Ingredients and chemical composition of the feed in the winter and summer.
In recent decades, research in dairy cattle has been focused on the evaluation of factors that may cause changes in the lipid composition of milk, due to the fact that unsaturated fatty acids, conjugated linoleic acids (CLA) and high monounsaturated fatty acids/saturated fatty acids (MUFA/SFA) and polyunsaturated fatty acids/saturated fatty acids (PUFA/MUFA) ratios in milk have shown beneficial properties, including antiatherogenic and anticarcinogenic effects in humans. The principal factors that determine these effects are the breed (Lawless et al., 1999; Wood et al., 1980) and feeding regime (Cooper et al., 2004) in addition to less-studied factors, such as the parity, days in milk (DIM) and extreme temperatures. CLA are a mixture of linoleic acid isomers that contain conjugated double bonds. Studies in experimental animals have demonstrated that CLA has properties that may be beneficial for humans, providing anticarcinogenic, antidiabetic, antiobesity, antiatherogenic and immune stimulatory effects (Huth et al., 2006; Nirvair et al., 2007; Pariza et al., 2001; Parodi., 1999). Milk and dairy products are the primary sources of CLA, and approximately 75 to 90% of the total CLA content in milk fat is represented by
2. Material and methods
2.1. Location and sampling
This study was carried out on a commercial dairy farm in Sonora, México, which was located at 29.07 ° longitude and 110.90 ° latitude. A total of 240 Holstein dairy cows were included in the study, and the parity of the cows ranged from 1 to 6. The cows were exposed to all of the environmental elements that can affect the behaviour and milk yield of dairy cattle, including solar radiation, rain, and wind, as shown in Figure 1. The cows were divided into parity groups, including the following: primiparous (P, 1 parity, n = 84); earlier multiparous (EM, from 2 to 3 parities, n = 96); and late multiparous (LM, from 4 to 6 parities; n = 60). The milk was sampled in proportion to the parity of the cows; exclusion criteria included the presence of mastitis and more than 350 DIM.
The diets were formulated using the Cornell-Penn-Miner Dairy model (Moate et al., 2004), and the ingredients and chemical compositions of summer and winter feed are shown in Table 1. Milking was carried out twice per day (0400 h and 1600 h), and the two milk samples (50 mL each) were combined. Milk samples were collected during the summer (from June to August 2006) and the winter (December 2006 to February 2007). The milk yield was measured using a Waikato MKV milk meter (Waikato MKV, Milking Systems, NZ) and evaluated on the last day of each month, which was standard practice on this dairy farm. The samples were transported on ice to the laboratory and stored at -20 °C for later analysis. Feed samples were collected on each sampling date. The DIM were also recorded.
The meteorological station “El Perico”, which was located 5 km from the farm, recorded the daily weather data, and this information was used to calculate the temperature-humidity index (THI) for the winter and summer seasons. The THI was calculated as follows: THI = td – (0.55 – 0.55RH) × (td – 58), where td = the dry bulb thermometer in °F and RH = the relative humidity expressed as a decimal (NOAA, 1976). A THI < 72 was taken as an indicator of zero stress for the dairy cows. THI values from 72 to 79 indicated that the dairy cows were under mild stress, whereas THI values from 79 to 88 indicated that cows were under high stress (Armstrong, 1994).
2.2. Feed analysis
2.2.1. Feed chemical composition
The chemical composition of the diets was analysed by the AgroLab México laboratory (Gómez Palacio, Dgo). The components of the feeds that were evaluated in winter and summer were the crude protein, neutral detergent fibre, acid detergent fibre, ether extract, and net absorbable energy.
2.2.2. Feed fatty acid analysis
Total lipids were extracted with a chloroform and methanol mixture (2:1, vol:vol), as described by Folch et al., (1957). Fatty acid methyl esters (FAMEs) were prepared by the standard method (AOAC, 1995). FAMEs were identified and quantified by gas chromatography using a Varian Star 3400 CX Gas Chromatograph (VARIAN Inc, Walnut Creek, CA) with a DB-23 (J & W Scientific, Folsom CA) capillary column (30 m x 0.25 mm). Helium was used as the carrier gas with a flow rate of 1 mL/min; the airflow was 300 mL/min, and the hydrogen flow was 30 mL/min. The column temperature was initially set at 50 °C and held for 1 min. It was then increased at 10 °C/min to 166 °C, and then at 1 °C/min to 174 °C and held for 30 s; next, it was increased at 2 °C/min to 194 °C and held for 30s. As a final step, the column temperature was increased at a rate of 3.5 °C/min to 215 °C and held for 5 min. The total running time was 42.6 min. Identification of the fatty acid profile was performed with an external standard FAME mix (C4-C24, Sigma, USA).
2.3. Milk quality
The milk composition (fat, protein, lactose, and total solids) was determined by near infrared spectrometry (Milkoscan Minor FT120).
2.3.1. Fatty acid analysis of the milk
Milk fat extraction was carried out according Luna et al., (2005). In brief, 30 mL of milk was centrifuged at 17,800 × g for 30 min at 8 °C (Beckman Coulter centrifuge, Mod. Allegra 64R). Approximately 350 mg of fat was subsequently removed for lipid extraction with 18 mL of a hexane:isopropanol mixture (3:2 v:v) per g of fat; next, 12 mL of sodium sulphate per g of fat was added as described by Hara & Radin (1978).
The milk fatty acids were transesterified with methoxide sodium according the method of Christie (1982) as modified by Chouinard et al., (1999). Briefly, the fatty acids were mixed with 2 mL of hexane per 40 mg of fatty acid, and 40 µL of methyl acetate was added. We then vortexed the mixture and added 40 µL of methylation reagent (1.75 mL of methanol mixed with 0.4 mL of 5.4 M sodium methoxide). The mixture was again vortexed and allowed to react for 10 min. We then added 60 µL of termination reagent (1 g of oxalic acid in 30 mL of diethyl ether). Next, the sample was centrifuged for 5 min at 2400 × g at 5 °C, and the hexane layer was removed and transferred to a new tube for evaporation with nitrogen. When the resulting methylated fatty acids had dried, 50 mg was transferred to an amber vial, and 200 µL of hexane was added. At that point, the sample was ready for quantification of fatty acids and 9-
188.8.131.52. Identification and quantification of FAMEs by gas chromatography
The FAMEs were identified and quantified by gas chromatography using a Varian Star 3400 CX Gas Chromatograph (VARIAN Inc., Walnut Creek, CA), with a DB-23 (J & W Scientific, Folsom, CA) capillary column (30 m x 0.25 mm). The procedure was identical to that described above for the
2.3.2. Desaturase index
The desaturase index was calculated as reported by Kelsey et al. (2003), using four pairs of fatty acids where each pair represented one product and one substrate of Δ9-desaturase. The fatty acid pairs (product and substrate) were as follows:
2.4. Statistical analysis
The PROC MIXED procedure provided in the SAS software (SAS Inst. Inc., Cary, NC, 2001) was used to adjust a model for analysing the fatty acid data. The model included the fixed effects of season and month within the season and parity; the random effects included milk yield and DIM (with linear and quadratic effects) and the residual term of animal per parity. The data were considered significant when P < 0.05. The response variable was reported as the least square mean ± SEM. The adjusted model was evaluated as follows:
When significant effects of the factors studied were found, mean comparisons of the response variables were performed by the LSMEANS procedure of SAS.
3. Results and discussion
3.1. Temperature-humidity index
In desert climates, the ambient temperatures are extreme and vary widely between winter and summer. To our knowledge, the previous studies that have evaluated variations in the milk 9-
3.2. Feed composition
According to the components and chemical analysis of the feed (Table 1), the summer and winter diets were similar in caloric, nitrogen, and fibre content. Nevertheless, changes in the ingredients were made according to the standard practice on the dairy farm. These changes included the use of whole cottonseeds, maize silage and less alfalfa content in the feed during the winter. The final forage-to-concentrate ratios of the diets were 58:42 and 59:41 for winter and summer, respectively. Accordingly, the fatty acid composition of the diets showed variations between the two seasons (Table 1). Because these compounds are precursors of CLA in both ruminal and mammary gland biosynthesis, the discussion is focused on the concentrations of
|Soya 47% Protein||3.53||5.59|
|Vitamins and minerals||2.28||2.08|
|Fatty acids of the diet4|
3.3. Milk yield and composition
The different climate conditions also had effects on the milk yield and composition (fat, protein, lactose, and total solids) as shown in Table 2. The milk yield was significantly lower (P < 0.05) in the summer (15.8 ± 0.5 kg/d) compared to the winter (18.9 ± 0.4 kg/d). This reduced yield may have been due, at least in part, to the reduced energy intake and the heat stress observed during the summer season, similar to the results reported by others (Fuquay, 1981; Granzin, 2006, Rhone et al., 2008; West, 2003). In addition, the reduction in milk yield agreed with the findings of West (2003), who has reported a reduction in the milk yield of 0.2 kg per unit increase in THI when the THI was above 72. Accordingly, in our study, the milk yield was 3.1 kg/d lower in the summer than in the winter; this reduction corresponded to the 16 THI units above 72 that were recorded during the summer (average 80 ± 5.1, range from 63 to 88). The percentages of fat, protein, lactose, and total solids in the milk were also lower during the summer season. This result may also have been attributable to the reduced feed intake induced by heat stress (Collier et al., 2006).
|Milk yield, kg/d||18.9a ± 0.40||15.8b ± 0.50|
|Milk fat, %||2.69a ± 0.10||1.91b ± 0.06|
|Milk protein, %||3.45a ± 0.03||3.34b ± 0.03|
|Lactose, %||4.41a ± 0.02||4.30b ± 0.02|
|Total solids, %||11.5a ± 0.11||10.4b ± 0.09|
3.4. Fatty acid profile in milk
Table 3 shows the significance, determined by the analysis of variance, of the effects of the season, month in the season, parity, DIM, and milk yield on the content of CLA, individual fatty acids, SFA, MUFAs, PUFAs, and the desaturase index. Most of the individual fatty acids in milk and the sum of the saturated fatty acids, the sum of the monounsaturated fatty acids and the sum of the polyunsaturated fatty acids, the MUFA/SFA and PUFA/SFA ratios and desaturase index were affected (P<0.05) by the season. In contrast, only the C14:0, C16:0, and unsaturated fatty acids, such as C16:1, C18:1 t11, C18:2 and 9-
Table 4 shows the fatty acid profiles of the milk during both seasons. A low content of saturated fatty acids (C14:0, C16:0, and C18:0) and a low sum of saturated fatty acids were observed during the summer. In contrast, the sum of monounsaturated fatty acids, the sum of polyunsaturated fatty acids, 9-
|Fatty acid||Model adjusted term|
|Season||Month (season)||Parity||DIM||DIM2, 1||Milk yield|
|Mean ± SE||CI (95%)||Mean ± SE||CI (95%)|
|C4:0||1.80 ± 0.18||1.44 – 2.14||0.90 ± 0.12||0.67 – 1.13||*|
|C6:0||9.69 ± 0.25||9.19 – 10.18||6.45 ± 0.30||5.86 – 7.03||**|
|C8:0||10.36 ± 0.14||10.07 – 10.64||8.28 ± 0.18||7.90 – 8.64||**|
|C10:0||26.10 ± 0.40||25.30 – 26.90||21.21 ± 0.41||20.40 – 22.02||**|
|C12:0||31.70 ± 0.48||30.74 – 32.65||27.74 ± 0.45||26.85 – 28.63||*|
|C14:0||109.05 ± 0.97||107.13 – 110.96||100.53 ±1.01||98.51 – 102.54||*|
|C14:1||8.71 ± 0.21||8.29 – 9.13||11.83 ± 0.28||11.27 – 12.39||**|
|C15:0||11.53 ± 0.13||11.28 – 11.78||11.69 ± 0.19||11.30 – 12.08||NS|
|C16:0||332.31 ± 2.37||327.62 – 337.00||299.90 ± 2.73||294.54 – 305.37||**|
|C16:1||14.04 ± 0.30||13.45 – 14.63||15.97 ± 0.38||15.22 – 16.72||NS|
|C17:0||6.09 ± 0.10||5.88 – 6.30||5.54 ± 0.11||5.32 – 5.75||NS|
|C18:0||125.18 ± 1.63||121.90 – 128.42||105.86 ± 1.68||102.50 – 109.21||*|
|C18:1, t11||9.64 ± 0.23||9.17 – 10.11||8.18 ± 0.28||7.62 – 8.73||*|
|C18:1, ||237.75 ± 2.50||232.80 – 242.70||288.23 ± 2.77||282.70 – 293.70||**|
|C18:2||22.79 ± 0.29||22.21 – 23.37||39.59 ± 0.84||37.92 – 41.26||**|
|C18:3||2.82 ± 0.08||2.65 – 2.98||4.37 ± 0.09||4.18 – 4.56||**|
|C20:0||1.25 ± 0.07||1.10 – 1.39||0.68 ± 0.08||0.51 – 0.84||*|
|Others||32.62 ± 0.30||32.01 – 33.22||33.12 ± 0.63||31.86 – 34.38||NS|
|Σ SFA4||665 ± 28.8||659.8 – 670.3||588.8 ± 41.4||581.3 – 596.3||**|
|Σ MUFA5||270.1± 25.3||265.6 – 274.7||324.2 ± 31.2||318.5 – 329.9||**|
|Σ PUFA6||32.3 ± 4.0||31.5 – 33.0||53.8 ± 11.2||51.7 – 55.8||**|
|MUFA/SFA ratio||0.408±0.007||0.398- 0.419||0.557±0.007||0.539-0.574||**|
These results show that the attributes of the milk were modified by the season, but this modification of the fatty profile and 9-
In contrast, it is well known that most CLA are produced in the mammary gland via Δ9 desaturase. As shown in Table 5, the activity of this enzyme was higher in summer than in winter for the four substrates (C14:0, C16:0, C18:1, and
|Mean ± SE||CI (95%)||Mean ± SE2||CI (95%)|
|cis-9 14:1||0.074 ± 0.001||0.071 – 0.077||0.104 ± 0.002||0.100 – 0.109||**|
|cis-9 16:1||0.040 ± 0.001||0.038 – 0.042||0.05 ± 0.001||0.048 – 0.052||**|
|cis-9 18:1||0.65 ± 0.002||0.65 – 0.66||0.73 ± 0.003||0.72 – 0.73||**|
|c-9, t-11 CLA||0.41 ± 0.007||0.39 – 0.42||0.55 ± 0.008||0.53 – 0.57||**|
Figure 3 shows the frequency distributions of the 9-
In the present study, parity affected the CLA content in milk fat as shown in Figure 4. For this analysis, dairy cows were grouped by parity status by season. In the summer, there were no differences in the 9-
The relationships between the milk CLA content and DIM, milk fat, and milk yield are shown in Figure 5 (Panels A, B, and C, respectively). The DIM had an effect on the milk CLAs with an R2 = 0.338, P < 0.05. In our study, the DIM explained a third (33.8%) of the variation in the CLA content. This result contrasted with previous reports that have shown that this factor explained less than 10% of the variation (Lock et al., 2005; Kelsey et al., 2003). The milk fat and milk yield did not have effects on the milk CLA content with R2 = 0.063 and R2 = 0.017, respectively; this result is in agreement with Kelsey et al., (2003).
We found that the practical management of the adjustment of the ingredients in the diet to maintain energy intake and avoid decreased milk yield during the summer (when temperatures may exceed 40 °C) was beneficial because this adjustment of the diet allowed an improvement in the unsaturated fatty acid profile and CLA content in the milk, despite the animals’ experiencing heat stress. The alterations in the fatty acid profile and CLA content in milk suggest that this milk could be healthier for the consumer in the summer, due to the increased MUFA/SFA and PUFA/SFA ratios.
The authors thank Dr. Arturo Madrid Lopez, Nutritionist, for his advice and assistance. We also thank Ing. Roy Swanson, the manager of the dairy farm where the present study was conducted. We also thank Amparo Nieblas Almada, Ingrid Rebeca Esquerra Brauer and Francisco A. Vazquez Ortiz for their excellent technical assistance.