Chemical composition and lipid profile of mare colostrum and milk of the quarter horse breed.

The objective of this study was to characterize the chemical composition and lipid profile of colostrum and milk of purebred Quarter Horse mares. Thirty-four (34) purebred mares were selected, which were then separated into groups according to age, birth order and lactation stage. Colostrum samples were collected in the first six hours after delivery and milk samples from the 7th postpartum day, with intervals of 14 days until the end of lactation. The samples were refrigerated and sent to the Milk Laboratory of the University (Laboleite-UFRN), where they were analyzed for chemical composition. Colostrum was assessed by refractometry. The lipid profile was determined by gas chromatography through a separation of methyl esters. The data were tabulated and subjected to descriptive statistics and analysis of variance by the F-Test, and the groups were compared by the Tukey test using a significance level of 5%. There was high protein content and reduced lactose content for the colostrum of the Quarter Horse mares, differing from other breeds. The milk composition was not influenced by the mares' age. However, variations in the lactation stage and in the birth order of the Quarter Horse mares altered the milk's chemical composition. There is variation in the lipid composition of milk according to the lactation stage, without changing the characteristic profile of the mares' milk or diminishing the nutritional quality of the lipid fraction.

Introduction

A considerable number of horses have been bred in several countries around the world in order to produce milk [1] because of its nutritional and therapeutic properties. According to Malacarne et al. [2] mares’ milk is consumed by 30 million people worldwide, and has been studied as a milk substitute in newborns and pre-mature humans. Furthermore this milk can be used as a dietary supplement for older adults, recovering patients, and mainly children allergic to cow milk [1].

On average, equine milk contains 6.5% lactose, 1.8% protein, 1.0% fat and 440 kcal/kg of energy [3]. It presents a desired protein profile in human food due to the whey:casein protein ratio and the spongiform structure of the micelles, which make it physiologically more digestible than cow milk [4]. The nutritional quality of the lipid fraction of equine milk is the result of small amounts of stearic and palmitic acids and large amounts of linoleic and linolenic acids [5], which has also supported indications for supplying equine milk to humans. Regarding colostrum, which has a dry matter much higher than milk (14 in milk to 29% in colostrum), it is important to highlight the high protein content (10%, on average), composed of immunoglobulins in 80% [6]. Colostrum fat is about 20% higher than milk fat produced in the initial third of lactation [7]. Bioactive peptide precursors, such as β-lactoglobulins and α-lactoalbumin, are present in colostrum from mares in considerable quantities [8]

Age, birth order, body weight of the mares, diet, environmental conditions and lactation stage have an influence on the chemical composition of milk [5, 9]. In addition to these factors, breed and genetics can change the composition of equine milk, especially the protein, fat and lactose levels [10]. Thus, the objective of this study was to characterize the chemical composition and lipid profile of colostrum and milk from purebred Quarter Horse mares of different ages, birth orders and lactation stages.

Materials and methods

Animal ethics and experimentation

The trial was submitted for evaluation by the Ethics Committee on the Use of Animals at Federal University of Rio Grande do Norte (protocol 062/2017), receiving approval registered under opinion number 058.062/2017. All animal management practices followed the recommendations of the National Council for the Control of Animal Experimentation (CONCEA) for the protection of animals used for animal experimentation and other scientific purposes, in accordance with the provisions of Law No. 11,794, of 8 October 2008, of Decree No. 6,899, of July 15, 2009.

Experimental animals, material sampling and laboratory analysis

Thirty-four (34) purebred mares were selected from three different stud farms specialized in breeding and selection of Quarter Horses in Rio Grande do Norte state. The mares were separated into groups according to age (young: 3–5 years; adults: 6–10 years; seniors 11–19 years old), birth order (number of births throughout life, ranging from 1 to 6) and lactation stage (initial, medium and final third, considering a 180-day lactation).” All evaluated mares in this test gave birth naturally without cases of distocya In addition, mares which gave birth to a dead foals were discarded from the experimental group as well, with 34 mares remaining in the experimental group at the end of the selection. The collections took place between the months of July/2017 and September/2018, because the 34 births were not concentrated in a single period.

Colostrum samples were collected after parturition by manual milking not exceeding six hours after the event, and were obtained by manual milking after cleaning the udder and stored in previously sterilized plastic bottles. The composition (fat, protein, casein, lactose, total solids and defatted dry extract), Brix percentage by refractometry determination and lipid profile of the colostrum samples were analyzed.

Milk samples were collected from the 7th postpartum day with 14-day intervals, and thus continued until the end of lactation (180 days after parturition). The foal remained separated from the mare for two hours preceding the procedure on the day determined for collection in order to guarantee sufficient volume of milk for sampling accumulated in the udder. The mares’ udders were sanitized with a compress soaked with 70% alcohol, while the milker’s hands were washed with clean water and neutral soap, dried with paper towels and also sanitized with 70% alcohol. The first three jets of milk were discarded at the time of collection, and then the cisterna udder was fully emptied by milking, with the milk being collected in a glass container previously sterilized in an autoclave.

The samples were identified, placed in an isothermal box containing artificial ice (4 to 8ºC) and sent to the UFRN Milk Quality Laboratory (LABOLEITE). The samples were subjected to electronic analysis by infrared absorption in DairySpec FT Bentley equipment to determine the chemical composition of the milk and colostrum. Qualitative analysis of colostrum was performed using a portable optical refractometer for sugar (Kasvi®, model K52-032, with a measurement range of 0 to 32% Brix and minimum division of 0.2%) after calibrating it with distilled water, as recommended by the manufacturer. One drop of colostrum was placed on the refractometer prism with the sample at room temperature and homogenized, and then reading was conducted through the monocular lens. The result in Brix% was obtained by the separation between the light area and the dark area formed on the equipment display after perpendicular disposition of the equipment to light.

The milk samples were lyophilized and the fatty acid methyl esters were obtained by adapting the methodology proposed by Kramer [11] to analyze the lipid profile. Approximately 0.8g of samples were weighed in glass tubes (16 x 150mm) with screw caps and septums in order to contain 15 to 30 mg of fat. Next, 2mL of hexane and 2mL of sodium methoxide (0.5M in methanol) were added to the tubes, followed by vortexing (30 seconds) and heating in a water bath (50°C for 10 minutes). The tubes were subsequently cooled in running water and 3 ml of acetyl chloride (5% in methanol) were added to each tube, after which the tubes were heated again (80°C for 10 minutes). Then, 1 ml of hexane and 10 ml of 6% K2CO3 were added, followed by vortexing for 1 minute and centrifugation (4,000 rpm for 2 minutes). The supernatant was transferred to 15mL Falcon tubes with approximately 1g of Na2SO4 mixture (previously oven dried) and activated carbon (1:1), followed by stirring (1 min.) and centrifugation (1 min. 4000 rpm). The supernatant was collected, transferred to an amber vial and then stored in a freezer at -20ºC.

The separation of the methyl esters from fatty acids was performed in a gas chromatograph (Focus GC—Thermo Scientific) equipped with flame ionization detector (CG-DIC) and SPTM-2560 capillary column (100m x 0.25mm x 0.20 μm—Supelco). The analysis parameters were: injector temperature of 250°C; detector temperature of 280°C; and 30:1 split ratio. The oven temperature was initially set at 140°C, increasing at a heating rate of 1°C/min to 220°C; then remaining at that temperature for 25 minutes. Hydrogen gas was used as carrier gas at a flow rate of 1.5 mL/minute. The injections were performed in duplicates for each extraction and the injection volume was 1μL. The identification of the fatty acid methyl esters was performed by comparing the peak retention times of the samples with the retention time of the esters of the reference standard (GLC-674, Nu-Chek Prep, Inc.), and the result was obtained through normalizing the areas with the results expressed as a percentage.

The atherogenicity index (AI) and thrombogenicity index (TI) were calculated using the equation described by Ulbricht and Southgate (1991):

Statistical procedures

Data were tabulated in spreadsheets and subjected to descriptive statistics and analysis of vaance by the F-test. The groups were divided according to age, birth order and lactation stage, and were then compared using the Tukey test at a significance level of 5% for type I error. Only the different lactation stages were compared for the lipid profile analysis of milk. The statistical analyses were performed using the SAS (Statistical Analysis System) statistical package, and the analysis of variance was performed according to the following model:

In which:

Y = Dependent variables;

μ = Overall mean;

group = Effect of the jth group (age, birth order and lactation stage) on dependent variables, being group 1 to 3;

residual = Residual effect.

Results and discussion

The values obtained in the colostrum composition (Table 1) confirm its nutritional richness. It is important to highlight the high percentage of protein found for the colostrum of Quarter Horse mares (18%), which was higher than the average of 15% reported by Csapó et al. [12] in Hungarian Draught, Haflinger, Breton and Boulonnaise mares; and the 16% found by Pecka et al. [10] when they evaluated the colostrum of Arabian mares. The lactose content of the colostrum evaluated in this study (1.53%) also differed from that presented by other authors, such as: 3.4% cited by Salimei et al. [13]; 2.95% found by Pikul & Wójtowski [14]; and 2.46% presented by Pecka et al. [7]. The results suggest that the colostrum of Quarter Horse mares may contain more protein and be less dense in energy when compared to other breeds. However, the high fat content of this secretion stands out, being 2.7 times greater than the milk fat of the initial third of lactation and exceeding the parameter mentioned by Pecka et al. [7].

Table 1

Chemical composition of colostrum and milk from purebred Quarter Horse mares.

Variable (%)	Colostrom		Milk
	Mean ± SD	CV (%)	Mean ± SD	CV (%)
Fat	1.70 ± 1.05	61.31	0.73 ± 0.45	61.30
Total protein	18.06 ± 2.00	11.09	1.68 ± 0.26	15.44
Casein	13.66 ± 2.00	14.63	1.26 ± 0.20	16.20
Lactose	1.53 ± 0.53	34.81	6.62 ± 0.30	4.45
Total solids	20.49 ± 2.36	11.53	10.00 ± 0.59	5.90
DDE1	19.95 ± 1.72	8.66	9.30 ± 0.27	2.89
Brix%	27.40 ± 4.15	15.18	-	-

Defatted dry extract; CV = coefficient of variation; SD: standard deviation.

The °Brix values obtained were high, in line with the high protein content of the evaluated material, since approximately 80% of the colostrum protein corresponds to immunoglobulins [12]. The analyzed colostrum samples fall within the range of 20 to 30% of the refractive index established by Nath et al. [15], which classifies them as good, and represents an important factor for the passive transfer of immunity and consequently for establishing the newborn’s health.

When analyzing the chemical characterization of milk (Table 1), a reversal between the protein and lactose concentrations is noticeable when the two secretions (colostrum and milk) are compared. However, as the lactose concentrations in milk are not as high as the protein concentration in colostrum, the levels of total solids and defatted dry milk extract are considerably lower than those observed for colostrum.

In studies conducted with Quarter Horse mares, Gibbs et al. [16] and Burns et al. [17] reported a variation of 1.8 to 2.9% for total milk protein, constituting values close to those found in this study. The lactose content of equine milk has previously reported to be higher than in other species [18] and the values obtained in the present study fall within the range reported in the literature for various horse breeds [7, 19–21], illustrating the importance of lactose as a source of carbohydrate in mares’ milk [7].

The Fat content found in this study was lower than the range of 1.0–1.5% reported by Gibbs et al. [14] for Quarter Horse mares, and also below the average value of 1.25% reported by Salamon et al. [5], but higher than the 0.62% reported by Reis et al. [20] for milk from Mangalarga mares. Equine milk has low levels of fat when compared to milk from other species [2]; however, the measurement of this component in mares’ milk is influenced by methodological details which are difficult to control and which translate into the high coefficient of variation (61.30%) presented in Table 1, and therefore deserve a brief discussion.

The small cistern of the mare’s udder requires frequent milking and/or nursing by the foal throughout the day. Healthy foals nurse/drink several times an hour [22] and the ejection of milk requires the release of oxytocin [23]. When extrapolated to the sample collection methodology, these anatomical and physiological peculiarities reflect the difficulty of completely emptying the udder, which is directly related to the fat content of milk [16], since the residual fraction milk is rich in fat. Therefore, it is possible that the low fat content of milk found in this study is not solely and exclusively explained by genetic variations, but also because there is not enough oxytocin release during the sample collection to remove the residual milk fraction, resulting in in low-fat samples.

There was a significant effect of the lactation stage on the fat, total protein and casein levels, with the effect of such variations also occurring on the defatted dry extract levels (Table 2).

Table 2

Effect of the lactation stage on the milk composition of purebred Quarter Horse mares (means ± standard deviation).

Variable (%)	Days in lactation
	7–60	61–120	121–180
Fat	0.61 ± 0.41b	0.85 ± 0.54a	0.70 ± 0.36ab
Total protein	1.94 ± 0.31a	1.69 ± 0.18b	1.53 ± 0.18c
Casein	1.47 ± 0.24a	1.27 ± 0.14b	1.15 ± 0.15c
Lactose	6.62 ± 0.23	6.60 ± 0.30	6.64 ± 0.32
Total solids	10.11 ± 0.50	10.09 ± 0.73	9.86 ± 0.46
Defatted dry extract	9.49 ± 0.26a	9.29 ± 0.25b	9.20 ± 0.24b

a, b, c Different letters on the same line indicate statistical difference by the Tukey test (p<0.05).

According to Markiewicz-Keszycka et al. [21], the evolution of lactation in mares leads to producing milk which is rich in lactose, but low in fat, protein and total solids. The fat levels at the end of lactation in this study were higher than those found at the beginning; Despite the tendency of increase observed for the fat content of milk according to the progress of lactation, it seems to be slightly lower than the 0.9% reported by Burns et al. [17] for milk from Quarter Horse mares at 150 days of lactation, thus reaffirming the difficulty of completely emptying the udder during collection and the permanence of alveolar milk in the mares evaluated in this study. Regarding protein contents and their fractions, there was a gradual reduction during lactation exactly as reported by Salimei and Fantuz [16]: a decrease of 20 to 25% of the total protein between the 28th and 150th days of lactation, accompanied by a 20 to 30% decrease of casein within the same period.

It is noteworthy that variations in the milk composition throughout lactation are essential for adjustments in the nutritional management of foals given the early development and rapid growth of horses at this stage of life (NRC, 1989). Add to this physiological nature, the expressive and particular muscular development of the Quarter Horse breed, which certainly requires an increase in protein intake so that there is no nutritional deficit or consequently losses in animal growth.

There was a difference (p<0.05) in the lactose levels in the effects of the birth order on the milk composition (Table 3), with a notable decrease in this component according to the maturity of the glandular tissue. Similar behavior was observed for defatted dry extract, most likely as a result of lactose variation, since this component is part of the dry extract.

Table 3

Effect of birth order on the milk composition of purebred Quarter Horse mares (means ± standard deviation).

Variable (%)	Birth order
	1	2	3	4	5	6
Fat	0.75 ± 0.52	0.73 ± 0.40	0.61 ± 0.42	0.84 ± 0.52	0.76 ± 0.39	0.76 ± 0.46
Prot	1.62 ± 0.14	1.62 ± 0.18	1.74 ± 0.33	1.61 ± 0.22	1.67 ± 0.23	1.74 ± 0.30
Cas	1.23 ± 0.12	1.22 ± 0.15	1.31 ± 0.24	1.21 ± 0.18	1.26 ± 0.19	1.30 ± 0.24
Lact	6.82 ± 0.24a	6.70 ± 0.30ab	6.67 ± 0.29 abc	6.48 ± 0.27c	6.52 ± 0.26bc	6.59 ± 0.29bc
TS	9.98 ± 0.89	10.09 ± 0.42	9.86 ± 0.68	10.06 ± 0.62	10.01 ± 0.43	10.02 ± 0.45
DDE	9.38 ± 0.16a	9.36 ± 0.20ab	9.34 ± 0.30ab	9.19 ± 0.05ab	9.26 ± 0.22ab	9.27 ± 0.29b

a, b, c Different letters on the same line indicate statistical difference by the Tukey test (p <0.05).

Fat = fat; Prot = total protein; Cas = casein; Lact = lactose; TS = total solids; DDE = defatted dry extract.

It is important to consider that the physiological variations that occur in the mammary gland with the advancement of the longevity of the matrix can provide maximum performance with the maturity of the animal, changing the contents of some constituents [24, 25]. This can be an important detail in the management of herds that specialize in producing milk, or even in foal nutrition.

In evaluating the milk production and composition of primiparous and multiparous Quarter Horse mares, Pool-Anderson et al. [26] found greater production in multiparous mares, but did not report any variation in the milk secretion composition, differently from what occurred in this study. The fact that the lactose content is linked to the osmotic function and the milk production of the mammary gland [27] generated contrary expectations to the observed result, since young mares have lower production, which would lead to lower lactose content in the milk of low birth order mares.

Although low birth order mares are generally younger animals, there was no effect (p>0.05) of age on milk composition (Table 4).

Table 4

Effect of age on the milk composition of purebred Quarter Horse mares (means ± standard deviation).

Variable (%)	Age (years)
	3–5	6–10	11–19
Fat	0.77 ± 0.43	0.71 ± 0.47	0.76 ± 0.41
Total protein	1.63 ± 0.11	1.67 ± 0.27	1.71 ± 0.31
Casein	1.23 ± 0.08	1.26 ± 0.21	1.29 ± 0.25
Lactose	6.64 ± 0.29	6.59 ± 0.30	6.66 ± 0.29
Total solids	9.99 ± 0.72	9.97 ± 0.61	10.04 ± 0.45
Defatted dry extract	9.32 ± 0.20	9.29 ± 0.29	9.29 ± 0.26

Milk production in mammals increases with age until physiological maturity is reached [28], when there is a tendency to a functional reduction of the mammary gland caused by aging of the glandular tissue [29]. In addition, there is the dilution effect, in which the highest production tends to dilute the dry extract components [27], and so variation in the milk composition from mares of different ages was expected, especially between the evaluated extremes, but this did not occur in this study.

Tables 5 and 6 show the lipid composition of colostrum and milk from mares and the effect of the lactation stage on the lipid profile for milk, while Table 7 shows the results of relationships between fatty acids.

Table 5

Means and standard deviations of saturated fatty acids (peak area %) in Quarter Horse mares’ colostrum and milk.

Fatty acid	Colostrom	Milk	Milk—days in milk
			7–60 d	61–120 d	121–180 d
C4:0	0.035±0.019	0.110±0.020	0.122±0.032a	0.110±0.008a	0.097±0.005a
C6:0	0.142±0.052	0.244±0.055	0.292±0.045a	0.255±0.017ab	0.185±0.288bc
C8:0	2.375±0.033	2.514±0.367	2.715±0.028a	2.747±0.101a	2.080±0.167b
C10:0	10.098±0.066	5.674±1.260	6.192±0.972a	6.685±0.045a	4.145±0.191b
C12:0	8.742±0.083	6.098±0.856	5.882±0.730b	7.075±0.025a	5.335±0.206b
C14:0	6.612±0.243	6.560±0.639	6.095±0.561b	7.312±0.123a	6.272±0.109b
C16:0	19.16±0.472	21.401±1.227	20.295±0.954ab	22.437±0.162a	21.485±1.229ab
C18:0	4.282±0.548	1.626±0.362	1.867±0.286a	1.827±0.080a	1.185±0.051b
∑AGS	53.652±1.364	46.491±3.818	45.322±3.632b	50.795±0.522a	43.357±0.628b

a,b,c Means followed by different letters on the same line differ statistically from each other using the Tukey test at the 5% significance level. ∑AGS = sum of saturated fatty acids.

Table 6

Means and standard deviations of unsaturated fatty acids (peak area %) in Quarter Horse mares’ colostrum and milk.

Fatty acid	Colostrom	Milk	Milk—days in milk
			7–60 d	61–120 d	121–180 d
C14:1	0.180±0.020	0.554±0.209	0.352±0.106b	0.497±0.005b	0.812±0.037a
C16:1	1.942±0.140	5.494±1.662	4.200±1.172b	4.882±0.056b	7.400±1.085a
C18:1n9cis	15.700±0.186	16.688±0.971	16.64±1.405a	16.600±0.367a	16.822±1.144a
C18:2n6cis	14.517±0.924	14.011±3.655	16.00±2.547a	16.585±0.416a	9.442±0.600b
C20:1n9	0.470±0.014	0.260±0.029	0.282±0.017a	0.245±0.005a	0.252±0.043a
C18:3n3	4.560±0.155	11.415±5.314	12.212±3.44a	5.387±0.075b	16.645±2.442a
C20:2n6	0.520±0.012	0.278±0.043	0.302±0.005a	0.322±0.009a	0.220±0.008b
C20:3n3	0.147±0.015	0.264±0.097	0.275±0.052b	0.150±0.000c	0.367±0.020a
∑AGM	18.697±0.295	23.099±2.547	21.547±2.636a	22.310±0.390a	25.440±2.312a
∑AGPI	19.980±1.041	26.105±3.236	28.985±0.985a	22.560±0.443b	26.770±2.971a

a,b Means followed by different letters on the same line differ statistically from each other using the Tukey test at the 5% level of significance. ∑AGM = sum of monounsaturated fatty acids; ∑AGP = sum of polyunsaturated fatty acids.

Table 7

Means and standard deviations of the relationships between saturated and unsaturated fatty acids in colostrum and milk of Quarter Horse mares.

Fatty acid ratios	Colostrom	Milk	Milk—days in milk
			7–60 d	61–120 d	121–180 d
∑n6	15.177±0.938	14.351±3.697	16.390±2.538a	16.947±0.410a	9.717±0.605b
∑n3	4.707±0.167	11.668±5.382	12.505±3.489a	5.537±0.075b	16.962±2.373a
n6:n3	3.225±0.155	1.692±1.122	1.437±0.603b	3.062±0.065a	0.577±0.044b
AGS:AGI	1.390±0.080	0.954±0.153	0.902±0.136b	1.130±0.0316a	0.830±0.023b
AI	1.415±0.085	1.105±0.173	1.010±0.150b	1.312±0.034a	0.995±0.040b
TI	0.962±0.069	0.597±0.215	0.512±0.129b	0.865±0.019a	0.415±0.058b

a,b Means followed by different letters on the same line differ statistically from each other using the Tukey test at the 5% level of significance. ∑n6 = sum of omega 6 fatty acids; ∑n3 = sum of omega 3 fatty acids; n6:n3 = ratio between omega 3 and omega 6 fatty acids; AGS:AGI = ratio between total saturated and unsaturated fatty acids; AI = atherogenicity index; TI = thrombogenicity index.

Saturated fatty acids prevailed over unsaturated faty acids in colostrum, with an emphasis on palmitic (C16:0), capric (C10:0), lauric (C12:0) and myristic (C14:0) acids, which increased the sum of saturated fatty acids (Table 5) and consequently the AGS:AGI ratio (Table 7). In the case of unsaturated acids, C18:1n9cis, C18:2n6cis and C18:3n3 stood out in comparison to the others. Similar behaviors for such acids have been reported by Pikul et al. [30] and Salamon et al. [5] for mares’ colostrum; however, the values obtained in the present study were lower than those reported by these authors.

There was a higher concentration of unsaturated fatty acids in milk compared to colostrum regarding the lipid profile of mature milk when compared to colostrum; however, fatty acids (saturated and unsaturated) which stood out in the lipid profile of colostrum also did so in milk, with the values obtained herein within the range presented by Claeys et al. [31]. According to these authors, equine milk has higher proportions of unsaturated fatty acids when compared to milk from other species (especially cattle), due to the minimal occurrence of biohydrogenation before the absorption of unsaturated fatty acids.

There was an effect of the lactation stage on the lipid profile of mares’ milk, with higher values of saturated fatty acids being obtained in the middle third of lactation. This is similar to the results presented by Orlandi et al. [32], especially in relation to C12:0, C14:0 and C16:0 acids. Pikul et al. [30] also found that the percentage of saturated acids decreased as lactation progressed. There was a significant reduction in linoleic acid in the final third of lactation, when investigating the most important unsaturated acids in our study, while linolenic acid concentrations were decreased in the middle of lactation. Consequently, the total amount of polyunsaturated fatty acids was also lower for the middle third of lactation.

The relationships between fatty acids presented in Table 7 show that even though some values obtained in this study are lower than those reported in the literature, there is a relevant nutritional advantage of the Quarter Horse mares’ milk compared to milk of other breeds. The high concentrations of linoleic and linolenic acids, which have important biological functions, ensure that the proportions of fatty acids are nearly ideal [2].

Lower atherogenicity and thrombogenicity indices indicate the potential for atheroma and thrombus prevention [33] and were very similar to those presented by Pikul et al. [30] for Konik mares. The reduction in indices in the final third of lactation was also observed by Markiewicz-Kęszycka et al. [34].

Conclusions

Quarter Horse mares produced colostrum with higher protein content and lower lactose content when compared to other horse breeds. The milk composition is not influenced by the mares’ age; however, the lactation stage and the birth order alter the chemical composition of the milk of Quarter Horse mares. There is variation in the milk’s lipid composition according to the lactation stage without changing the characteristic profile of mares’ milk and without negative effects on the nutritional quality of the lipid fraction.

19 May 2020

PONE-D-20-04990

Chemical composition and lipid profile of mare colostrum and milk of the quarter horse breed

PLOS ONE

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3. Please include a copy of Table 8 which you refer to in your text on line 269.

Additional Editor Comments (if provided):

There is a major flaw in the interpretation of the data, methodology, the statistical analysis, English language and redaction style. It is necessary to improve the manuscript by examining the questions that need to be clarified in a way. For your guidance, you can check the reviewers' comments. Thank you for giving us the opportunity to consider your work.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: No

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In fact, the authors characterized the chemical composition and lipid profile of colostrum and milk from purebred Quarter Horse mares. The authors introduced that the body weight of the mares, diet, environmental conditions have an influence on the chemical composition of mare’s milk (page 3, line 74). However, the study tested only the impact of age, birth order, and lactation stage effect on the milk composition.

Therefore, are the other factors (body weight, diet, environmental condition) unified for all the groups from the three different stud farms? This need to be clarified.

Moreover, the authors reviewed in the introduction the importance of the mare’s milk and its composition and there wasn’t any information on the research background on the mare’s colostrum even though it is tested in the study simultaneously with the mare's milk. Therefore, I think it’s better to introduce this as well.

Anyway, the authors reported important results that the Quarter Horse mare’s colostrum is of higher protein content and lower lactose content when compared to other breeds. The authors present that mares’ age does not impact the milk composition; however, the lactation stage and the birth order influence the chemical composition. The authors stated that the lactation stage alters the milk’s lipid composition without any changes in the milk profile or nutritional value.

On page 7, the authors discussed the results of the colostrum composition, where the fat value was not discussed even though it is presented in table 1, page 8.

On page 10 (lines 226-227) the authors stated ´´The fat levels at the end of lactation in this study were higher than those found at the beginning…..``, while in table 2 on page 10 the fat content was the highest in the middle lactation stage not the end stage?

On page 9 (lines 207-216) & on page 10 (lines 229-231) the authors argue that the reduction in the fat content in the mare’s milk is mainly attributed to the inability of the total evacuation of the mares udder, while in page 5 (line 109) they acknowledge that: ´´ the udder was fully milked…..``. This controversy need to be explained.

Reviewer #2: Dear authors,

The paper describes a fairly straight-forward (no disrespect intended) study on the composition of QH colostrum and milk, with a comparison to that of other horse breeds. The paper generally reads well, although specific suggestions have been made below.

A concern is that the reference numbers do not correspond to the appropriate references, which could indicate a lack of thorough checking of the manuscript before submission.

Specific comments/suggestions/questions:

Title: Quarter Horse with capital letters

41: subjected instead of submitted

43: reduced suggests a comparison – compared to what? (presumably other breeds)

68: I think contains would be better than is composed of

72: is the result of

72: large amounts rather than high amounts

76: breed rather than race

84: for instead of to

97: parturition rather than delivery

100: it was the Brix percentage that was analysed (not refractometry – that is the method of analysis)

103: parturition

104: for two hours, instead of during the last two hours

106: remove previously

109: collected instead of placed

128: added to each tube, after which the tubes were heated again….

151: subjected rather than submitted

155: analyses

167: obtained rather than verified

168: in instead of for

169: 18% or 18.1% would seem accurate enough (certainly when compared to whole percentages reported for other breeds)

169: (18.06%), which was higher than…

172: add the lactose content in the present study (in parentheses)

172: that instead of those

177: in line with instead of following

180: …as good, and represents an important factor for the passive transfer…

188: remove milk

189: However, as the lactose concentration in milk is not as high as the protein concentration in colostrum, the levels…..

194: The lactose content of equine milk has previously reported to be higher than in other species [16] and the values obtained in the present study fall within the range reported in the literature for various horse breeds [10, 17-19], illustrating the importance of lactose as a source….

199: remove ‘value regarding’ and lower instead of less

201: remove are

202: remove certainly

207: nursing by the foal instead of breastfeeding

208: healthy foals nurse/drink several times an hour

208: …and the ejection of milk requires the release of oxytocin.

211: consider ‘peculiarities’ instead of particularities and remove ‘on’

226: remove ‘a’

240: I’m not sure we can speak of ‘animal performance’ in foals

241: in instead of for

241: explain what you mean by ‘birth order’. Presumably this refers to the number of foals that the mare has had.

243: remove ‘breast’ (not a term used in horses)

246: table 3 – please include what the measure of variation is (presumable standard deviation)

I understand the lactose and DDE % in milk may differ statistically, depending on the number of foals, but I find it hard to believe that such a small difference would be (clinically/biologically) relevant and therefore question whether this should be discussed (at length). At least a comment could be added about any biological significance.

255: lower instead of smaller

264: In addition, there is the dilution effect, in which…

267: remove ‘fact’

276: the present instead of this (to make it clear to which study is being referred)

284: concentration instead of prevalence

284: …fatty acids in milk when compared….

287: range instead of threshold

288: remove ‘in fact’

The numbers of (many of the) references do not correspond to the correct papers. Please check and amend.

292: for instead of ‘of’

293: results

294: also found that the concentration (or percentage) of saturated acids decreased as lactation progressed. (Remove ‘in evaluating the lipid profile of mares’ milk throughout lactation in evaluating the lipid profile of mares’ milk throughout lactation.’)

296: …third of lactation, when investigating the most important…..

297: …linolenic acid concentrations were decreased….

298: Consequently, the total amount of…

301: Table instead of Tabela

307: reported instead of contained

308: species or breeds?

308: The high concentrations of linoleic and linolenic acids, which have important biological functions [2], ensure that the proportions of fatty acids are nearly ideal. Please provide reference for ideal values.

320: similar instead of close

326: other horse breeds

326: The milk composition is not influenced by…

330: ‘negative effects on the nutritional…’ instead of harming

335: horses

Reviewer #3: I can see that there has been a very good effort during this study. Here some advices to the authors:

I recommend an English review. There are some English mistakes that I have mentioned but there are other ones to be checked.

I understand that it is not easy to work with randomized conditions as mares were coming from different stables. This is why I consider that the composition of the food that they received should be added as an annex to cover all the possibilities of different compositions in colostrum and milk.

Were all the deliveries at term? If there were changes in the scheduled birth date (early delivery), the differences in the udder physiology may have produced alterations in the milk composition. I recommend to mention this but also to discuss it.

Lines 64-67: The phrase is incredibly long. Divide it to make easier the understanding.

Line 70: makes

Line 79 Birth order. What do you mean? Its position compared to the rest of individuals? The number of pregnancies?

Line 93. Were there any selection criteria?

Line 98: Colostrum samples were collected after delivery by manual milking.

Line 104: Erase last: If they were on the two hours preceding the procedure, It cannot be any others.

Line 105: In order to guarantee sufficient volume of milk

Results and discussion. I highly recommend to separate the results from the discussion. If results are separated from the discussion, the reader can obtain its own conclusions without being guided and then compare them with the manuscript.

Besides, I consider that this way results are very poorly presented

Lines 167-168. Separate them. It is a very strange sentence. I guess you mean: ...confirm its nutritional richness. It is important to highlight the high percentage...

Table 1: Colostrum instead colotrom. To be corrected in the other tables as well.

Line 189: I guess you mean that there are not high concentrations of lactose in milk. Rephrase

Line 199: Fat content found in this study...

References: Some references like 29 has not the abbreviation: Acta universitaria should be abbreviated to Acta univ. I did not check all the references.

Reviewer #4: Authors wanted to describe the composition of the quarter horse colostrum and milk. They seem to collect one sample of colostrum within 6h after birth and then, they collect milk samples, with no information about previous foal suckling, every 14 days. Analysis were performed on sample to give general data on milk (protein, lactose, caseins, lactose) and a detailed analysis of lipids.

General comments:

- The abstract should be totally re-written to explain the main method and results obtained in the study.

- They are few references and they are quite old: the newest one is from 2016 (only one) and all the others are older than 2014. Moreover, some links to reference are not accurate (example in line 287: reference 7 seems to be linked to a first author named Claeys and it’s not the case).

- Sometimes, English used in the paper is very strange, it should be reviewed by a native English speaker.

- The methods used and groups definition are poorly described:

o Until the results & discussion parts, it is impossible to know what are the group of ages, of birth orders, and of lactation stages are not defined by the authors. All along the paper, the size of the different groups is never mentioned. Statistical validity of this study is thus poor.

o Sampling of the colostrum is difficult to understand. What was sampled in each mare: Only one sample in each mare within 6 hours? Did the foal suckle before or after? Many questions that interfere with results.

o Some methods (example: infrared absorption in DairySpec FT Bentley) are poorly described, giving only the name of the manufacturer: rationale and descriptions are lacking.

o Statistical procedures are poorly described. Most of the time, it is impossible to understand what is compared with what. There is no information about normality of data, how this has been tested and the potential use of non-parametric test if the data don’t follow a Gaussian distribution. In the end, the general impression is that job has not been done properly.

- Results and Discussion

o The choice of the structure that associate results and discussion is inappropriate because it is already very difficult to understand the groups (not) defined in the methods section. Giving clear results would help to understand how samples and groups have been used through the study.

o Data observed are in quarter horses, but they may be similar in other horses: conclusion about the high protein level in quarter horse milk could be generalised to other breed if the same assays were used in other breeds.

o Frequently, it’s difficult to know if the results/discussion part is talking about colostrum, milk or both. It should be stated each time!

o Some statements are not statistically proven by the presented data or are not coming from references. We don’t care about what can be thought or imagined.

o In all presented tables, variables are expressed in % with no mention of percent of what: total milk, dry matter, defatted dry extract.

o Presentation of results is very poor, only giving tables that are difficult to read. Some graphs showing evolution or differences could help the reader.

o There is a major confusing factor that has not been statistically studied. It is obvious that mares with higher “birth order” (I would prefer using “lactation number”) will be older than primiparous one. A complete statistical model should have been realised to elude this confusion.

Conclusion

As the methods are poorly described and most of the time seem incomplete or inadequate, it is difficult to trust in the accuracy of the presented data. Thus, this paper is not suitable for publication.

Reviewer #5: Although the topic may be of interest,the manuscript lacks key elements:

The abstract contains unnecessary information; the keywords are not appropriate; the material and methods are not clear and lacking in information (eg. at 95 the authors do not explain how the groups were divided, they do not report the separation times of the foal for the colostrum; the amount of milk collected, at the time of samples, was not reported).

The statistical analysis should have consisted in ANOVA for repeated measurement and to include the effect of the farm. Furthermore, the two variables considered (age and parity (birth order) give similar information and one of the two variables should be removed from the model.

The references are not appropriated and are also dated (eg Cit 1, 2). In addition, eight tables are too numerous and the titles of the tables are not self-explanatory (eg Table 7).

Superfluous information is often reported in the text (e.g. l 106-110).

Finally, the results and conclusions are not satisfactory and should be reviewed in the light of the suggested considerations.

**********

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Reviewer #1: Yes: Manal Bakry Mohamed Hemida

Reviewer #2: Yes: Dr. Robin van den Boom, DVM, PhD, Dip.ECEIM

Reviewer #3: No

Reviewer #4: Yes: Jérôme Ponthier

Reviewer #5: No

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Submitted filename: PONE-D-20-04990_reviewer.pdf

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5 Jul 2020

The authors thank the Reviewers and Editor for the considerations. The research group has been working hard to get the work published in the journal.

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2. Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary).

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Additional Editor Comments (if provided):

There is a major flaw in the interpretation of the data, methodology, the statistical analysis, English language and redaction style. It is necessary to improve the manuscript by examining the questions that need to be clarified in a way. For your guidance, you can check the reviewers' comments. Thank you for giving us the opportunity to consider your work.

The manuscript has been reviewed as suggested by editor and reviewers.

Reviewer #1:

In fact, the authors characterized the chemical composition and lipid profile of colostrum and milk from purebred Quarter Horse mares. The authors introduced that the body weight of the mares, diet, environmental conditions have an influence on the chemical composition of mare’s milk (page 3, line 74). However, the study tested only the impact of age, birth order, and lactation stage effect on the milk composition.Therefore, are the other factors (body weight, diet, environmental condition) unified for all the groups from the three different stud farms? This need to be clarified.

The intention of introduction was to make it clear that there are other sources of variation in the quality of water milk. On this occasion, only the objectives of the study were investigated.

Moreover, the authors reviewed in the introduction the importance of the mare’s milk and its composition and there wasn’t any information on the research background on the mare’s colostrum even though it is tested in the study simultaneously with the mare's milk. Therefore, I think it’s better to introduce this as well.

The text was inserted (in the end of line 73) as follow: “Regarding colostrum, which has a dry matter much higher than milk (14 to 29%), it is important to highlight the high protein content (10%, on average), composed of 80% immunoglobulins (Csapó et al. 1995). Colostrum fat is about 20% higher than milk fat produced in the initial third of lactation (Pecka et al. 2012). Bioactive peptide precursors, such as β-lactoglobulins and α-lactoalbumin, are present in colostrum from mares in considerable quantities (Fessas et al. 2001). “

Anyway, the authors reported important results that the Quarter Horse mare’s colostrum is of higher protein content and lower lactose content when compared to other breeds. The authors present that mares’ age does not impact the milk composition; however, the lactation stage and the birth order influence the chemical composition. The authors stated that the lactation stage alters the milk’s lipid composition without any changes in the milk profile or nutritional value.

On page 7, the authors discussed the results of the colostrum composition, where the fat value was not discussed even though it is presented in table 1, page 8.

The text was inserted (Line 176) as follow:

“However, the high fat content of this secretion stands out, being 2.7 times greater than the milk fat of the initial third of lactation and exceeding the parameter mentioned by Pecka et al. 2012.

On page 10 (lines 226-227) the authors stated ´´The fat levels at the end of lactation in this study were higher than those found at the beginning…..``, while in table 2 on page 10 the fat content was the highest in the middle lactation stage not the end stage?

The text has been rewritten (Page 19, Line 226 – 227) as follow:

"Despite the tendency of increase observed for the fat content of milk according to the progress of lactation, the values were still lower than the 0.9% reported by Burns et al. 1992"

On page 9 (lines 207-216) & on page 10 (lines 229-231) the authors argue that the reduction in the fat content in the mare’s milk is mainly attributed to the inability of the total evacuation of the mares udder, while in page 5 (line 109) they acknowledge that: ´´ the udder was fully milked…..``. This controversy need to be explained.

On page 9, lines 229-230, we refer to the difficulty of extracting the milk contained in the breast alveoli, which, as a rule, only happens in mares by suctioning the foal or applying oxytocin for removal by milking. In the methodology, when we mention total emptying of the udder, we speak of the udder cistern, of the milk already released from the alveoli.

In the methodology (on line 109), the text “and then the udder was fully milked” has been rewritten as follow:

“and then the cisterna udder was fully emptied by milking”.

Reviewer #2:

Dear authors,

The paper describes a fairly straight-forward (no disrespect intended) study on the composition of QH colostrum and milk, with a comparison to that of other horse breeds. The paper generally reads well, although specific suggestions have been made below.

A concern is that the reference numbers do not correspond to the appropriate references, which could indicate a lack of thorough checking of the manuscript before submission.

References and citations have been corrected

Specific comments/suggestions/questions:

Title: Quarter Horse with capital letters

It was done

41: subjected instead of submitted

It was done

43: reduced suggests a comparison – compared to what? (presumably other breeds)

In the sbstract (line 43) the text has been rewritten) as follow:

“There was a high protein and fat content, and low lactose for the colostrum of the Quarter Horse mares.”

68: I think contains would be better than is composed of

It was done

72: is the result of

It was done

72: large amounts rather than high amounts

It was done

76: breed rather than race

It was done

84: for instead of to

It was done

97: parturition rather than delivery

It was done

100: it was the Brix percentage that was analysed (not refractometry – that is the method of analysis)

The refractometry have been corrected as follow:

“Brix percentage (refratometry determination)”

103: parturition

It was done

104: for two hours, instead of during the last two hours

It was done

106: remove previously

It was done

109: collected instead of placed

It was done

128: added to each tube, after which the tubes were heated again….

It was done

151: subjected rather than submitted

It was done

155: analyses

It was done

167: obtained rather than verified

It was done

168: in instead of for

It was done

169: 18% or 18.1% would seem accurate enough (certainly when compared to whole percentages reported for other breeds)

The value 18.04% was rounded to 18.1%.

169: (18.06%), which was higher than…

It was done

172: add the lactose content in the present study (in parentheses)

The lactose content “(1,53%) has been inserted on line 172 as suggested.

172: that instead of those

It was done

177: in line with instead of following

It was done

180: …as good, and represents an important factor for the passive transfer…

It was done

188: remove milk

It was done It was done

189: However, as the lactose concentration in milk is not as high as the protein concentration in colostrum, the levels…..

It was done

194: The lactose content of equine milk has previously reported to be higher than in other species [16] and the values obtained in the present study fall within the range reported in the literature for various horse breeds [10, 17-19], illustrating the importance of lactose as a source….

It was done

199: remove ‘value regarding’ and lower instead of less

It was done

201: remove are

It was done

202: remove certainly

It was done

207: nursing by the foal instead of breastfeeding

It was done

208: healthy foals nurse/drink several times an hour

It was done

208: …and the ejection of milk requires the release of oxytocin.

It was done

211: consider ‘peculiarities’ instead of particularities and remove ‘on’

It was done

226: remove ‘a’

It was done

240: I’m not sure we can speak of ‘animal performance’ in foals

“Performance” has been replaced by “growth”.

241: in instead of for

It was done.

241: explain what you mean by ‘birth order’. Presumably this refers to the number of foals that the mare has had.

The description “birth order (number of births throughout life)” has been inserted on line 95.

243: remove ‘breast’ (not a term used in horses)

It was done.

246: table 3 – please include what the measure of variation is (presumable standard deviation)

The description “(means ± standard deviation)” has been included on caption of Table 2, 3 and 4.

I understand the lactose and DDE % in milk may differ statistically, depending on the number of foals, but I find it hard to believe that such a small difference would be (clinically/biologically) relevant and therefore question whether this should be discussed (at length). At least a comment could be added about any biological significance.

The following text has been included on line 250:

“It is important to consider that the physiological variations that occur in the mammary gland with the advancement of the longevity of the matrix can provide maximum performances with the animal's maturity, changing the contents of some constituents (Ribeiro et al., 2008; Rangel et al., 2008 ). ”

255: lower instead of smaller

It was done.

264: In addition, there is the dilution effect, in which…

It was done.

267: remove ‘fact’

It was done.

276: the present instead of this (to make it clear to which study is being referred)

It was done.

284: concentration instead of prevalence

It was done.

284: …fatty acids in milk when compared….

It was done.

287: range instead of threshold

It was done.

288: remove ‘in fact’

It was done.

The numbers of (many of the) references do not correspond to the correct papers. Please check and amend.

All references have been checked and They were corrigited when necessary.

292: for instead of ‘of’

It was done.

293: results

It was done.

294: also found that the concentration (or percentage) of saturated acids decreased as lactation progressed. (Remove ‘in evaluating the lipid profile of mares’ milk throughout lactation in evaluating the lipid profile of mares’ milk throughout lactation.’)

It was done.

296: …third of lactation, when investigating the most important…..

It was done.

297: …linolenic acid concentrations were decreased….

It was done.

298: Consequently, the total amount of…

It was done.

301: Table instead of Tabela

It was done.

308: species or breeds?

It was done.

308: The high concentrations of linoleic and linolenic acids, which have important biological functions [2], ensure that the proportions of fatty acids are nearly ideal. Please provide reference for ideal values.

The Reference “Malacarne et al. 2002” has been added as suggested.

320: similar instead of close

It was done

326: other horse breeds

It was done

326: The milk composition is not influenced by…

It was done

330: ‘negative effects on the nutritional…’ instead of harming

It was done

335: horses

It was done

Reviewer #3: I can see that there has been a very good effort during this study. Here some advices to the authors:

I recommend an English review. There are some English mistakes that I have mentioned but there are other ones to be checked.

I understand that it is not easy to work with randomized conditions as mares were coming from different stables. This is why I consider that the composition of the food that they received should be added as an annex to cover all the possibilities of different compositions in colostrum and milk.

Unfortunately this point is delicate for our research. The three stud farms that were willing to receive the University are involved in equestrian competitions and keep a considerable level of competitiveness among themselves. Considering the direct correlation between diet and performance, the owners did not provide information related to the nutritional management of the breeding stock.

Were all the deliveries at term? If there were changes in the scheduled birth date (early delivery), the differences in the udder physiology may have produced alterations in the milk composition. I recommend to mention this but also to discuss it.

The text “All evaluated mares in this test gave birth naturally without cases of dystopian parts. In addition, mares which gave birth to a dead foals were discarded from the experimental group as well” has been inserted on line 95 as suggested.

Lines 64-67: The phrase is incredibly long. Divide it to make easier the understanding.

The text (line 64-67) has been divided and writeen and as suggested

Line 70: makes

It was done

Line 79 Birth order. What do you mean? Its position compared to the rest of individuals? The number of pregnancies?

Yes, the number of pregnancies. Here in Brazil, the transport of embryos has become frequent, as the number of foals produced by a matrix is often not the number of times that the mammary gland went into activity. So we evaluate this parameter

Line 93. Were there any selection criteria?

The reproduction reports of the properties were monitored and matrices that would give birth within the experimental period were selected. Fort this study, just those mares which had no complications during pregnancy and delivery remained under evaluation

Line 98: Colostrum samples were collected after delivery by manual milking.

The term “by manual milking” has been inserted to text (Line 98) as suggested.

Line 104: Erase last: If they were on the two hours preceding the procedure, It cannot be any others.

“Last” was erased.

Line 105: In order to guarantee sufficient volume of milk

“there being a” was erased.

Results and discussion. I highly recommend to separate the results from the discussion. If results are separated from the discussion, the reader can obtain its own conclusions without being guided and then compare them with the manuscript.

Besides, I consider that this way results are very poorly presented

Make the separation of the results from the discussion is not requirement from Plos one jornal so we decided keep the results and discussion together.

Lines 167-168. Separate them. It is a very strange sentence. I guess you mean: ...confirm its nutritional richness. It is important to highlight the high percentage...

It has been correct as suggested.

Table 1: Colostrum instead colotrom. To be corrected in the other tables as well.

Colotrom was replaced by colostrum.

Line 189: I guess you mean that there are not high concentrations of lactose in milk. Rephrase

It has been correct as suggested.

Line 199: Fat content found in this study...

It has been correct as suggested.

References: Some references like 29 has not the abbreviation: Acta universitaria should be abbreviated to Acta univ. I did not check all the references.

All references were checked and corrected when necessary.

Reviewer #4: Authors wanted to describe the composition of the quarter horse colostrum and milk. They seem to collect one sample of colostrum within 6h after birth and then, they collect milk samples, with no information about previous foal suckling, every 14 days. Analysis were performed on sample to give general data on milk (protein, lactose, caseins, lactose) and a detailed analysis of lipids.

General comments:

- The abstract should be totally re-written to explain the main method and results obtained in the study.

- They are few references and they are quite old: the newest one is from 2016 (only one) and all the others are older than 2014. Moreover, some links to reference are not accurate (example in line 287: reference 7 seems to be linked to a first author named Claeys and it’s not the case).

- Sometimes, English used in the paper is very strange, it should be reviewed by a native English speaker.

We appreciate the criterion with which our article was reviewed. The language and references have been checked and adjusted when needed as suggested.With regard to the bibliography consulted, for various reasons equine milk has not yet been extensively revised, which makes the number of articles on the subject limited. Even so, we can guarantee that our review included classic and important references on the topic, at the same time that we always try to cite the most recent studies as a way of showing the progress of science.

- The methods used and groups definition are poorly described:

The result tables show the well-characterized groups.

“(number of births in the matrix)” has been inserted on line 95 after birth order

Until the results & discussion parts, it is impossible to know what are the group of ages, of birth orders, and of lactation stages are not defined by the authors. All along the paper, the size of the different groups is never mentioned. Statistical validity of this study is thus poor.

o Sampling of the colostrum is difficult to understand. What was sampled in each mare: Only one sample in each mare within 6 hours? Did the foal suckle before or after? Many questions that interfere with results.

Yes. Each mare was evaluated only once in the postpartum period, given a small amount of colostrum and its importance for the newborn.

Some methods (example: infrared absorption in DairySpec FT Bentley) are poorly described, giving only the name of the manufacturer: rationale and descriptions are lacking.

Statistical procedures are poorly described. Most of the time, it is impossible to understand what is compared with what. There is no information about normality of data, how this has been tested and the potential use of non-parametric test if the data don’t follow a Gaussian distribution. In the end, the general impression is that job has not been done properly.

We are very sorry to have caused such an impression. The team worked hard throughout the research to expose the results so that it could contribute to the science.

- Results and Discussion

The choice of the structure that associate results and discussion is inappropriate because it is already very difficult to understand the groups (not) defined in the methods section. Giving clear results would help to understand how samples and groups have been used through the study.

Data observed are in quarter horses, but they may be similar in other horses: conclusion about the high protein level in quarter horse milk could be generalised to other breed if the same assays were used in other breeds.

Frequently, it’s difficult to know if the results/discussion part is talking about colostrum, milk or both. It should be stated each time!

The paragraphs that discuss colostrum and milk have been subtly flagged. Our intention was to produce a text with pleasant Reading.

Some statements are not statistically proven by the presented data or are not coming from references. We don’t care about what can be thought or imagined.In all presented tables, variables are expressed in % with no mention of percent of what: total milk, dry matter, defatted dry extract.

The percentages are for total milk. Such form of expression is recommended by the International Dairy Federation.

Presentation of results is very poor, only giving tables that are difficult to read. Some graphs showing evolution or differences could help the reader.

There is a major confusing factor that has not been statistically studied. It is obvious that mares with higher “birth order” (I would prefer using “lactation number”) will be older than primiparous one. A complete statistical model should have been realised to elude this confusion.

Here in Brazil it is not as obvious as it seems. If all mares started their reproductive life after puberty and had a birth each year, it would be as mentioned. However, here in Brazil equestrian sports strongly move the economy and many mares athletes start breeding only after retirement, while others, who were never athletes, start at three years of age. Thus, the wear and activity of the mammary gland are not similar in mares of the same age, which is why we decided to investigate the age and also the birth order of each matrix.

Conclusion

As the methods are poorly described and most of the time seem incomplete or inadequate, it is difficult to trust in the accuracy of the presented data. Thus, this paper is not suitable for publication.

Reviewer #5: Although the topic may be of interest,the manuscript lacks key elements:

The abstract contains unnecessary information; the keywords are not appropriate; the material and methods are not clear and lacking in information (eg. at 95 the authors do not explain how the groups were divided.

The text “...to age (young: 3 – 5 years; adults:6 – 10 years; seniors 11 – 19 years old), birth order (number of births throughout life, ranging from 1 to 6) and lactation stage (initial, medium and final third, considering a 180-day lactation).” has been added on line 95 in methodolgy section.

They do not report the separation times of the foal for the colostrum; the amount of milk collected, at the time of samples, was not reported).

The statistical analysis should have consisted in ANOVA for repeated measurement and to include the effect of the farm. Furthermore, the two variables considered (age and parity (birth order) give similar information and one of the two variables should be removed from the model.

A similar question was raised by one of your peers, however, age and birth order do not provide similar answers.

If all mares started their reproductive life after puberty and had a birth each year, it would be as mentioned. However, here in Brazil equestrian sports strongly move the economy and many mares athletes start breeding only after retirement, while others, who were never athletes, start at three years of age. Thus, the wear and activity of the mammary gland are not similar in mares of the same age, which is why we decided to investigate the age and also the birth order of each matrix.

The references are not appropriated and are also dated (eg Cit 1, 2). In addition, eight tables are too numerous and the titles of the tables are not self-explanatory (eg Table 7).

Superfluous information is often reported in the text (e.g. l 106-110).

All references and tables has been reviewed as suggested. The number of tables has been reduced from 8 to 7.

Finally, the results and conclusions are not satisfactory and should be reviewed in the light of the suggested considerations.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

31 Jul 2020

PONE-D-20-04990R1

Chemical composition and lipid profile of mare colostrum and milk of the quarter horse breed

PLOS ONE

Dear Dr. Chagas,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Needs to be checked native speaker English and all suggestions of reviewers should be addressed (even when you claim to have made changes). Please review the referee comments again and make your final revision.

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We look forward to receiving your revised manuscript.

Kind regards,

Arda Yildirim, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Needs to be checked native speaker English and all suggestions of reviewers should be addressed (even when you claim to have made changes). Thanks

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: I Don't Know

Reviewer #3: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Dear authors,

I feel the paper could still benefit from further English editing and the revision has introduced sections that are difficult to read/comprehend. I have included some specific comments below, but am somewhat concerned that quite a few of my suggestions have not been addressed (even when you claim to have made changes). You have also referenced a number of papers that I cannot access and/or read. I assume these are relevant and have been cited appropriately.

Specific comments/suggestions:

Title: Quarter Horse (captal Q and H)

43: just 'subjected' (not 'subjected instead of submitted')

45: the sentence has not been changed and I (still) think a comment should be added that the protein and lactose content are different to those (reported) in other breeds.

71: just 'contains' (not 'is contains')

75: remove 'composed of'

78: is it 14% in milk and 29% in colostrum, please clarify

91: 'submitted for' (rather than 'submitted to')

92: please explain UFRN

104: dystocia instead of dystopian parts

105: were these removed from the group of 34 mares, or never included? please clarify

106: presumably collections took place within two seasons within this time frame. Please clarify

180: 'in the colostrum' (rather than 'for the colostrum')

182: 'in Hungarian'

184: 'differed from that presented' (remove 'instead of those')

213: 'The fat content found in this study was lower...'

218: influenced or complicated rather than surrounded

222: 'nursing by the foal throughout'

242: I don't think it is correct to conclude that the fat% in your study is lower than the 0.9% reported by Burns, as both values will be a mean (with standard deviation) and you have not statistically compared the data. Maybe: seems to be slightly lower than the 0.9%.....

267-270: I was hoping for a statement to indicate that not all changes found are likely to be biologically relevant (because they are so small, even if they are statistically relevant). It is not clear (to me) what you are trying to say in this sentence.

298: 'the present study' (instead of 'this study')

304: 'concentration' (instead of 'prevalence')

304: '...fatty acids in milk compared to colostrum'

307: 'range' (instead of 'threshold')

308: remove 'in fact'

314: 'results' (instead of 'result')

350: 'The milk composition is not influenced by...'

359: 'horses' instead of 'horse'

Reviewer #3: Authors did correct most of the proposed comments/changes.

However, I still consider (as two of my peers) that results should be separated from the discussion as explained before. When there are several peers that are addressing this poorly presentation of results, the author should consider to change it even it is not a mandatory requirement of PlosOne as answered by the authors.

This is why, I let the decision of accepting the article with a gathered results/discussion section to the editors.

**********

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Reviewer #1: Yes: Manal Bakry Mohamed Hemida

Reviewer #2: No

Reviewer #3: No

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24 Aug 2020

The authors thank the Reviewers and Editor for the considerations. The research group has been working hard to get the work published in the journal. The manuscript has been reviewed as suggested by editor and reviewers.

Editor Comments:

Needs to be checked native speaker English and all suggestions of reviewers should be addressed (even when you claim to have made changes). Thanks

The manuscript has been revided as suggested.

Reviewer #1: (No Response)

Reviewer #2: Dear authors,

I feel the paper could still benefit from further English editing and the revision has introduced sections that are difficult to read/comprehend. I have included some specific comments below, but am somewhat concerned that quite a few of my suggestions have not been addressed (even when you claim to have made changes). You have also referenced a number of papers that I cannot access and/or read. I assume these are relevant and have been cited appropriately.

The manuscript has been revided as suggested.

Specific comments/suggestions:

Title: Quarter Horse (captal Q and H)

The correction has been made.

43: just 'subjected' (not 'subjected instead of submitted')

The correction has been made.

45: the sentence has not been changed and I (still) think a comment should be added that the protein and lactose content are different to those (reported) in other breeds.

The correction has been made.

In the lines 42-43 the text has been modified as follow:

“There was a high protein content and reduced lactose content for the colostrum of the Quarter Horse mares, differing from other breeds”

71: just 'contains' (not 'is contains')

The correction has been made.

75: remove 'composed of'

The correction has been made.

78: is it 14% in milk and 29% in colostrum, please clarify

In the lines 79-80 the text has been modified as follow:

Regarding colostrum, which has a dry matter much higher than milk (14 in milk to 29% in colostrum)...

91: 'submitted for' (rather than 'submitted to')

The correction has been made.

92: please explain UFRN

The correction has been made.

104: dystocia instead of dystopian parts

The correction has been made.

105: were these removed from the group of 34 mares, or never included? please clarify

In the lines 110-112 the text has been modified as follow:

…In addition, mares, which gave birth to dead foals, were discarded from the experimental group as well, with 34 mares remaining in the experimental group at the end of the selection...

106: presumably collections took place within two seasons within this time frame. Please clarify

In the lines 112-114 the text has been modified as follow:

...The collections took place between the months of July/2017 and September/2018, because the 34 births were not concentrated in a single period...

180: 'in the colostrum' (rather than 'for the colostrum')

The correction has been made.

182: 'in Hungarian'

The correction has been made.

184: 'differed from that presented' (remove 'instead of those')

The correction has been made.

213: 'The fat content found in this study was lower...'

The correction has been made.

218: influenced or complicated rather than surrounded

The correction has been made.

222: 'nursing by the foal throughout'

The correction has been made.

242: I don't think it is correct to conclude that the fat% in your study is lower than the 0.9% reported by Burns, as both values will be a mean (with standard deviation) and you have not statistically compared the data. Maybe: seems to be slightly lower than the 0.9%.....

The correction has been made.

The sentence has been rewritten in line 263 as follow:

… of lactation, it seems to be slightly lower than the 0.9% reported by Burns et al. [17]...

267-270: I was hoping for a statement to indicate that not all changes found are likely to be biologically relevant (because they are so small, even if they are statistically relevant). It is not clear (to me) what you are trying to say in this sentence.

The correction has been made.

The sentence has been added in line 293 as follow:

...This can be an important detail in the management of herds that specialize in producing milk, or even in foal nutrition.

298: 'the present study' (instead of 'this study')

The correction has been made.

304: 'concentration' (instead of 'prevalence')

The correction has been made.

304: '...fatty acids in milk compared to colostrum'

The correction has been made.

307: 'range' (instead of 'threshold')

The correction has been made.

308: remove 'in fact'

The correction has been made.

314: 'results' (instead of 'result')

The correction has been made.

350: 'The milk composition is not influenced by...'

The correction has been made.

359: 'horses' instead of 'horse'

The correction has been made.

Reviewer #3: Authors did correct most of the proposed comments/changes.

However, I still consider (as two of my peers) that results should be separated from the discussion as explained before. When there are several peers that are addressing this poorly presentation of results, the author should consider to change it even it is not a mandatory requirement of PlosOne as answered by the authors.

This is why, I let the decision of accepting the article with a gathered results/discussion section to the editors.

The authors of this manuscript has been decided to keep the resultas and discussion together but it can modified as the Reviewer 2 suggested if it is a requirement of the Plos one journal.

Submitted filename: Response to Reviewers 24.08.docx

Click here for additional data file.

27 Aug 2020

Chemical composition and lipid profile of mare colostrum and milk of the quarter horse breed

PONE-D-20-04990R2

Dear Dr. Chagas,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Arda Yildirim, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thanks for your hard work.

Reviewers' comments:

3 Sep 2020

PONE-D-20-04990R2

Chemical composition and lipid profile of mare colostrum and milk of the quarter horse breed

Dear Dr. Chagas:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Dr. Arda Yildirim

Academic Editor

PLOS ONE