Changes in Total Antioxidant Contents of Colostrum of Honamli and Hair Goat Breeds During the First 5 Days of Parturition

Colostrum is the exclusive provider of natural host defense system molecules and antioxidants or antioxidative system for the offspring’s. Goat neonates are highly susceptible to infection and colostrum nutrients must be acquired by the neonates through suckling during the early hours of life (Castro et al., 2005). Most of studies focused on the presence of antioxidative factors in colostrum and milk in cow (Kankofer and Lipko-Przybylska, 2008) and sow (Lipko-Przybylska and Kankofer, 2012). Generally, colostrum is defined as secretion of mammary glands as the first 4 days after post-parturient period in bovine (Gopal and Gil, 2000). Therefore, colostrum samples from two goat breeds were collected during initial five days postpartum. In this present study, we have employed spectrophotometric ABTS-based method for measurement of the antioxidant efficacy and the Folin-Ciocalteu method for testing total phenolic content in colostrum samples. One of the distinctive features of this work is used goat breeds in present study; Honamli goat which is indigenous, unique and it has been recently registered by the Ministry of Agriculture and studies mostly focused on the determination of its morphological and production traits. Honamli goats are massive, taller and larger and are bred at the plateau of Taurus Mountains, around Burdur, Antalya, Isparta and Konya for both milk and meat yields (Elmaz et al., 2012). The other goat breed is the Hair goat (Anatolian Black) breed is more prevalent and is the major goat breeds in Turkey (Akbas and Saatci, 2016).
        
The determination and comparison of TAC values in Honamli and Hair goat colostrum on daily basis following parturition may likely contribute literature for description of antioxidant capability of both indigenous goat breeds. We found differences between two breeds and significantly higher total antioxidant capacity value of colostrum’s of both breeds compared to those of mature milk in both goat breeds.

Animal procurement
 
The Burdur Mehmet Akif Ersoy University Research Ethics Committee approved all procedures in this study (permission number:291, 03/05/2017). 10 Honamli and 8 Hair goats of 2-2.5 years old in their 2^nd pregnancy were used because, the Honamlý goat flocks were consisting of goats with 2-2.5-year-old goats readily available at Burdur Mehmet Akif Ersoy University, Agriculture, Livestock and Food Application and Research Centre. The average weight of 72 ± 5 kg for Honamli and 60 ± 4 kg for Hair goats. Hair goat group was consisting of goat with similar age to that of Honamli goats and owned by private producer. Both Honamli and Hair Goat Flocks fed in a semi-extensive herd (pasture and integration with concentrate). The both farms were located in the Burdur province where in Western Mediterranean Region of Turkey. The colostrum samples were collected within an hour 1^st sample after parturition and were subsequently taken at daily intervals from the goats for 5 days between March and April 2018.
Collection of colostrum samples
 
The animals were fed ad libitum with grass silage and with concentrates or hay and had water from the pen system. Also, the animals were not given nutritional supplements before parturition and selected animals were separated from the rest of flock with single birth. Before collecting colostrum samples, udders of each goat washed with water and dried it using paper towels. The colostrum samples were taken with hand milked and were stored in clean and dry sterile 50 ml Falcon tubes and were transported under refrigeration conditions to the laboratory where samples were frozen -80°C until analysis. Before analysis, colostrum samples were thawed after attaining 37°C in water. Control milk samples were collected approximately 20th day postpartum.
 
Measurement of TAC levels in goat colostrum by the ABTS method
 
Total antioxidant activity of colostrum samples was measured using the ABTS test (Re et al., 1999) with slight modifications. Trolox equivalent antioxidant capacity (TEAC) is one of the assays applied for the determination of the antioxidant capacity of variety of food stuff including milk (Zulueta et al., 2007). The stock solution of ABTS radicals was prepared by mixing potassium persulfate solution (Merck, Darmstadt, Germany) (2.6 mM) with the ABTS solution (7 mM) (Merck, Darmstadt, Germany) and this mixture was stored in dark at room temperature for 12 hours. The working solution was prepared by diluting the stock solution with chromatographic grade methanol (Sigma, St. Louis, MO, USA) to a final absorbance of 1.1 ± 0.002 at 734 nm. After colostrum samples (0.3 mL) were mixed with 2.7 mL of working solution, they were incubated at room temperature for 30 min. Immediately after centrifugation at 12,000 ´ g for 2 min at room temperature. Absorbance’s of clear solutions were measured at 734 nm against methanol (blank) by a spectrophotometer (Optizen Pop, Mecasys Co., Ltd., Korea). The antioxidant capacity of the goat colostrum samples was expressed as Trolox®equivalent (TE) (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Fluka, St. Louis, MO, USA) antioxidant capacity (TEAC). TEAC was measured duplicate and mean value was given for each colostrum sample. Calibration curve was obtained with the average of two parallels corresponding to blank, 25, 20, 16.6, 10 and 5 µM μmol / L Trolox solutions. The equation of the line used was y = -0,0347x + 1,0853 with an R² = 0,9997. The absorbance readings were recorded at 734 nm after the 30 min of inhibition period. The results were expressed as mmol of Trolox equivalent (TE) per liter of colostrum sample.
 
TPCs analysis of colostrum samples
 
The spectrophotometric Folin-Ciocalteu method (Folin and Ciocalteu, 1927) according to Singleton and Rossi (Singleton and Rossi, 1965) was used to determine the total phenolic contents of colostrum samples, using gallic acid (Fluka, St. Louis, MO, USA) as a standard. The working solution included the Folin-Ciocalteu (FC) reagent (Merck, Darmstadt, Germany) and distilled water (1:10, v/v). After sample or standard (1 mL) was mixed with FC working solution (5 mL) and incubated for 3 min, 4 mL of sodium carbonate (Sigma, St. Louis, MO, USA) (75 g/L) was added. This mixture was incubated in the dark for 2 hours at room temperature, followed by the centrifugation for 2 min at 12,000 g. Gallic acid (GA) was used to obtain the calibration curve which was prepared using as standard with concentrations of blank, 25, 50, 62 and 100 mg/L. The linear equation was y= 0,026x + 0,0215, with a R²= 0.995. Absorbance values were determined by a spectrophotometer at 760 nm against distilled water. Results were expressed as mg GA equivalents (GAE) per liter of colostrum samples. Both the tests were carried out in duplicate on each goat colostrum and milk sample.
 
Statistical analysis
 
Statistically significant differences among means were determined by the analysis of variance (ANOVA) using the SAS software program (The SAS System for Windows 9.0, Chicago, USA). The Duncan’s multiple range test was conducted for the separation of means with significant differences at α=0.05 level.

Changes in TAC and TPCs values of Honamli and Hair goat’s colostrum in the first five days after parturition are presented in Table 1 and 2. In Honamli and Hair goat groups, both TAC and TPC contents were the highest in the first day colostrum; it markedly decreased with days and the lowest in mature milk. Honamli goat colostrum samples were to have the higher TAC values than those of Hair goat (P<0.05). With 1^st colostrum, the TAC concentration of Honamli colostrum was 107.09 µmol TE/L and in Hair goat colostrum was 85.97 µmol TE/L respectively. The average TAC value decreased to its lowest value of 70.12 ± 24.23 µmol TE/L on the fifth day in Honamli and 56.01 µmol TE/L at the fourth day in Hair goat following parturition. The average TAC value of Honamli and Hair goat milk was 35.09 and 32.11 µmol TE/L.
 



       
At calving, there was significant (P<0.05) difference in TACs concentration between Honamli and Hair goat colostrum (Table 2). The TPCs was the highest at 2815 mg/GAE/L in Hair goat and 2728 mg/GAE/L in Honamli goat colostrum on first colostrum respectively. The concentration of TPCs decreased in both Hair and Honamli goat colostrum and it reached to the lowest values of 2117 mg GAE/L in Hair and 1814 mg GAE/L in Honamli goat colostrum samples after five days of postpartum respectively. Hair goat colostrum samples were to have the higher TPCs values than those of Honamli goat (P<0.05). The average total phenolic content of milk was 191.88 mg GAE/L in Honamli and 217.69 mg GAE/L in Hair goat colostrum samples. The average TAC recovery was 99.97% in the spectrophotometric ABTS method and 99.23% in the total phenolic content assays.
       
The present study showed that the presence of significant breed differences between colostrum samples TAC and TPCs contents of both goat breeds (P<0.01). In the present study, we had collected colostrum samples from Honamli and Hair goats at similar age, single birth, grown at the same altitude and fed under the similar conditions to reduce the impact of such factors. The higher values of standard deviation observed in both the assays in colostrum sample measurements were strictly related to colostrum chemistry composition, which likely affect on individual characteristics of goat breeds (Doreau et al., 1990). The ABTS values of Honamli and Hair goat milk measured in the prior studies were much lower than the results in the present study. The difference in method adopted for measurement of TAC in milk (Lipko-Przybylska et al., 2010; Albera and Kankofer, 2011) are in agreement with the present study. In the present study, average total phenolic value of milk was 191.88 ± 23.52 mg GAE/L in Honamli and 217.69 ± 26.32 mg GAE/L in Hair goats which were higher than those reported by Chavez-Servin (Chavez-Servin et al., 2018). He reported that, TPCs values in Alpine goat milk as 87.90 ± 30.19 mg GAE/L in free-ranged and 65.26 ± 11.26 mg GAE/L in confinement goat. A study suggested that the concentration of antioxidant compounds in goat milk slightly depends on forage flavonoid content (De Foe et al., 2006). Therefore, present study was planned to reduce the factors such as seasons, farming system, grazing system, feeding practice and altitude on colostrum TPCs, by choosing goats farmed in similar altitude (37^o 43' 6.01 N 30^o 16' 56.399" E), farming system and pasture quality. Also, both goat breeds consumed a similar pasture plants as well. According to our findings, colostrum TAC and TPCs were seem to be more influenced by the breed than factors mentioned above and vary across breeds as well. Previously, it was showed that the presence of difference on the basis of significant antioxidant capacity among the Jamnupari, crossbreed of the Jamnupari and Saanen goat’s milk samples (Alyaqoubi et al., 2014). They concluded that the Jamnupari goat breed exhibited the highest capacity parity in total phenol content (TPC), ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. In the present study, total antioxidant capacity of the goat breeds colostrum samples showed significant (P<0.05) lower values during the course of lactation, which can be a natural result of a decline in antioxidant reservoirs of mothers.

The present study concluded that Honamli and Hairy goat colostrum have a great antioxidant capacity as demonstrated using ABTS and Folin-Ciocalteu assays. From the overall results, it is concluded that there were major differences between the goat breeds and colostrum TAC and TPC values. The highest values of TAC and TPCs occurred in the first hours after calving and after which those contents were decreased in both breeds. In the present study, it was confirmed that the secretion of antioxidants in high levels via colostrum continued as long as for five or longer days of parturition, therefore the intake of colostrum by kids can be extended for a longer period.

We thank Professor Dr. Yusuf Yilmaz, Department of Food Engineering, Faculty of Engineering and Architecture, Burdur Mehmet Akif Ersoy University for allocating bench space for colostrum antioxidant content analysis in his laboratory and valuable discussions on the topic. We also acknowledged the support from The University of Burdur Mehmet Akif Ersoy, Burdur, Turkey. This work was supported by the Burdur Mehmet Akif Ersoy University Scientific Research Projects Coordinator Office under Postgraduate Education Projects (No: 0479-YL-17). The funded agency did not involved in the study design, collection of colostrum samples, laboratory analysis, data analysis, documentation of the data and decision of article submission for publication.

The authors report no conflicts of interest.