the improvement points to be made in the Materials and Methods.
Response to Reviewer 3 Comments
Point 1: “Line 88 and elsewhere, Materials and Methods: Rumen fermentation characteristics in vitro, although it is debatable, have been a standard procedure for years to examine any changes in a small bottle over time. Fermentation interval varies depending on the purpose of the experiment, but 70 hr is not a regular fermentation interval found occasionally in the literature. Please provide a decent explanation of why 70 hr incubation period was chosen for this study.”
and
“Lines 176`178: In the Materials and Methods, the addition of trace minerals during in vitro rumen fermentation at 24 or 48 h was performed without an appropriate explanation. The authors must explain in the text why such experimental treatments were established in the Materials and Methods section. Another point to highlight was that without appropriate substrates supplying energy and protein to the incubation bottle, only trace minerals were supplemented, which requires proper explanation.”
Response 1: In vitro incubations with rumen fluid that asses trace-mineral effects on fermentation and rumen solubility parameters have longer incubation periods (ex: in vitro with Zn sources by Eryavuz and Dehority 2009). This enables the assessment of solubility of mineral sources that have a low solubility (ex -oxide sources). Furthermore, the implementation of 70 hours incubations were also in close relation with the addition of minerals after 24 or 48 hours of fermentation, which aimed to assess the effects of trace-element supplementation of a highly mineral-depleted medium: supplying the trace-minerals after the start of fermentations (24 or 48 hours) could allow rumen microbes to assimilate the soluble or available minerals provided by the substrate and the rumen fluid before the supplementation.
Point 2: “Line 90: For Mn and Zn treatment, 4 replications was employed. However, for Cu, 6 replications were employed. Is there any specific reason for this? An appropriate explanation is needed in the text.”
Response 2: with the experimental design applied in this study (using simultaneously two Gas Endeavour® units), a total of 30 gas production measuring cells were available, allowing the use of 4 replicates/treatment during incubations with Mn and Zn (as there were two sources, -oxide and -sulfate, for Mn and Zn). For treatments with Cu, as only one mineral source (Cu-sulfate) was used, more gas production measuring cells were available, hence we increased the number of replicates in order to increase the statistical power.
Point 3: “Line 135: Line 135: Appropriate permission to use rumen-cannulated animals should be described in the text.”
Response 3: The following statement was added in the text “The use of rumen-cannulated dairy cows in this study was approved by the Ethics Committee no052 (France) and the study protocol was registered under the number APAFIS#28768-2020122108098663 v3 by the French Ministri of Scientific Research.”
Point 4: “Line 189-190: The nylon bags containing undegraded substrate were frozen at -18°C for 24 h after being removed from the flask and briefly rinsed with cold water. Is there any specific reason to freeze the nylon bags before washing and oven-dried for 48 h at 60°C? If nylon bags were frozen, then the pore of the nylon bag is highly likely to be damaged during the freezing process, and the rinsing procedure after thawing may overestimate the true digestibility of the substrate. Please explain the reason in the text.”
Response 4: For the dry matter degradability assessment, we adapted the standard in sacco procedure developed and currently used by INRAE (French National Research Institute for Agriculture, Food and the Environment), based on the work realized by Michalet-Doreau et al. (1987) and Dulphy et al. (1999). Indeed, in the scientific literature there are multiple procedures for treatments of nylon bags (in situ studies) after the removal from the rumen (incubation flask in our study): washing in cold water, introduction in iced water or freezing. All these procedures aim to stop the fermentation/degradation as fast as possible. In addition of stopping the fermentation, another purpose of freezing the nylon bags after removal from the rumen fluid, was to detach the bacteria (at least partially) from fiber particles.
References :
- Jean-Pierre Dulphy, Camille Demarquilly, René Baumont, Marie Jailler, Louis L'Hotelier, Catalin Dragomir, 1999. Study of modes of preparation of fresh and conserved forage samples for measurement of their dry matter and nitrogen degradations in the rumen. Ann. Zootech. 48 (4) 275-288 (1999). DOI: 10.1051/animres:19990404
- Michalet-Doreau, B., R. Verite & P. Chapoutot, 1987. Methodologie de Mesure de la Degradabilite In Sacco de l'Azote des Aliments dans Ie Rumen. Bulletin Technique No 69, Centre de Recherches Zootechniques et Veterinaires, INRA, Theix, pp. 5-7.
Specific comments: the acronym forms, teminology and typos were revised as idicated.
Kind regards,
Round 2
Reviewer 3 Report
No further comments
