Consumption of Purple Neem Foliage Rich in Anthocyanins Improves Rumen Fermentation, Growth Performance and Plasma Antioxidant Activity in Growing Goats

Round 1

Reviewer 1 Report

Taethaisong et al.

This study is dealing with an interesting question regarding the rumen fermentation, growth performance, and plasma antioxidant activity effect of purple neem foliage consumption by Goats.

Although the subject is interesting, there are some serious issues and shortcomings in the way the manuscript is written. First of all- the manuscript has to go through serious English editing and scientific editing, since there are many sentences that just don’t make sense. In addition, there are significant missing details in the materials and methods - including the study design, the sample collection, following by issues with interpretation of data and more. I have listed the main limitations in detail below.

 

Introduction：

Line45：Thailand is currently a tropical country with a unique environment；currently？I think this sentence is inaccurate.

Line55-58：Purple Neem foliage (Azadirachta indica A. Juss var. siamensis Valeton) is a popular crop in Thailand and is one of the world's most significant cereal crops, has it been cereal crops？

Because of its potential biological and pharmacological uses, neem shoots and leaves are regarded one of the most essential features for improving grain quality. Neem shoots and leaves improving grain quality？What is your basis for this assertion?

Line74-78：The information of anthocyanin ……stress in ruminants. From the introduction of purple neem foliage in the manuscript, I didn't find any relevant reports about its strong oxidation, and also improve rumen fermentation, rumen digestibility in ruminants, and improve plasma antioxidant of ruminants. Please supplement the corresponding literature reports

Materials and Methods:

Line 45：45 c, I think it should be 65℃ for 48 h. The method description is not clear enough

Line 89-99：Re describe this paragraph.

Line 92: 1 ml. The international unit should be 1 mL

Line 101: Can the author provide nutritional data of normal neem and purple neem?

Line 104: 2*2+1, I think it might be 2×3, How did you think about it?

Line 120: There are no statistical results about the nutritional components of each group in Table 2. The principle of feed formula is equal energy and nitrogen, but there is no relevant energy data in the results.

Line 120: In Table 2, the content of premix in each group changes greatly, and the change of premix between the treatment groups in the normal feed formula is almost similar. I don't quite understand your practice of transferring premix. How did you think about it?

Line 143: Is the calculation formula correct? Please supplement references.

Results

Line 236: Please supplement the relevant calculation formula (% BW, g / kgBW^0.75)

Line 255: Table 6 contains data about feces and urine, but there is no collection method about feces and urine in the material method. Please supplement relevant methods.

I'm not sure if your statistical method is wrong. Please check it carefully.

Discussion

Line 311-313: Our current study….. high quality of the basal diet. It might be attributed to the relatively high quality of the basal diet. The nutrient content of each group is similar, but how does the high-quality diet base reflect? Is there any relevant application of purple neem in other animals? Please supplement relevant literature.

 

There is no unified format for writing in many parts of the manuscript; such as 72 hours or h; ml or mL; C or ℃ or ºc

Author Response

"Please see the attachment" in word file below

Author Response File: Author Response.pdf

Reviewer 2 Report

......with a 30 m 0.25 mm 0.25m 172

column (DB-FFAP), and peak detection was compared and estimated as previously stated 173

by [11]..........

Error in reading. The SUPELCO standard should be applied. On yours GC

Methodology error.

4 hours of fermentation is too short.

ON what basis did you do that ????

A minimum of 8 hours and preferably 12 or even 24 hours

 

Author Response

Response to Reviewer 2 Comments

Point 1: 4 hours of fermentation is too short. ON what basis did you do that ???? A minimum of 8 hours and preferably 12 or even 24 hours

 

Response 1: From my experiment about fermentation.

1. The in vivo study is limited in scope, and samples cannot be collected at all times. Because it will have a negative impact on the animal and produce stress.

2.In this experiment, samples were taken 4 h after feeding. Fermentation in the rumen was high at 4 h. Following that, it will slowly diminish. As a result, we do not require frequent measurements. In addition, we were fed animal two time per day, in the morning and evening, during this trial.

3. The fermentation in the rumen was measured at the 4 h after feeding. Because this is the period with the highest fermentation process. and is the period with the highest amount of absorption. Furthermore, the fermentation in the rumen evaluated after 4 h after feeding will correspond with the amount of substance in the blood

"Please see the attachment" in word file below 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have made many of the necessary changes to the manuscript. However, there are some areas that need to be revised by the author with more detailed explanation for good.

Table 1 I think it would be more appropriate to place the “(mg/g DM)” after the " Purple Neem foliage".

Table 2 and 3 needs reworking in structure and formatting. And please unify the forms of writing: such as NDF or ADF.

Table 3. The author has revised as per my suggestion, but no statistical analysis between the groups.

 

I think “Line 187:50 ml and line 191 20 µl” should be replaced with “mL and µL”. Standard abbreviations should be followed.

 

Author Response

Response to Reviewer 1 Comments

 

Point 1: Table 1 I think it would be more appropriate to place the “(mg/g DM)” after the " Purple Neem foliage".

 

Response 1: Table 1. Anthocyanin composition (mg/g DM) in Purple Neem foliage.

Anthocyanin composition (mg/g DM)	Purple Neem foliage (mg/g DM)
Total Anthocyanin Cyanidin	132.89 39.96
Delphinidine	26.12
Petunidine	32.60
Pelagonodine	10.93
Peonidine	9.492
Malvidine	19.67

 

Point 2: Table 2 and 3 needs reworking in structure and formatting. And please unify the forms of writing: such as NDF or ADF. 

Response 2: Table 2. Chemical composition of Normal Neem foliage and Purple Neem foliage.

Ingredient
	Purple Neem foliage	Normal Neem foliage
Chemical composition (% DM)
Dry matter	57.37	51.10
Crude protein	9.24	9.20
Ash	7.45	6.20
Ether extract	1.80	1.70
Non-fibrous carbohydrate	25.00	31.90
Neutral detergent fiber	56.51	51.00
Acid detergent fiber	44.85	37.50

Calculated as: NFC =100 − (% NDF + % CP + % EE + % ash).

 

Point 3: Table 3. The author has revised as per my suggestion, but no statistical analysis between the groups.

Response 3: Table 3. Feed ingredients and chemical composition of experimental diets.

		Diet
Items		Control	3%Normal Neem foliage	6%Normal Neem foliage	3%Purple Neem foliage	6%Purple Neem foliage	SEM		p-Value
Ingredients (% air-dry basis)
soybean meal		18.00	19.00	17.00	19.00	17.00
Rice bran		27.00	21.00	20.80	23.00	22.00
cassava chip		27.00	30.00	32.30	35.00	33.10
corn		26.40	22.40	22.80	18.40	20.80
salt		0.40	0.40	0.40	0.40	0.40
limestone		0.20	0.20	0.20	0.20	0.20
premix		1.00	4.00	0.50	1.00	0.50
Purple Neem		0.00	3.00	6.00	3.00	6.00
Chemical composition (% DM)
Dry matter		74.06	75.32	74.57	75.37	74.84	0.24		0.43
Ash		6.17d	6.95a	6.19b	6.10cd	6.42cb	0.008		0.01
Crude protein		16.71a	16.01e	16.29c	16.27d	16.62b	0.07		0.01
Ether extract		4.27d	4.59c	5.57a	4.57c	5.08b	0.13		0.01
Non-fibrous carbohydrate		28.21	31.12	38.13	38.55	34.49	2.49		0.69
Neutral detergent fiber		44.64	41.33	33.82	34.51	37.39	2.50		0.67
Acid detergent fiber		30.70	33.22	30.87	32.60	32.15	2.83		1.00
TDN, %		88.69a	84.77c	87.18ab	87.37ab	86.91b	0.39		0.005
Metabolizable energy, Mcal / kg DM		3.21a	3.06b	3.15a	3.16a	3.14ab	0.01		0.005

Contains per kilogram premix: 10,000,000 IU vitamin A; 70,000 IU vitamin E; 1,600,000 IU vitamin D; 50 g iron; 40 g zinc; 40 g manganese; 0.1 g cobalt; 10 g copper; 0.1 g selenium; 0.5 g iodine; Calculated as: NFC =100 − (% NDF + % CP + % EE + % ash); Estimated by the equation TDN = (%) DCP + DNFC) + DEE × 2.25 + (DNDF); Estimated by the equation ME (Mcal / kg DM) = (TDN × 0.04409 × 0.82).

 

Point 4: I think “Line 187:50 ml and line 191 20 µl” should be replaced with “mL and µL”. Standard abbreviations should be followed.

Response 4: Furthermore, the second sub-sample was extracted at 50 ℃ for 24 hours with 0.01 N hydrochloric acid (HCl) dis-solved in an 80% methanol solution, and the supernatant was collected and transferred into a 50 mL volumetric flask for HPLC determination of anthocyanin [11]. The chromatographic separation was performed on a reversed-phase column Zorbax SB-C18 column (3.5 µm particle size, i.d. 4.6 mm × 250 mm, Agilent Technologies, Santa Clara, CA, USA) for 65 min at 28 ℃. The injection volume was fixed at 20 µL.

Author Response File: Author Response.docx

Reviewer 2 Report

I accept the introduced changes, the publication may appear in its current form

Author Response

Response to Reviewer 2 Comments

 

Point 1: Table 1 I think it would be more appropriate to place the “(mg/g DM)” after the " Purple Neem foliage".

 

Response 1: Table 1. Anthocyanin composition (mg/g DM) in Purple Neem foliage.

Anthocyanin composition (mg/g DM)	Purple Neem foliage (mg/g DM)
Total Anthocyanin Cyanidin	132.89 39.96
Delphinidine	26.12
Petunidine	32.60
Pelagonodine	10.93
Peonidine	9.492
Malvidine	19.67

 

Point 2: Table 2 and 3 needs reworking in structure and formatting. And please unify the forms of writing: such as NDF or ADF. 

Response 2: Table 2. Chemical composition of Normal Neem foliage and Purple Neem foliage.

Ingredient
	Purple Neem foliage	Normal Neem foliage
Chemical composition (% DM)
Dry matter	57.37	51.10
Crude protein	9.24	9.20
Ash	7.45	6.20
Ether extract	1.80	1.70
Non-fibrous carbohydrate	25.00	31.90
Neutral detergent fiber	56.51	51.00
Acid detergent fiber	44.85	37.50

Calculated as: NFC =100 − (% NDF + % CP + % EE + % ash).

 

Point 3: Table 3. The author has revised as per my suggestion, but no statistical analysis between the groups.

Response 3: Table 3. Feed ingredients and chemical composition of experimental diets.

		Diet
Items		Control	3%Normal Neem foliage	6%Normal Neem foliage	3%Purple Neem foliage	6%Purple Neem foliage	SEM		p-Value
Ingredients (% air-dry basis)
soybean meal		18.00	19.00	17.00	19.00	17.00
Rice bran		27.00	21.00	20.80	23.00	22.00
cassava chip		27.00	30.00	32.30	35.00	33.10
corn		26.40	22.40	22.80	18.40	20.80
salt		0.40	0.40	0.40	0.40	0.40
limestone		0.20	0.20	0.20	0.20	0.20
premix		1.00	4.00	0.50	1.00	0.50
Purple Neem		0.00	3.00	6.00	3.00	6.00
Chemical composition (% DM)
Dry matter		74.06	75.32	74.57	75.37	74.84	0.24		0.43
Ash		6.17d	6.95a	6.19b	6.10cd	6.42cb	0.008		0.01
Crude protein		16.71a	16.01e	16.29c	16.27d	16.62b	0.07		0.01
Ether extract		4.27d	4.59c	5.57a	4.57c	5.08b	0.13		0.01
Non-fibrous carbohydrate		28.21	31.12	38.13	38.55	34.49	2.49		0.69
Neutral detergent fiber		44.64	41.33	33.82	34.51	37.39	2.50		0.67
Acid detergent fiber		30.70	33.22	30.87	32.60	32.15	2.83		1.00
TDN, %		88.69a	84.77c	87.18ab	87.37ab	86.91b	0.39		0.005
Metabolizable energy, Mcal / kg DM		3.21a	3.06b	3.15a	3.16a	3.14ab	0.01		0.005

Contains per kilogram premix: 10,000,000 IU vitamin A; 70,000 IU vitamin E; 1,600,000 IU vitamin D; 50 g iron; 40 g zinc; 40 g manganese; 0.1 g cobalt; 10 g copper; 0.1 g selenium; 0.5 g iodine; Calculated as: NFC =100 − (% NDF + % CP + % EE + % ash); Estimated by the equation TDN = (%) DCP + DNFC) + DEE × 2.25 + (DNDF); Estimated by the equation ME (Mcal / kg DM) = (TDN × 0.04409 × 0.82).

 

Author Response File: Author Response.docx