The current study was undertaken to study the effect of lactation number on quality of bovine colostrum under temperate conditions. It was found that the specific gravity, fat, total protein, lactose and total solids content of the colostrum samples from second lactation cows were significantly (p ≤ 0.05) lower than other lactations. Casein protein, whey proteins, solids not fat and electrical conductivity were comparable among different lactations. The specific gravity of the colostrum samples from first lactation cows was significantly (p ≤ 0.05) higher than other lactation cows. The ash content of the colostrum samples of fourth lactation was significantly (p ≤ 0.05) higher than first and second lactation however it was comparable other lactation samples. The pH the colostrum samples from fifth lactation cows was significantly (p ≤ 0.05) lower than other lactations which among themselves possessed comparable (p > 0.05) values. The total plate count (TPC) of the colostrum samples from sixth lactation cows was significantly (p ≤ 0.05) higher than other lactations which among themselves possessed comparable (p > 0.05) values.
Colostrum is not only a good source of nutrients, it also has biologically active substances which are important for nutrition and health. Colostrum an early milk secreted at the time of parturition and is rich in antibodies which acts as an immune booster for post-natal calf health (Verma et al., 2018). The colostrum composition changes with a number of factors. The composition of mammary secretion varies during the whole lactation period to fulfill the changing metabolic needs of the new born from birth till weaning. Colostrum is the mammary secretion produced shortly after parturition (Levieux and Ollier, 1999), through first 24 h after parturition or during the first few days after calving (Tsioulpas et al., 2007). Bovine colostrum has numerous purported health benefits, if harvested immediately after calving to maintain its quality. The concentration of total solids is higher in bovine colostrum compared to bovine milk (27.6%, w/w, versus 12.3%, w/w), because of greater protein content (14.9% versus 2.8%) and more fat content (6.7% versus 4.4%), whereas the concentration of lactose is lower (2.5% versus 4.0%) as reported by Fox and McSweeney (2003); Kehoe et al. (2007). The concentration of growth factors and immunoglobulins is highest in the first portion of colostrum, which decrease rapidly thereafter (Playford et al., 2000; Blum, 2006). Immunoglobulins consist of greater than 50% of the total proteins present in colostrum, which comprise almost all antibodies, present in maternal blood. IgG1 constitutes about 90% of colostrum Ig (Gapper et al., 2007).
The growth factors and immune factors present in bovine colostrum are similar to those present in human colostrum but in higher quantities: IgG concentration in human colostrum is 2% while in bovine colostrum it is 86% (Wilson, 1997). Bovine colostrum rebuilds the immune system, destroys viruses, bacteria and fungi, accelerates healing of all body tissue, helps lose weight, burn fat, increase bone and lean muscle mass and slows down and even reverses aging. There is variation of colostral Ig and lactoferrin concentrations of dairy cows with lactation number. The primiparous cows have lower values than multiparous cows. Plasma Ca and inorganic P (PI) concentration of dairy cows decreased around parturition because of the large transfer of Ca and P to colostrum, and older cows have a greater risk of developing milk fever. The lactation number is a factor altering mineral concentration of colostrum, concurrently shifting the mineral status of newborn calves and postpartum cows (Kume and tanabe,1993). Colostrum quality is higher in the animals which give birth to male calves than in animals which give birth to female calves (Nazir et al, 2018).
Materials and Methods
Source of Colostrum
Colostrum samples were collected from the MLRI, SKUAST-Kashmir and various field locations. A total of ninety-nine samples were collected. The samples were collected in sterile containers and transported to the laboratory in ice cool totes, thereafter the samples were analyzed for the following parameters for three consecutive days post parturition as per approved procedures-
All the chemicals used were of analytical grade and were obtained from standard firms (Qualigens Fine Chemicals, Nice Chemicals Pvt. Ltd., Hi Media Lab. Pvt. Ltd. etc.).
Preparation of Samples
Colostrum was warmed and thoroughly mixed by pouring into the clean receptacle and back repeatedly and whenever needed with plunger/stirrer to reincorporate any material adhering to containers in order to make sure that the samples collected were representative of the entire batch of colostrum that was being sampled. After thorough mixing about 200ml of colostrum was taken in sampling bottles with the help of colostrum sampler and the analysis was carried out immediately.
All the analytical procedures required for the analysis of colostrum were carried out in the laboratory of the Division of Livestock Products Technology, Faculty of Veterinary Sciences and Animal Husbandry, SKUAST-Kashmir, Shuhama Alusteng, Ganderbal. For physico-chemical analysis about 200ml of colostrum was used for the determination of various parameters.
pH of Colostrum
The pH of colostrum samples was recorded by directly dipping the combined electrode of digital pH meter (Tanco Lab. Equipments), after proper calibration of the instrument, into the samples. Two readings were taken for each sample and average pH recorded.
Specific Gravity of Colostrum
For determination of specific gravity of colostrum, Zeal type lactometer was used. After recording the temperature of the sample correctly lactometer reading was recorded. The corrected lactometer reading was calculated to arrive at the correct specific gravity
Electrical Conductivity (EC) of Colostrum
Electrical conductivity of the samples was taken by dipping the electrode of electrical digital conductivity meter (brand “TANCO, India Lab. Equipment’s”) into the sample after proper calibration of instrument. Two or three readings were taken for each sample and average electric conductivity was calculated.
The colostrum samples were analyzed for determination of various physico-chemical parameters using the standard procedures of Association of Official Analytical Chemists (A.O.A.C., 1995). Brief description of the methods is outlined below-
Total Solids (TS) of Colostrum
For the determination of total solids about 10g of the colostrum sample in duplicate was weighed accurately on electronic balance, corrected up to 0.1mg, in a dry, pre-weighed, flat bottomed moisture cups and kept in hot air oven at 102 ±1°C for 4 hours. Then moisture cups were transferred immediately to a desiccator to cool to the room temperature (at least 30 minutes). The process of drying, cooling and weighing was repeated at 30 minutes interval until the difference between the two consecutive weighing readings was less than one milligram. Weight loss of the cup after drying was recorded and expressed in terms of total solids percent.
% Total solids = [(W1– W)/ (W2 – W)] x 100
W = weight of empty dried cup (g)
W1 = weight of cup + sample after drying (g)
W2 = weight of cup + sample (g)
Solids Not Fat (SNF) (Colostrum)
SNF of the colostrum was calculated by indirect method. The difference between total solids (%) and fat (%) gave the SNF content in colostrum.
SNF (%) = TS% – Fat%
Fat of colostrum was estimated by Gerber’s method (IS: 1224 (1977). 10ml of Gerber’s sulphuric acid (90ml of concentrated sulphuric acid added to 10ml of distilled water) was taken carefully in a clean dry butyrometer (ISI marked) with the help of automatic dispenser (tilt measure) without wetting the neck. To this 10.75 ml of thoroughly mixed colostrum sample was added with the help of milk pipette on the side walls of the butyrometer. Then 1ml of amyl alcohol was added to the butyrometer on the sides. Dry rubber lock stopper was used to close the butyrometer. These were then shaken and inverted 2-3 times till complete dissolution of the acid and colostrum contents. Then tubes were placed in water bath for 5 minutes at 65±2°C to ensure that all the casein particles were dissolved. The butyrometer tubes were then placed in a centrifuge in a radial symmetry and as evenly spaced as possible. Centrifugation was done for 4 minutes at 1100 rpm. Butyrometer tubes were then removed from centrifuge and placed again in water bath for 5 minutes at 65±2°C. With the help of stopper and key, the fat level was adjusted in such a way that scale reading corresponds to the lowest point of the fat meniscus and the surface of separation of the fat and acid. The observed fat level was recorded as percent fat of test sample.
Micro-kjeldahl method was followed for determination of protein content of colostrum (AOAC, 2000).
For determination of ash about 10 ml of colostrum samples in duplicate were accurately weighed on electronic balance, corrected upto 0.1 mg, in dried and preweighed crucibles and kept in hot air oven at 102 ±1°C for 4 hours. The sample in the crucible was subjected to carbonization followed by incineration of the sample by placing the crucible in muffle furnace at 550°C – 600°C for about 2 hours (AOAC, 2000).
Lane-Eynon Oxidation–Reduction Reaction method was followed for determination of lactose content of colostrum samples. About 25 ml of colostrum was taken in a 500 ml conical flask and diluted with distilled water to about 200 ml. About 3.75 ml of 10 per cent acetic acid solution were added to it and then subjected to boiling. On cooling, it was transferred quantitatively to a 250 ml volumetric flask and the volume was made up to mark with distilled water. It was then filtered through a filter paper and the filtrate was collected in a dry conical flask. The burette was filled with this filtrate. 5 ml of each of Fehling solution A and B were pipetted into 250 ml of conical flask and preliminary titration was made by adding the filtrate containing lactose, from the burette, 1 ml at a time, to the Fehling solution kept boiling till the blue colour changes to red. About 5 drops of methylene blue indicator were added to the boiling mixture and titration was completed within a total boiling time of 3 minutes by additions of 4 to 6 drops of the filtrate till end point was reached indicated by the change of blue colour to colourless supernatant.
Lactose (%) = W/V x 250 x 100/25 x 1/1000
V = Volume of filtrate required for complete reduction of 10 ml of Fehling solution
W = Lactose equivalent in mg for V ml
The colostrum samples were collected in sterile containers and bought under hygienic conditions to the laboratory of Division of LPT, F. V. Sc. and A. H., SKUAST-K, were subjected to microbiological analysis for total plate count using standard plate count technique as per APHA (2004).
Sample Preparation and Serial Dilution
About 10ml of colostrum was aseptically transferred to a pre-sterilized volumetric flask and 90ml of peptone water was added to it to get solution of 10-1 dilution. About 1ml of this diluted solution was transferred to another tube containing 9ml of sterile 0.1 percent peptone water (peptone from Qualigens Fine Chemicals) to get 10-2 dilution. This procedure was repeated to obtain 10-3 dilution and so on, until appropriate dilution was achieved which yielded plates with 25 to 250 colony forming units (cfu). All the procedures were performed in the sterilized environmental conditions of laminar air flow (NSW-201 Horizontal Laminar Flow cabinet).
Total Plate Count
For determination of TPC, total plate count agar (Hi-Media Laboratories, Pvt. Ltd., Mumbai) was used. About 17.5g of it was dissolved in 1000ml of distilled water followed by sterilization in an autoclave at 15 lb pressure (121oC) for 15 minutes and cooled to remain at 45oC. With the help of sterile pipette serial dilutions of sample were made and 1ml from each test tube was inoculated into a double set of pre-sterilized petridishes. Pour plate technique were followed for plating. The innoculum and media in petridishes were mixed thoroughly and uniformly by rotating the plates alternatively in clockwise and anticlockwise directions followed by back and forth motion on level surface. When media in plates solidified, they were inverted and incubated aerobically at 35±1°C for 24±3 hours. The number of micro-organisms per ml of sample was calculated by selecting plates containing 25 to 250 cfu/ml or selecting plates with count closest to this range. The cfu/ml was calculated by using the formula-
N = ∑C/ [(1 x n1) + (0.1 x n2)] d
N = number of colonies per milliliter of product
∑C = sum of all colonies on all plates counted
n1 = number of plates in lower dilution counted
n2 = number of plates in next higher dilution counted
d = dilution from which the first counts were obtained
Finally, the cfu/ml was expressed as log10 cfu/ml of sample
The data obtained from duplicate samples were averaged and the data so generated were analyzed statistically following the method of Snedecor and Cochran (1980), Gomez and Gomez (1984) and Steel and Torrie (1984). The data was processed in a computer using SPSS software package. The analysis of variance of group mean was computed and significance of means tested by using least significant difference test at 5 per cent level of significance. One-way and two-way analysis of variance with all possible interactions was carried out. The nested means were compared when the interaction was found to be significant. In the absence of such significance the overall means were compared.
Results and Discussion
The data pertinent to the study related to the effect of the Lactation number and transition period on various physico-chemical, compositional and microbiological characteristics of bovine colostrum has been outlined in Table 1 and graphically portrayed in Fig.1 and 2. As indicated by the result, irrespective of the lactation number of the animal, the day 1 postpartum colostrum samples had significantly (p ≤ 0.05) higher specific gravity than day 2 and day 3 colostrum samples and between the latter two samples the day two samples had significantly (p ≤ 0.05) higher specific gravity compared to day three samples. The results agree favourably with those of Foley and Otterby (1978), Quigley III et al (1994), Morin et al (2001) and Sobczuk-Szul et al (2013). Without regard to the days of transition, the specific gravity of the colostrum samples from second lactation cows was significantly (p ≤ 0.05) lower than first lactation cows. The findings are parallel to the findings of Quigley III et al. (1994). The specific gravity of the colostrum samples from first lactation cows was significantly (p ≤ 0.05) higher than other lactation cows. These results corroborate the findings of Morin et al. (2001). The fat content of the colostrum samples during days periods postpartum showed a declining trend with values being significantly (p ≤ 0.05) different from one another, irrespective of the lactation number of the animal under study. The values are close to the values reported by Foley and Otterby (1978), Klimes et al. (1986) and Raducan et al. (2013). As far as the fat values of the colostrum samples from different lactation numbers are concerned, without regard to the days of transition, the fat content of the colostrum samples from second lactation cows was significantly (p ≤ 0.05) lower than other lactations.
The results are parallel to the findings as reported by Morill et al. (2012). Total protein content of the colostrum during the transition period postpartum declined progressively with values at each day under study being significantly (p ≤ 0.05) different from one another. Similar results are reported by Foley and Otterby (1978), Klimes et al. (1986), Elfstrand et al. (2002) and Raducan et al. (2013). Regardless of the days of transition, the total protein content of the colostrum samples from second lactation cows was significantly (p ≤ 0.05) lower than other lactations. Similar results have been cited by Quigley III et al. (1994). The casein protein content of the colostrum samples on day 1 were significantly lower than day 2 and day 3 while the values at day 2 were significantly higher than day 3. Similar trend was reported by Benheng and Chengxiang (1996). Without regard to the days of transition, the Casein protein content of the colostrum samples among different lactations was comparable. The results agree favourably with the findings of Quigley III et al. (1994).
Table 1: Effect of the lactation number during transition phase on various physico-chemical, compositional and microbiological characteristics of bovine colostrum (Mean ±S.E.)
|Days||Lactation number||Overall mean|
|D2||1.048 ±0.003||1.043 ±0.002||1.046 ±0.004||1.042 ±0.003||1.042±0.005||1.040±0.003||1.044±0.0012|
|D3||1.041 ±0.002||1.039 ±0.001||1.041 ±0.003||1.037 ±0.002||1.036 ±0.004||1.036 ±0.002||1.039±0.0013|
|Total protein (%)|
|Casein protein (%)|
|Whey protein (%)|
|Total solids (%)|
|Solids not fat (%)|
|Electrical conductivity (mScm-1)|
|Total plate count (log10cfu/ml)|
Mean±SE with different superscripts row-wise (alphabets) and column wise (numerals) differ significantly (p≤0.05)
The whey protein content of the colostrum samples during various periods postpartum showed a declining trend with the values at day 1 being significantly (p ≤ 0.05) higher compared to either day 2 or day 3 which within themselves were comparable. These results corroborate the findings of Klimes et al (1986) and Benheng and Chengxiang (1996). Without regard to the days of transition, the whey protein content of the colostrum samples showed no significant (p > 0.05) difference among different lactation.
Fig. 1: Effect of the lactation number during transition phase on specific gravity (a), fat (b), total protein (c), casein protein (d), whey protein (e) and lactose (f) of bovine colostrum.
Fig. 2: Effect of the lactation number during transition phase on total solids (a), solids not fat (b), ash (c), pH (d), electrical conductivity (e) and total plate count (f) of bovine colostrum
Lactose while opposing the decreasing tendency of other parameters, went on increasing with each passing day post-partum having lowest value at day 1 and highest at day 3 postpartum, all the three values were significantly (p ≤ 0.05) different from one another. The findings of Foley and Otterby (1978), Benheng and Chengxiang (1996), Elfstrand et al. (2002) and Kleinsmith (2011) are in close conformity with the outcome of the current study. Irrespective of the days of transition the lactose content of colostrum samples of second lactation was significantly (p ≤ 0.05) lower than the lactose content of colostrum samples of first, third, fourth and fifth lactation however the values compared favourably with sixth lactation values. The values are more or less close to the values reported by Morill et al. (2012). Total solids of the colostrum samples during various periods postpartum went on decreasing with values being significantly (p ≤ 0.05) different from one another, irrespective of the age of the animal under study. Similar trend has been reported by Foley and Otterby (1978), Klimes et al. (1986) and Raducan et al. (2013). As far as the total solids value of the colostrum samples from different lactation numbers is concerned, the colostrum samples from second lactation cows had significantly (p ≤ 0.05) lower total solids than other lactations. Similar results are reported by Quigley III et al. (1994) and Morill et al. (2012).
Irrespective of the different lactation numbers the day 1 postpartum colostrum samples had significantly (p ≤ 0.05) higher solids not fat than day 2 and day 3 colostrum samples and between the latter two samples, the day 2 samples had significantly (p ≤ 0.05) higher solids not fat compared to day 3 samples. Similar trend has been reported by Raducan et al. (2013). As far as the solids not fat value of the colostrum samples from different lactation numbers are concerned, there was no significant (p > 0.05) difference among them. Without taking into account different lactation numbers, the day 1 postpartum colostrum samples had significantly (p ≤ 0.05) higher ash content than day 2 and day 3 colostrum samples which within themselves possessed comparable ash content. Findings reported by Klimes et al. (1986) and Tsioulpas et al. (2007) corroborate the findings in the current study. Without regard to the days of transition, the ash content of the colostrum samples of fourth lactation was significantly (p ≤ 0.05) higher than the lactose content of colostrum samples of first and second lactation. The pH the colostrum samples from fifth lactation cows was significantly (p ≤ 0.05) lower than other lactations. The latter among themselves possessed similar (p > 0.05) values. Irrespective of the different lactation numbers pH values increased significantly (p ≤ 0.05) with every passing day post-partum upto day 3 post-partum. The day 1 postpartum colostrum samples had significantly (p ≤ 0.05) lower pH value than day 2 and day 3 colostrum samples and between the latter two samples, the day 2 samples had significantly (p ≤ 0.05) lower pH values compared to day 3 samples. Similar increase has been reported by Klimes et al. (1986) and Elfstrand et al. (2002).
The electrical conductivity of the colostrum samples, without regard to the days of transition possessed comparable values having no significant (p > 0.05) difference among different lactation numbers whatsoever. Irrespective of the different lactation numbers the day 1 postpartum colostrum samples had significantly (p ≤ 0.05) higher values than day 2 and day 3 colostrum samples and between the latter two samples, the day 2 samples had significantly (p ≤ 0.05) higher value compared to day 3 samples. The results at day 1 uphold the findings of Raimondo et al. (2009) and Bar et al. (2010). Without taking into consideration different lactation numbers, the total plate count (TPC) showed an increasing trend with the passage of post-partum period with values being significantly (p ≤ 0.05) lower at day 1 followed by a significant (p ≤ 0.05) increase at day 2 and further significant (p ≤ 0.05) increase at day 3. The findings are close to the findings of Morill et al. (2012). Irrespective of the days of transition, the total plate count (TPC) of the colostrum samples from sixth lactation cows was significantly (p ≤ 0.05) higher than other lactations. Lower values with regard to the major colostrum components in second lactation compared to preceding or succeeding lactations has also been reported by Quigley III et al. (1994) and Morill et al. (2012). The precise reason to explain the situation is currently not available however.
The animals in second lactation possessed lower values for various physico-chemical, compositional and microbiological characteristics than the animals in first and third or higher lactations.
I am highly thankful to my Advisor and whole staff of Division of LPT, F.V.Sc. & A.H. Shuhama, SKUAST-K for being with me during my research