日粮脂肪和能量水平对奶牛氧化应激、生产性能的影响及抗氧化剂添加效果研究
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摘要
饲料中的油脂在加工、贮存和饲喂过程中无时不存在被氧化的危险,脂质氧化是导致机体氧化应激的重要原因。本研究分三部分共五个试验,首先从日粮脂肪和奶牛机体的氧化潜在性着手,分析了奶牛场常用饲料原料的稳定性,评价了抗氧化剂(AOX)对油脂的保护效果(试验一)；调查了围产期中国荷斯坦奶牛脂质氧化状态以及脂质代谢和氧化应激相关性(试验二)。在此基础上选取两种稳定性差别较大的不同饱和度的脂肪酸饲喂高产奶牛,评估其对生产性能和抗氧化状态的影响及AOX的添加效果(试验三),并从对瘤胃抗氧化状态和微生物区系变化方面进行深入研究(试验四)。基于围产期奶牛脂质代谢和氧化应激的相关性,通过能量饲喂水平及添加AOX调控围产期奶牛脂质代谢和抗氧化状态(试验五)。
第一部分日粮脂肪和奶牛机体的潜在氧化特性研究(试验一和试验二)
试验一选取奶牛场常用的高脂饲料原料,分别添加0或500ppm的AOX。采用活性氧法(AOM)测定了其室温下的过氧化物值(POV),脂肪氧化测定仪测定其100℃时的诱导时间。试验结果：以籽实的形式存在的脂肪其POV较低,籽实中POV值最高的DDGS为2.27meq/kg,POV值最低的膨化大豆为0.67 meq/kg。依据日粮不同,AOX降低籽实中的POV值32%-68%。不饱和脂肪酸钙的POV较高,而饱和脂肪粉的值较低(33.13 vs 4.53)。不饱和脂肪酸钙的诱导时间显著低于脂肪粉,添加AOX的脂肪酸钙其稳定性提高至原来的3倍,而饱和脂肪粉添加AOX仅提高50%左右。AOX可延长饲用油脂诱导时间,其中对棉籽中脂肪的诱导时间延长幅度最大,可提高其稳定性7-8倍以上。这提示AOX可显著提高脂肪的稳定性,尤其是不饱和脂肪酸含量高的脂肪。
为揭示奶牛机体脂质氧化的潜在性,试验二调查了围产期奶牛的脂质代谢及与氧化应激状态的相关性。选取10头具有相近体况(BCS=3)、胎次(3-5胎)预产期(±2天)的经产中国荷斯坦奶牛,从产前3周至产后3周,每周尾静脉采血分离血浆检测脂质代谢与氧化应激指标；另选取10头处于泌乳后期的健康、正常体况牛作为对照组(泌乳量低于20kg/d)。结果发现：整个围产期奶牛血浆葡萄糖的浓度显著高于对照组,分娩后葡萄糖的浓度比分娩前降低32.6%(2.58 vs3.83)。产前3周至产后3周,血浆游离脂肪酸(NEFA)浓度先上升后下降,在分娩当天和产后1周达到最大峰值,与其他各组差异显著(p<0.05)。而甘油三酯的浓度在分娩后与对照组相似,均显著低于分娩前的水平(p<0.05)。围产期奶牛血浆超氧化物歧化酶(SOD)的活性均低于对照组,分娩前后1周和分娩时达到最低值,与对照组差异显著(p<0.05)。分娩后1周,血浆丙二醛(MDA)浓度达到最高峰,同时清除活性氧的能力降至最低(p<0.05)。在所检测的代谢指标中,NEFA与氧化应激指标相关性最大,其与MDA浓度的相关系数达0.74,而与清除活性氧能力及总抗氧化能力呈负相关,相关系数分别为-0.61和-0.56。这些试验结果表明围产期奶牛同样存在脂质过氧化的现象,围产期奶牛能量负平衡可导致机体脂肪动员的增加,脂质过氧化物生成增多,而分娩后血浆SOD、清除活性氧能力降至最低,氧化应激的发生与脂质代谢有很强的相关性。
第二部分饲喂不同饱和度的脂肪及添加抗氧化剂对奶牛生产性能、抗氧化状态和瘤胃发酵的影响(试验三和试验四)
试验三的目的是研究不同饱和度脂肪酸添加AOX对奶牛生产性能和血液代谢的影响。低饱和脂肪酸以53.9%饱和度的美加力形式与精料均匀混合,高饱和脂肪酸以88.6%饱和度的棕榈酸脂肪粉的形式补充,AOX的添加浓度为0或0.025%。试验结果：脂肪酸类型及AOX处理对奶牛的干物质采食量(DMI)无显著影响。补充不饱和脂肪酸的奶牛乳产量与4%校正乳产量显著低于补充饱和脂肪酸组(P<0.05)；乳脂及乳蛋白含量不受脂肪酸类型和AOX处理的影响。AOX添加显著提高了LS组奶牛的乳产量和4%校正乳产量(P<0.05),而对HS组没有显著影响(P>0.05)。不考虑AOX的效应,与补充饱和脂肪酸的奶牛相比,补充不饱和脂肪酸奶牛血浆SOD的活性显著降低,MDA含量显著升高,而血浆葡萄糖浓度有降低的趋势(P≦0.10)。AOX添加降低了奶牛血浆NEFA和H202含量,同时提高了奶牛的总抗氧化能力。不饱和脂肪酸补充提高了奶牛红细胞膜C12:0,C18:0,cis-9 C18:1,trans-11 C18:1,C18:2, C20:5和C22:6的比例,同时降低了C14:0,C16:0和C16:1的比例。AOX添加显著提高奶牛红细胞膜中链脂肪酸,如C14:0,C14:1和C16:1比例,减少长链脂肪酸中C18:0,cis-9 C18:1,C20:5和C22:6比例。补充不饱和脂肪酸显著增加奶牛乳中cis-9 C18:1, trans-10, cis-12 C18:2比例同时降低乳C18:0的比例,AOX添加显著增加乳中cis-9 C18:1比例,降低乳中C12:0,C16:0和trans-10, cis-12C18:2的比例。以上结果表明补充不饱和脂肪酸的奶牛生产性能及血液代谢要劣于饱和脂肪酸组；AOX一定程度上可改善不饱和脂肪酸对奶牛的生产性能及血液代谢的副作用,而对饱和脂肪酸添加效果不显著。
试验四利用体外产气法分别评估了高饱和脂肪酸及低饱和脂肪酸添加AOX(0或500 ppm)对湖羊瘤胃体外发酵参数的影响。试验结果：脂肪酸类型不影响瘤胃发酵参数和抗氧化状态(P>0.05)；与饱和脂肪酸相比,补充低饱和脂肪酸可显著降低瘤胃液中产琥珀酸丝状杆菌相对于总菌的比例(P<0.05)、显著提高原虫比例(P<0.05)。添加AOX可提高体外24h产气量和有机物消化率(P<0.05),并有减少乙酸摩尔比、增加丙酸摩尔比的趋势(P<0.10),同时AOX添加也有降低瘤胃液中MDA浓度、提高SOD活性的趋势(P<0.10)。AOX对黄色瘤胃球菌和白色瘤胃球菌的影响依赖于脂肪酸的类型,低饱和脂肪酸添加AOX可显著提高黄色瘤胃球菌和白色瘤胃球菌相对于总菌的比例,而高饱和脂肪酸添加无显著效果。这说明日粮添加不饱和脂肪酸不利于纤维分解菌的生长,添加AOX可改善不饱和脂肪酸对瘤胃微生物的不利影响,而对饱和脂肪酸影响较小。
第三部分产前能量水平对围产期奶牛脂质代谢和氧化应激的影响及抗氧化添加效果研究(试验五)
本试验研究了产前能量水平和AOX添加对围产期奶牛脂质代谢和氧化应激状态的调控。产前3周,40头经产中国荷斯坦奶牛按照胎次、体况、预产期和上泌乳期产奶量一致原则随机分入4个组,每组10头。试验采用2×2因子设计,处理因素包括AOX (0或5g/d per cow)和产前能量水平(1.43或1.28 Mcal/kg DM)。产后所有牛饲喂相同的泌乳日粮,AOX添加至产后3周。试验结果显示：产前饲喂高能日粮显著降低奶牛产前及产后的DMI(P<0.05)。能量水平和AOX处理不影响奶牛的能量平衡和胎牛初生重(P>0.05)。与低能日粮相比,产前高能日粮饲喂降低了奶牛产后3周的乳产量,围产期添加AOX有提高泌乳早期乳产量的趋势(P<0.10)。产前高能饲喂增加整个围产期奶牛血浆葡萄糖和β-羟丁酸的浓度,而AOX添加显著降低围产期血浆β-羟丁酸浓度(P<0.05)。处理与时间之间对谷胱甘肽过氧化物酶(GSH-Px)活性和MDA浓度的影响有交互作用,即在某些特定时间点,如产前3d,产前高能日粮饲喂显著提高奶牛血浆GSH-Px的活性和MDA的浓度,而AOX添加显著降低产前10d和3d血浆GSH-Px浓度,同时显著降低奶牛分娩时血浆MDA的浓度(P<0.05)。产前能量水平显著影响奶牛红细胞膜中脂肪酸含量(P<0.05)。产前采食高能日粮的奶牛产后3d红细胞膜脂肪酸的含量显著高于采食低能组,而AOX添加组奶牛红细胞膜脂肪酸含量仅在数值上有提高,但与未添加组差异不显著(P>0.05)。能量水平和AOX对红细胞膜中饱和脂肪酸和不饱和脂肪酸比例影响不显著(P>0.05)。产前饲喂不同能量水平的日粮可导致奶牛产后红细胞膜的组成显著不同,而AOX添加仅增加cis-9,trans-11C18:2的比例。与低能日粮相比,产前高能日粮显著提高了奶牛产后3d红细胞膜的流动性(1.51 vs 1.41,P<0.05),但AOX对红细胞膜的流动性影响不显著。以上结果提示产前低能饲喂更有利于产后采食量的恢复和生产性能的提高。添加AOX可以提高脂质代谢和抗氧化能力从而改善血液代谢,提高生产性能。
综上所述,高脂饲料原料和动物机体的脂质都存在被氧化的潜在危险,机体氧化应激的发生与脂质代谢有很强的相关性。补充不饱和脂肪酸的奶牛生产性能及血液代谢要劣于饱和脂肪酸,这可能与不饱和脂肪酸对瘤胃纤维分解菌的抑制有关。AOX一定程度上改善不饱和脂肪酸对瘤胃微生物的不利影响,提高瘤胃抗氧化状态和有机物的消化率,而对饱和脂肪酸效果不显著。产前高能饲喂不利于奶牛采食量和产后生产性能的提高,同时不利于奶牛的血浆脂质代谢和抗氧化应激状态。添加AOX可通过提高脂质代谢和抗氧化能力在一定程度上改善奶牛的健康状况和生产性能。
Dietary fat is susceptible to oxidation during any stage of processing, storage and feeding. Lipid oxidation also often occurs in animal body when the production of free radicals exceeds the capacity of antioxidant system. This study contained three parts including five experiments. In the first part, the stability of different ingredients rich in fat was evaluated, in the absence or presence of antioxidant (AOX) (Expt.1). To evaluate the potential oxidation of lipid in dairy cows, the lipid metabolism character and its correlation to anti-oxidative status was investigated in Chinese Holstein cows (Expt.2). Then, in the second part, two types of fatty acids that differ in stability and saturated degree were selected to evaluate their effects on performance and anti-oxidative status in high lactating cows, in the absence or presence of antioxidant (AOX) (Expt.3). An in vitro gas test trial was carried out to investigate the effect of different rumen inert fatty acids with a dietary antioxidant on rumen fermentation, anti-oxidative status and microflora (Expt.4). In the last part, two pre-partum diets with different energy density were formulated to study the effect of energy level on lipid metabolism and anti-oxidative status, in the absence or presence of AOX (Expt. 5).
Part one:Evaluation of stability in the diets rich in fat and lipid oxidation in dairy cows (Expt.1 and 2).
In Expt.1, Ingredients that rich in fat were selected and mixed with 0 or 500 ppm antioxidant (AOX). Peroxide values (POV) in the room temperature were determined by active oxygen method (AOM), and induction time was determined by ML OXIPRESTM at 100℃. The POV value in the whole seed was lower, ranging from 0.67meq/kg in extruded soybean to 2.27meq/kg in DDGS. Addition of AOX decreased the POV by 32 to 68% based on the ingredients. However, the POV was higher in unsaturated calcium salt of long chain fatty acids, compared with saturated fatty acid (33.13 vs 4.53 meq/kg), while the induction time showed the opposite results. The AOX could improve the stability of unsaturated calcium salt of long chain fatty acids by 3 times, but only 50% improvement was found in saturated fatty acid. The stability of cotton seed with AOX was 7-8 times of that without AOX. It is indicated that AOX could improve the stability of the diets, especially when polyunsaturated fatty acids were included in the diets.
The objective of Expt.2 was to evaluate the potential oxidation of lipid in dairy cows. The lipid metabolism and its correlation to oxidative status were investigated in Chinese Holstein cows during transition period. Ten cows with similar parity, expected calving date, and body condition score were selected and blood samples collected weekly by coccygeal vein from-3 to 3 weeks relative to calving. Another ten cows in late-lactating were selected as control. The concentration of plasma glucose was higher in transition cows, compared to late-lactating cows. After calving, plasma glucose decreased to a lower level, compared to that at calving and prepartum (2.58 at 1 week postpartum vs 3.83 mmol/L at calving). The nonesterified fatty acid (NEFA) peaked 1 week postpartum, significantly higher than that in prepartum and control cows. Similar to the control, plasma triglyceride postpartum was lower than that prepartum. Activity of superoxide dismutase (SOD) in transition cows was lower than late-lactating cows, especially 0 and 1 week relative to calving. Meanwhile, malondialchehyche (MDA) reached to the highest level 1 week postpartum, contrasting with activity of antisuperoxide anion free radical (ASAFR), which decreased to the lowest level. Concentration of NEFA was related to most of anti-oxidative parameters (R= 0.74,-0.61 and-0.56 for MDA, ASAFR and TAOC, respectively). In summary, transition cows are susceptible to oxidative stress, indicated by lower SOD and ASAFR activity after calving. The increased requirement to glucose during the initial step of lactation cows will make cows mobilize adipose tissues. The oxidative status may have a close relationship with lipid metabolism.
Part two:Effect of diets supplemented with fatty acids of different degrees of saturation, in the absence or presence of AOX, on lactation performance and rumen fermentation in dairy cows (Expt.3 and 4).
The objective of Expt.3 was to evaluate the effect of diets supplemented with fatty acids of different degrees of saturation, in the absence or presence of AOX, on lactation performance of dairy cow. Calcium salt of long-chain fatty acids was supplemented as a source of lower saturation fatty acid (LS,53.9% SFA), and palmitic acid was supplemented as the higher saturation fatty acid source (HS,88.6% SFA). The AOX was added at 0.025% in the ration. Neither fatty acid type nor AOX supplementation showed significant effect on dry matter intake (DMI) during the study. Compared with those in HS, yield of milk and 4% FCM were lower in the LS-fed cows. Milk fat and milk protein concentrations were not affected by fatty acid type or AOX supplementation. Adding AOX increased the yield of milk and 4% FCM in the LS-fed cows, but did not affect those fed HS. Plasma SOD activity was significantly lower, plasma glucose tended to be lower, and plasma MD A was higher in the LS-fed animals, compared with those fed HS. Addition of AOX decreased both plasma NEFA and H2O2 contents and increased TAOC activity across the fatty acid types. Composition of C12:0, C18:0, cis-9 C18:1, trans-11 C18:1, C18:2, C20:5 and C22:6 in the erythrocyte membrane was higher, while C14:0, C16:0 and C16:1 was lower for cows fed diets supplemented with LS, compared with those with HS. Addition of AOX increased C14:0, C14:1 and C16:1 and decreased C18:0, cis-9 C18:1, C20:5 and C22:6-composition in the erythrocyte membrane. Cows fed LS had higher cis-9 C18:1 and trans-10, cis-12 C18:2 in milk at the expense of C18:0, whereas AOX addition increased milk cis-9 C18:1 at the expense of milk C12:0, C16:0, and trans-10, cis-12 C18:2. It is inferred that dietary inclusion of unsaturated fatty acids resulted in inferior lactation performance. Whereas, these negative effects may be partially alleviated by the addition of antioxidant.
Experiment 4 was carried out to evaluate the effect of LS and HS supplementation on rumen fermentation in vitro, in the absence or presence of AOX. The experiment was carried out in a 2×2 factorial design, with fatty acids type as one factor and AOX as another one. Fermentation patterns and anti-oxidative status were not affected by different fatty acids supplementation. Supplementation of LS significantly increased the populations of protozoa relative to total bacterial 16S rDNA, but showed negative effect on fibrobacter succinogenes. Addition of AOX significantly increased gas production at 24h incubation. Molar proportion of propionate tended to increase at the expense of acetate due to AOX addition. AOX tended to decrease MDA value and increase SOD activity. An interaction between AOX and fat type was observed on ruminococcus flavefaciens and ruminococcus albus. Inclusion of AOX increased these two bacteria in LS group, but not in HS. It is concluded that unsaturated fatty acids inclusion in the diets may result in inferior effect on cellulolytic bacteria in the rumen, while these negative effects may be partially alleviated by the addition of antioxidant.
Part three:Effect of energy density prepartum on performance and anti-oxidative status in transition cows, in the absence or presence of antioxidant (Expt.5)
In Expt.5, a 2 x 2 factorial design trial was conducted to evaluate the effect of dietary antioxidant and energy density on performance and anti-oxidative status in transition cows. Forty cows were randomly allocated to 4 dietary treatments. High or low energy density diets prepartum (1.43 or 1.28 Mcal NEL/kg DM, respectively) were formulated with or without AOX (0 or 5 g/d per cow). These diets were fed to cows for 21 days pre-partum. During the post-partum period, all cows were fed the same lactation diets, and AOX treatment followed as for the pre-partum period. Feeding a high energy diet depressed the DMI, milk yield, and 4% FCM of cows. However, AOX inclusion in the diet improved the milk and 4% FCM yields. There was an interaction of energy density by AOX on milk protein, milk fat and total solids contents. Feeding a high energy diet pre-partum significantly increased plasma glucose andβ-hydroxybutyrate (P＜0.05), whereas dietary AOX significantly decreased plasmaβ-hydroxybutyrate value during the transition period (P<0.05) There were also interactions between time and treatment for plasma glutathione peroxidase activity and MDA content during the study. Cows fed high energy diets pre-partum had higher plasma glutathione peroxidase activity 3 days prior to parturition, compared with those on low energy diets. Inclusion of AOX in diets' decreased plasma glutathione peroxidase activity in cows 3 and 10 days pre-partum. Addition of AOX significantly decreased MDA values at calving (P＜0.05). Energy density induced marginal changes in fatty acid composition in the erythrocyte membrane 3 days post-partum, while AOX only significantly increased cis-9, trans-11 C18:2 conjugated linoleic acid composition. The increase in fluidity of the erythrocyte membrane was only observed in the high energy treatment. The above results indicated that feeding low energy density prepertum could improve the postpartum DMI and lactation performance. The addition of AOX is beneficial to the lactation performance by improving the lipid metabolism and antioxidative status.
In summary, both the fat in the diet and lipid in the body are potentially oxidized, which could induce oxidative stress in dairy cows. The oxidative status may have a close relationship with energy metabolism. Feeding unsaturated fatty acids may result in inferior lactation performance, which is associated with inferior effect on rumen cellulolytic bacteria. Whereas, these negative effects may be partially alleviated by the addition of AOX. The effect of AOX depends on types of fatty acids. When unsaturated fatty acids were included in the diets, a significant benefit is shown by addition of AOX, but this is not the case with saturated fatty acid inclusion. A diet containing high energy density pre-partum may negatively affect the anti-oxidative status, DMI and subsequent performance. Addition of AOX may improve the anti-oxidative status and lipid metabolism, eventually resulting in improved lactation performance.
第一部分日粮脂肪和奶牛机体的潜在氧化特性研究(试验一和试验二)
试验一选取奶牛场常用的高脂饲料原料,分别添加0或500ppm的AOX。采用活性氧法(AOM)测定了其室温下的过氧化物值(POV),脂肪氧化测定仪测定其100℃时的诱导时间。试验结果：以籽实的形式存在的脂肪其POV较低,籽实中POV值最高的DDGS为2.27meq/kg,POV值最低的膨化大豆为0.67 meq/kg。依据日粮不同,AOX降低籽实中的POV值32%-68%。不饱和脂肪酸钙的POV较高,而饱和脂肪粉的值较低(33.13 vs 4.53)。不饱和脂肪酸钙的诱导时间显著低于脂肪粉,添加AOX的脂肪酸钙其稳定性提高至原来的3倍,而饱和脂肪粉添加AOX仅提高50%左右。AOX可延长饲用油脂诱导时间,其中对棉籽中脂肪的诱导时间延长幅度最大,可提高其稳定性7-8倍以上。这提示AOX可显著提高脂肪的稳定性,尤其是不饱和脂肪酸含量高的脂肪。
为揭示奶牛机体脂质氧化的潜在性,试验二调查了围产期奶牛的脂质代谢及与氧化应激状态的相关性。选取10头具有相近体况(BCS=3)、胎次(3-5胎)预产期(±2天)的经产中国荷斯坦奶牛,从产前3周至产后3周,每周尾静脉采血分离血浆检测脂质代谢与氧化应激指标；另选取10头处于泌乳后期的健康、正常体况牛作为对照组(泌乳量低于20kg/d)。结果发现：整个围产期奶牛血浆葡萄糖的浓度显著高于对照组,分娩后葡萄糖的浓度比分娩前降低32.6%(2.58 vs3.83)。产前3周至产后3周,血浆游离脂肪酸(NEFA)浓度先上升后下降,在分娩当天和产后1周达到最大峰值,与其他各组差异显著(p<0.05)。而甘油三酯的浓度在分娩后与对照组相似,均显著低于分娩前的水平(p<0.05)。围产期奶牛血浆超氧化物歧化酶(SOD)的活性均低于对照组,分娩前后1周和分娩时达到最低值,与对照组差异显著(p<0.05)。分娩后1周,血浆丙二醛(MDA)浓度达到最高峰,同时清除活性氧的能力降至最低(p<0.05)。在所检测的代谢指标中,NEFA与氧化应激指标相关性最大,其与MDA浓度的相关系数达0.74,而与清除活性氧能力及总抗氧化能力呈负相关,相关系数分别为-0.61和-0.56。这些试验结果表明围产期奶牛同样存在脂质过氧化的现象,围产期奶牛能量负平衡可导致机体脂肪动员的增加,脂质过氧化物生成增多,而分娩后血浆SOD、清除活性氧能力降至最低,氧化应激的发生与脂质代谢有很强的相关性。
第二部分饲喂不同饱和度的脂肪及添加抗氧化剂对奶牛生产性能、抗氧化状态和瘤胃发酵的影响(试验三和试验四)
试验三的目的是研究不同饱和度脂肪酸添加AOX对奶牛生产性能和血液代谢的影响。低饱和脂肪酸以53.9%饱和度的美加力形式与精料均匀混合,高饱和脂肪酸以88.6%饱和度的棕榈酸脂肪粉的形式补充,AOX的添加浓度为0或0.025%。试验结果：脂肪酸类型及AOX处理对奶牛的干物质采食量(DMI)无显著影响。补充不饱和脂肪酸的奶牛乳产量与4%校正乳产量显著低于补充饱和脂肪酸组(P<0.05)；乳脂及乳蛋白含量不受脂肪酸类型和AOX处理的影响。AOX添加显著提高了LS组奶牛的乳产量和4%校正乳产量(P<0.05),而对HS组没有显著影响(P>0.05)。不考虑AOX的效应,与补充饱和脂肪酸的奶牛相比,补充不饱和脂肪酸奶牛血浆SOD的活性显著降低,MDA含量显著升高,而血浆葡萄糖浓度有降低的趋势(P≦0.10)。AOX添加降低了奶牛血浆NEFA和H202含量,同时提高了奶牛的总抗氧化能力。不饱和脂肪酸补充提高了奶牛红细胞膜C12:0,C18:0,cis-9 C18:1,trans-11 C18:1,C18:2, C20:5和C22:6的比例,同时降低了C14:0,C16:0和C16:1的比例。AOX添加显著提高奶牛红细胞膜中链脂肪酸,如C14:0,C14:1和C16:1比例,减少长链脂肪酸中C18:0,cis-9 C18:1,C20:5和C22:6比例。补充不饱和脂肪酸显著增加奶牛乳中cis-9 C18:1, trans-10, cis-12 C18:2比例同时降低乳C18:0的比例,AOX添加显著增加乳中cis-9 C18:1比例,降低乳中C12:0,C16:0和trans-10, cis-12C18:2的比例。以上结果表明补充不饱和脂肪酸的奶牛生产性能及血液代谢要劣于饱和脂肪酸组；AOX一定程度上可改善不饱和脂肪酸对奶牛的生产性能及血液代谢的副作用,而对饱和脂肪酸添加效果不显著。
试验四利用体外产气法分别评估了高饱和脂肪酸及低饱和脂肪酸添加AOX(0或500 ppm)对湖羊瘤胃体外发酵参数的影响。试验结果：脂肪酸类型不影响瘤胃发酵参数和抗氧化状态(P>0.05)；与饱和脂肪酸相比,补充低饱和脂肪酸可显著降低瘤胃液中产琥珀酸丝状杆菌相对于总菌的比例(P<0.05)、显著提高原虫比例(P<0.05)。添加AOX可提高体外24h产气量和有机物消化率(P<0.05),并有减少乙酸摩尔比、增加丙酸摩尔比的趋势(P<0.10),同时AOX添加也有降低瘤胃液中MDA浓度、提高SOD活性的趋势(P<0.10)。AOX对黄色瘤胃球菌和白色瘤胃球菌的影响依赖于脂肪酸的类型,低饱和脂肪酸添加AOX可显著提高黄色瘤胃球菌和白色瘤胃球菌相对于总菌的比例,而高饱和脂肪酸添加无显著效果。这说明日粮添加不饱和脂肪酸不利于纤维分解菌的生长,添加AOX可改善不饱和脂肪酸对瘤胃微生物的不利影响,而对饱和脂肪酸影响较小。
第三部分产前能量水平对围产期奶牛脂质代谢和氧化应激的影响及抗氧化添加效果研究(试验五)
本试验研究了产前能量水平和AOX添加对围产期奶牛脂质代谢和氧化应激状态的调控。产前3周,40头经产中国荷斯坦奶牛按照胎次、体况、预产期和上泌乳期产奶量一致原则随机分入4个组,每组10头。试验采用2×2因子设计,处理因素包括AOX (0或5g/d per cow)和产前能量水平(1.43或1.28 Mcal/kg DM)。产后所有牛饲喂相同的泌乳日粮,AOX添加至产后3周。试验结果显示：产前饲喂高能日粮显著降低奶牛产前及产后的DMI(P<0.05)。能量水平和AOX处理不影响奶牛的能量平衡和胎牛初生重(P>0.05)。与低能日粮相比,产前高能日粮饲喂降低了奶牛产后3周的乳产量,围产期添加AOX有提高泌乳早期乳产量的趋势(P<0.10)。产前高能饲喂增加整个围产期奶牛血浆葡萄糖和β-羟丁酸的浓度,而AOX添加显著降低围产期血浆β-羟丁酸浓度(P<0.05)。处理与时间之间对谷胱甘肽过氧化物酶(GSH-Px)活性和MDA浓度的影响有交互作用,即在某些特定时间点,如产前3d,产前高能日粮饲喂显著提高奶牛血浆GSH-Px的活性和MDA的浓度,而AOX添加显著降低产前10d和3d血浆GSH-Px浓度,同时显著降低奶牛分娩时血浆MDA的浓度(P<0.05)。产前能量水平显著影响奶牛红细胞膜中脂肪酸含量(P<0.05)。产前采食高能日粮的奶牛产后3d红细胞膜脂肪酸的含量显著高于采食低能组,而AOX添加组奶牛红细胞膜脂肪酸含量仅在数值上有提高,但与未添加组差异不显著(P>0.05)。能量水平和AOX对红细胞膜中饱和脂肪酸和不饱和脂肪酸比例影响不显著(P>0.05)。产前饲喂不同能量水平的日粮可导致奶牛产后红细胞膜的组成显著不同,而AOX添加仅增加cis-9,trans-11C18:2的比例。与低能日粮相比,产前高能日粮显著提高了奶牛产后3d红细胞膜的流动性(1.51 vs 1.41,P<0.05),但AOX对红细胞膜的流动性影响不显著。以上结果提示产前低能饲喂更有利于产后采食量的恢复和生产性能的提高。添加AOX可以提高脂质代谢和抗氧化能力从而改善血液代谢,提高生产性能。
综上所述,高脂饲料原料和动物机体的脂质都存在被氧化的潜在危险,机体氧化应激的发生与脂质代谢有很强的相关性。补充不饱和脂肪酸的奶牛生产性能及血液代谢要劣于饱和脂肪酸,这可能与不饱和脂肪酸对瘤胃纤维分解菌的抑制有关。AOX一定程度上改善不饱和脂肪酸对瘤胃微生物的不利影响,提高瘤胃抗氧化状态和有机物的消化率,而对饱和脂肪酸效果不显著。产前高能饲喂不利于奶牛采食量和产后生产性能的提高,同时不利于奶牛的血浆脂质代谢和抗氧化应激状态。添加AOX可通过提高脂质代谢和抗氧化能力在一定程度上改善奶牛的健康状况和生产性能。
Dietary fat is susceptible to oxidation during any stage of processing, storage and feeding. Lipid oxidation also often occurs in animal body when the production of free radicals exceeds the capacity of antioxidant system. This study contained three parts including five experiments. In the first part, the stability of different ingredients rich in fat was evaluated, in the absence or presence of antioxidant (AOX) (Expt.1). To evaluate the potential oxidation of lipid in dairy cows, the lipid metabolism character and its correlation to anti-oxidative status was investigated in Chinese Holstein cows (Expt.2). Then, in the second part, two types of fatty acids that differ in stability and saturated degree were selected to evaluate their effects on performance and anti-oxidative status in high lactating cows, in the absence or presence of antioxidant (AOX) (Expt.3). An in vitro gas test trial was carried out to investigate the effect of different rumen inert fatty acids with a dietary antioxidant on rumen fermentation, anti-oxidative status and microflora (Expt.4). In the last part, two pre-partum diets with different energy density were formulated to study the effect of energy level on lipid metabolism and anti-oxidative status, in the absence or presence of AOX (Expt. 5).
Part one:Evaluation of stability in the diets rich in fat and lipid oxidation in dairy cows (Expt.1 and 2).
In Expt.1, Ingredients that rich in fat were selected and mixed with 0 or 500 ppm antioxidant (AOX). Peroxide values (POV) in the room temperature were determined by active oxygen method (AOM), and induction time was determined by ML OXIPRESTM at 100℃. The POV value in the whole seed was lower, ranging from 0.67meq/kg in extruded soybean to 2.27meq/kg in DDGS. Addition of AOX decreased the POV by 32 to 68% based on the ingredients. However, the POV was higher in unsaturated calcium salt of long chain fatty acids, compared with saturated fatty acid (33.13 vs 4.53 meq/kg), while the induction time showed the opposite results. The AOX could improve the stability of unsaturated calcium salt of long chain fatty acids by 3 times, but only 50% improvement was found in saturated fatty acid. The stability of cotton seed with AOX was 7-8 times of that without AOX. It is indicated that AOX could improve the stability of the diets, especially when polyunsaturated fatty acids were included in the diets.
The objective of Expt.2 was to evaluate the potential oxidation of lipid in dairy cows. The lipid metabolism and its correlation to oxidative status were investigated in Chinese Holstein cows during transition period. Ten cows with similar parity, expected calving date, and body condition score were selected and blood samples collected weekly by coccygeal vein from-3 to 3 weeks relative to calving. Another ten cows in late-lactating were selected as control. The concentration of plasma glucose was higher in transition cows, compared to late-lactating cows. After calving, plasma glucose decreased to a lower level, compared to that at calving and prepartum (2.58 at 1 week postpartum vs 3.83 mmol/L at calving). The nonesterified fatty acid (NEFA) peaked 1 week postpartum, significantly higher than that in prepartum and control cows. Similar to the control, plasma triglyceride postpartum was lower than that prepartum. Activity of superoxide dismutase (SOD) in transition cows was lower than late-lactating cows, especially 0 and 1 week relative to calving. Meanwhile, malondialchehyche (MDA) reached to the highest level 1 week postpartum, contrasting with activity of antisuperoxide anion free radical (ASAFR), which decreased to the lowest level. Concentration of NEFA was related to most of anti-oxidative parameters (R= 0.74,-0.61 and-0.56 for MDA, ASAFR and TAOC, respectively). In summary, transition cows are susceptible to oxidative stress, indicated by lower SOD and ASAFR activity after calving. The increased requirement to glucose during the initial step of lactation cows will make cows mobilize adipose tissues. The oxidative status may have a close relationship with lipid metabolism.
Part two:Effect of diets supplemented with fatty acids of different degrees of saturation, in the absence or presence of AOX, on lactation performance and rumen fermentation in dairy cows (Expt.3 and 4).
The objective of Expt.3 was to evaluate the effect of diets supplemented with fatty acids of different degrees of saturation, in the absence or presence of AOX, on lactation performance of dairy cow. Calcium salt of long-chain fatty acids was supplemented as a source of lower saturation fatty acid (LS,53.9% SFA), and palmitic acid was supplemented as the higher saturation fatty acid source (HS,88.6% SFA). The AOX was added at 0.025% in the ration. Neither fatty acid type nor AOX supplementation showed significant effect on dry matter intake (DMI) during the study. Compared with those in HS, yield of milk and 4% FCM were lower in the LS-fed cows. Milk fat and milk protein concentrations were not affected by fatty acid type or AOX supplementation. Adding AOX increased the yield of milk and 4% FCM in the LS-fed cows, but did not affect those fed HS. Plasma SOD activity was significantly lower, plasma glucose tended to be lower, and plasma MD A was higher in the LS-fed animals, compared with those fed HS. Addition of AOX decreased both plasma NEFA and H2O2 contents and increased TAOC activity across the fatty acid types. Composition of C12:0, C18:0, cis-9 C18:1, trans-11 C18:1, C18:2, C20:5 and C22:6 in the erythrocyte membrane was higher, while C14:0, C16:0 and C16:1 was lower for cows fed diets supplemented with LS, compared with those with HS. Addition of AOX increased C14:0, C14:1 and C16:1 and decreased C18:0, cis-9 C18:1, C20:5 and C22:6-composition in the erythrocyte membrane. Cows fed LS had higher cis-9 C18:1 and trans-10, cis-12 C18:2 in milk at the expense of C18:0, whereas AOX addition increased milk cis-9 C18:1 at the expense of milk C12:0, C16:0, and trans-10, cis-12 C18:2. It is inferred that dietary inclusion of unsaturated fatty acids resulted in inferior lactation performance. Whereas, these negative effects may be partially alleviated by the addition of antioxidant.
Experiment 4 was carried out to evaluate the effect of LS and HS supplementation on rumen fermentation in vitro, in the absence or presence of AOX. The experiment was carried out in a 2×2 factorial design, with fatty acids type as one factor and AOX as another one. Fermentation patterns and anti-oxidative status were not affected by different fatty acids supplementation. Supplementation of LS significantly increased the populations of protozoa relative to total bacterial 16S rDNA, but showed negative effect on fibrobacter succinogenes. Addition of AOX significantly increased gas production at 24h incubation. Molar proportion of propionate tended to increase at the expense of acetate due to AOX addition. AOX tended to decrease MDA value and increase SOD activity. An interaction between AOX and fat type was observed on ruminococcus flavefaciens and ruminococcus albus. Inclusion of AOX increased these two bacteria in LS group, but not in HS. It is concluded that unsaturated fatty acids inclusion in the diets may result in inferior effect on cellulolytic bacteria in the rumen, while these negative effects may be partially alleviated by the addition of antioxidant.
Part three:Effect of energy density prepartum on performance and anti-oxidative status in transition cows, in the absence or presence of antioxidant (Expt.5)
In Expt.5, a 2 x 2 factorial design trial was conducted to evaluate the effect of dietary antioxidant and energy density on performance and anti-oxidative status in transition cows. Forty cows were randomly allocated to 4 dietary treatments. High or low energy density diets prepartum (1.43 or 1.28 Mcal NEL/kg DM, respectively) were formulated with or without AOX (0 or 5 g/d per cow). These diets were fed to cows for 21 days pre-partum. During the post-partum period, all cows were fed the same lactation diets, and AOX treatment followed as for the pre-partum period. Feeding a high energy diet depressed the DMI, milk yield, and 4% FCM of cows. However, AOX inclusion in the diet improved the milk and 4% FCM yields. There was an interaction of energy density by AOX on milk protein, milk fat and total solids contents. Feeding a high energy diet pre-partum significantly increased plasma glucose andβ-hydroxybutyrate (P＜0.05), whereas dietary AOX significantly decreased plasmaβ-hydroxybutyrate value during the transition period (P<0.05) There were also interactions between time and treatment for plasma glutathione peroxidase activity and MDA content during the study. Cows fed high energy diets pre-partum had higher plasma glutathione peroxidase activity 3 days prior to parturition, compared with those on low energy diets. Inclusion of AOX in diets' decreased plasma glutathione peroxidase activity in cows 3 and 10 days pre-partum. Addition of AOX significantly decreased MDA values at calving (P＜0.05). Energy density induced marginal changes in fatty acid composition in the erythrocyte membrane 3 days post-partum, while AOX only significantly increased cis-9, trans-11 C18:2 conjugated linoleic acid composition. The increase in fluidity of the erythrocyte membrane was only observed in the high energy treatment. The above results indicated that feeding low energy density prepertum could improve the postpartum DMI and lactation performance. The addition of AOX is beneficial to the lactation performance by improving the lipid metabolism and antioxidative status.
In summary, both the fat in the diet and lipid in the body are potentially oxidized, which could induce oxidative stress in dairy cows. The oxidative status may have a close relationship with energy metabolism. Feeding unsaturated fatty acids may result in inferior lactation performance, which is associated with inferior effect on rumen cellulolytic bacteria. Whereas, these negative effects may be partially alleviated by the addition of AOX. The effect of AOX depends on types of fatty acids. When unsaturated fatty acids were included in the diets, a significant benefit is shown by addition of AOX, but this is not the case with saturated fatty acid inclusion. A diet containing high energy density pre-partum may negatively affect the anti-oxidative status, DMI and subsequent performance. Addition of AOX may improve the anti-oxidative status and lipid metabolism, eventually resulting in improved lactation performance.
引文
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