Impacts of the unsaturation degree of long-chain fatty acids on the volatile fatty acid profiles of rumen microbial fermentation in goats in vitro

This study investigated the impacts of the degree of unsaturation （unsaturity） of long-chain fatty acids on volatile fatty acid （VFA） profiles of rumen fermentation in vitro. Six types of long-chain fatty acids, including stearic acid （C18：0, control group）, oleic acid （C18：1, n-9）, linoleic acid （C18：2, n-6）, a-linolenic acid （C18：3, n-3）, arachidonic acid （C20：4, n-6） and eicosapentaenoic acid （C20：5, n-3）, were tested. Rumen fluid from three goats fitted with ruminal fistulae was used as inoculum and the inclusion rate of long-chain fatty acid was at 3% （w/w） of substrate. Samples were taken for VFA analysis at 0, 3, 6, 9, 12, 18 and 24 h of incubation, respectively. The analysis showed that there were significant differences in the total VFA among treatments, sampling time points, and treatment×time point interactions （P〈0.01）. a-Linolenic acid had the highest total VFA （P〈0.01） among different long-chain fatty acids tested. The molar proportion of acetate in total VFA significantly differed among treatments （P〈0.01） and sampling time points （P〈0.01）, but not treatment×time point interactions （P〉0.05）. In contrast, the molar proportion of propionate did not differ among treatments during the whole incubation （P〉0.05）. However, for butyrate molar proportions, significant differences were found not only among sampling time points but also among treatments and treatment×time point interactions （P〈0.01）, with eicosapentaenoic acid having the highest value （P〈0.01）. Additionally, no statistically significant differences were found in the acetate to propionate ratios among treatments groups （P〉0.05）, even the treatments stearic acid and a-linolenic acid were numerically higher than the others. The inclusion of 3% long-chain unsaturated fatty acids differing in the degree of unsaturation brought out a significant quadratic regression relation between the total VFA concentration and the double bond number of fatty acid. In conclusion, the a-linolenic acid with 3 double bonds appeared better for improving rumen microbial fermentation and the total VFA concentration.

Identification of a Long-Chain Fatty Acid Elongase from Nannochloropsis sp. Involved in the Biosynthesis of Fatty Acids by Heterologous Expression in Saccharomyces cerevisiae

The marine microalga Nannochloropsis sp.contains various elongases and desaturases that are critical for biosynthesis of polyunsaturated fatty acids.A full-length cDNA encoding a long-chain fatty acid elongase,named Ns FAE,was cloned from Nannochloropsis sp..The open reading frame of Ns FAE(GenBank accession no.MF680548)consisted of 1068 bp and encoded a predicted protein of 355 amino acids with molecular mass 38.8 k Da.The deduced polypeptide showed 43%–44%identity to fatty acyl elongases from other algae.RT-PCR experiments indicated that the Ns FAE gene exhibited the highest expression in Nannochloropsis sp.at 72 h(i.e.,during the third growth stage)and the expression was significantly lower in the other four growth stages.Plasmid p Ns FAE-CRISPR and a recombinant DNA fragment(ADH1p-Ns FAE-CYCt)were transformed into Saccharomyces cerevisiae strain BY4742 using the CRISPR-Cas system.Yeast transformants containing Ns FAE produced three fatty acids not normally present in wild-type BY4742-linoleic acid,linolenic acid and eicosadienoic acid-indicating that Ns FAE encodes a functional elongase enzyme.

The effects of the unsaturated degree of long-chain fatty acids on the rumen microbial protein content and the activities of transaminases and dehydrogenase in vitro

This study investigated the effects of the degree of unsaturation（unsaturity） of long-chain fatty acids on microbial protein content and the activities of transaminases and dehydrogenase in vitro using goat rumen fluid as the cultural medium.Six types of fatty acids,stearic acid（C18：0,group A,control group）,oleic acid（C18：1,n-9,group B）,linoleic acid（C18：2,n-6,group C）,α-linolenic acid（C18：3,n-3,group D）,arachidonic acid（C20：4,n-6,group E）,and eicosapentaenoic acid（C20：5,n-3,group F）,were tested,and the inclusion ratio of each fatty acid was 3%（w/w） in total of culture substrate.Samples were taken at 0,3,6,9,12,18 and 24 h,respectively,during culture for analyses.Compared with stearic acid,linoleic acid,a-linolenic acid,and arachidonic acid increased the bacterial protein content,while oleic acid and eicosapentaenoic acid had no significant effects;the protozoal protein content was reduced for all the unsaturated fatty acids,and the magnitude of the reduction appeared to be associated with the degree of unsaturity of fatty acids.The total microbial protein content was dominantly accounted by the protozoal protein content（about 4-9 folds of the bacterial protein）,and only increased by linoleic acid,but reduced by oleic acid,arachidonic acid and eicosapentaenoic acid.There were no significant effects in the activities of both glutamic oxaloacetic transaminase（GOT） and glutamic-pyruvic transaminase（GPT） for all the other fatty acids,except for arachidonic acid which enhanced GOT activity and oleic acid which enhanced GPT activity.The total dehydrogenase activity was positively correlated with the degree of unsaturation of fatty acids.In conclusion,the inclusion of 3%of long-chain unsaturated fatty acids increased bacterial protein content,whereas reduced protozoal protein content and enhanced dehydrogenase activity.The fatty acids with more than three double bonds had detrimental effects on the microbial protein content.This work demonstrates for the first time the effect rule of the unsaturation degree of long-chain fatty acids on the rumen microbial protein in vitro.

CONVERSION OF UNSATURATED LONG－CHAIN FATTY ACIDS TO 10－HYDROXY FATTY ACIDS BY RESTING CELLS OF Nocardia sp

Resting cells of Nocardia sp.were used to convert a series of unsaturated long-chain fatty acids to 10-hvdroxy fatty acids.Structures of all metabolites were suggested by 1Hnuclear magnetic resonance and mass spectrum.The results showed that the hydroxylation sterospecificity happened at the cis-9 double bond with other position unaffected.

Long-chain acyl-CoA synthetase in fatty acid metabolism involved in liver and other diseases:An update

Long-chain acyl-Co A synthetase(ACSL) family members include five different ACSL isoforms, each encoded by a separate gene and have multiple spliced variants. ACSLs on endoplasmic reticulum and mitochondrial outer membrance catalyze fatty acids with chain lengths from 12 to 20 carbon atoms to form acyl-Co As, which are lipid metabolic intermediates and involved in fatty acid metabolism, membrane modifications and various physiological processes. Gain- or lossof-function studies have shown that the expression of individual ACSL isoforms can alter the distribution and amount of intracellular fatty acids. Changes in the types and amounts of fatty acids, in turn, can alter the expression of intracellular ACSLs. ACSL family members affect not only the proliferation of normal cells, but the proliferation of malignant tumor cells. They also regulate cell apoptosis through different signaling pathways and molecular mechanisms. ACSL members have individual functions in fatty acid metabolism in different types of cells depending on substrate preferences, subcellular location and tissue specificity, thus contributing to liver diseases and metabolic diseases, such as fatty liver disease, obesity, atherosclerosis and diabetes. They are also linked to neurological disorders and other diseases. However, the mechanisms are unclear. This review addresses new findings in the classification and properties of ACSLs and the fatty acid metabolismassociated effects of ACSLs in diseases.

Effects of long-chain fatty acid supplementation on the growth performance of grower and finisher pigs: a meta-analysis

Background: Supplementation of feed with long-chain fatty acids(LCFAs) during the grower and finisher phases has long been discussed as a growth promotion strategy in pigs, but its effects are inconsistent. The purpose of our study was to comprehensively evaluate its effects on the growth performance based on the average daily gain(ADG), average daily feed intake(ADFI) and gain: feed(G:F) ratio and to unveil the roles of the basal diet, LCFA concentration and LCFA saturation.Results: We searched the Pub Med and Web of Science databases(articles published from Jan 1 st, 2000, to Sep 30 th,2018;restricted to English) and compared LCFA-supplemented diets with control diets. We retrieved 2346 studies, 18 of which(1314 pigs, 26 records) were eligible for our analysis. We used a random-effects model to calculate the weighted mean differences(WMDs) and 95% confidence intervals(CIs). LCFA supplementation in the grower-finisher phase improved the ADG(WMD = 41.74 g/d, 95% CI: 8.81 to 74.66, P = 0.013) and G:F ratio(WMD = 0.019, 95% CI: 0.006 to 0.032, P = 0.003). For supplementation solely in the finisher phase, we found a similar performance in the ADG(WMD = 39.93 g/d, 95% CI: 26.48 to 53.38, P < 0.001) and G:F ratio(WMD = 0.019, 95% CI: 0.006 to 0.032, P < 0.001) but a reduction in the ADFI(WMD =-83.863 g/d, 95% CI:-156.157 to-11.569, P = 0.023). Specifically, approximately 5%LCFA supplementation in the finisher phase had significant effects on the ADG(WMD = 51.385 g/d, 95% CI: 35.816 to66.954, P < 0.001), ADFI(WMD =-102.869 g/d, 95% CI:-189.236 to-16.502, P = 0.02) and G:F ratio(WMD = 0.028, 95%CI: 0.018 to 0.039, P < 0.001), whereas a concentration of approximately 1% exhibited no effects.Conclusions: Overall, regardless of the basal diet and saturation, LCFA supplementation greatly improves the growth performance of grower and finisher pigs, primarily by increasing the energy density.

Roles of Long-chain Acyl Coenzyme A Synthetase in Absorption and Transport of Fatty Acid

Long-chain acyl coenzyme A synthetase(ACSL) is a member of the synthetase family encoded by a multigene family;it plays an important role in the absorption and transport of fatty acid.Here we review the roles of ACSL in the regulating absorption and transport of fatty acid,as well as the connection between ACSL and some metabolic diseases.

Evaluation of Fourier Transform Infrared Spectroscopy for Diagnosis of Peroxisomal Diseases with Abnormal Very-Long-Chain Fatty Acid Metabolism

Very long chain fatty acids (VLCFAs) are accumulated in cells and blood in patients with peroxisomal diseases, such as adrenoleukodystrophy (ALD) and Zellwger Syndrome (ZS). The purpose of this study is to investigate usefulness of Fourier transform infrared spectroscopy (FTIR) with attenuated total reflection (ATR) analysis method for clinical diagnosis of those diseases, thereby we measured the infrared spectra of the sera of patients and healthy controls. Correlation coefficients between 2nd derivative FTIR spectra of the serum samples and the VLCFA content ratio which is used as a clinical parameter to date were comprehensively calculated to investigate which wavenumber showed high correlation with the VLCFA ratio. Multiple regression analysis using the serum FTIR spectra showed that high correlations were observed with VLCFA ratios (C26:0/C22:0 ratio), and we could construct a suitable regression model (R2 = 0.97, p ﹣19). In addition, the model system using various VLCFAs in newborn bovine serum also showed that several FTIR peaks in 800 ~ 900 cm﹣1 region were found to have good correlation with VLCFA ratios. Our results support that FTIR analysis is useful for diagnosis of peroxisomal diseases.

Impact of Chain Length of Saturated Fatty Acids during Their Heterogeneously Catalyzed Deoxygenation

Fatty acids with different chain length were deoxygenated in the absence of hydrogen (caprylic acid (CA), lauric acid (LA) and stearic acid (SA)). The catalytic tests were carried over Pd-containing catalysts out in a batch reactor under inert gas for 6 h at 250&degC to 350&degC and pressures from 18 to 75 bar in the absence of additionally fed hydrogen. Pd-containing catalysts were tested;the best performing catalyst was 10% Pd/C with 63% undecane yield at 327&degC. These catalysts were used for a comparative decarboxylation of CA, LA and SA. At equal reaction conditions (300&degC, 6 h), the chain length of the fatty acid had a strong impact on the conversion, which was steadily increasing, whereas the alkane selectivity ran through a maximum. This work demonstrated the usability of Pd-containing catalysts for the decarboxylation of various fatty acids in the absence of additionally fed hydrogen with respect to the manufacture of hydrocarbons that can be used as blending components for fuels.

LCFAs降解动力学特性及对厌氧消化各阶段的影响

针对长链脂肪酸(LCFAs)影响厌氧消化体系稳定性的问题,研究了不同浓度下3种典型LCFAs(棕榈酸C16:0、硬脂酸C18:0、油酸C18:1)的甲烷化特性及其对厌氧消化的影响.甲烷化结果表明,油酸实现完全降解明显更加缓慢(26d),但比产甲烷活性(SMA)更高,分别为棕榈酸、硬脂酸的1.2、2.7倍,并且延滞期(t0)和最大产甲烷速率(R_(max))随浓度升高呈线性增加,表明饱和度较碳链长度对LCFAs降解动力学影响更大.微生物群落分析表明,油酸组中Clostridium_sensu_stricto、Petrimonas丰度更高(10.38%和10.03%),在β-氧化前可能饱和化产生了多羟基LCFAs,降解相对缓慢;油酸的加入还促进了Methanosarcina、Methanobacterium及Methanomassiliicoccus的富集,强化了氢营养产甲烷路径.此外,3种LCFAs对厌氧消化各阶段均产生抑制作用,产甲烷阶段的抑制程度最强(分别为16.51%、40.49%、63.77%),且受碳链长度与饱和度共同影响.

Effect of TRH and TSH on Circulatory Glucose and Fatty Acids Responses in Hypoinsulenemic Male Dwarf Goats

Diabetes mellitus or hypoinsulinemia was induced successfully in the male dwarf goats aged be-tween 2 - 3 years with 2 consecutive administrations of streptozotocin. A comparable group of intact control goats was also maintained. In ruminants including goats unlike non-ruminants, insulin generally displays ineffectiveness or resistance in their biochemical setup to facilitate gluco-neogenesis, the only source of glucose in these animals. In present study almost in the absence of insulin through induced hypoinsulinemia the effects of thyrotropin releasing hormone (TRH) (30 μg/kg body weight) and thyroid stimulating hormone (TSH) (2.5 μg/kg body weight) on circulatory glucose and different fatty acid fractions were studied in insulin resistant ruminant model. Fatty acid fractions were estimated by gas chromatography. Both TRH and TSH administration lowered glycemia in insulin deficient goats compared to the controls but significantly with TSH dose only. In intact goats the detectable circulating long chain fatty acids (LCFAs) fractions of lauric, myristic, palmitic, stearic, oleic and linoleic acid were undetected except linoleic acid in the hypoinsulinemic state, however were found restored following TRH and TSH administrations and some of LCFAs;stearic (6417%), oleic (1676%) and linoleic acid (1225%) increased exceptionally with TSH dose. In Intact goats however the hormones variedly increased the fractions. The volatile fatty acid fractions (VFAs) of formic, acetic, propionic, iso-butyric, n-butyric, iso-valeric, n-valeric, iso-caproic, n-caproic and heptanoic acid were detected in the goats. The most VFAs fractions markedly increased in hypoinsulinemic goats compared to the control goats following TRH and TSH infusion. These results have indicated that endogenously stimulated thyroid hormones with TRH and TSH in insulin deficient state inhibit the mechanisms of utilizing the fatty acids in glucose production. Therefore the study reveals thyroid hormones inhibitory effects on gluconeogenesis in insulin resistance and hyperglycemia.

磁性固相萃取-气质联用-同位素内标法精准检测白酒中8种高级脂肪酸乙酯

高级脂肪酸乙酯是引起酒体浑浊的关键因素之一。为系统分析白酒中的高级脂肪酸乙酯以改善白酒生产过程中的浑浊问题,建立了一种基于氨基化四氧化三铁(Fe_(3)O_(4)-NH_(2))磁性固相萃取结合气质联用技术的方法,用以同时测定白酒中8种高级脂肪酸乙酯。通过考察酒样的pH值、离子强度、吸附剂用量、吸附时间、解吸溶剂种类、解吸溶剂酸碱度、解吸时间等因素,确定了较佳的萃取条件。结果表明:这8种高级脂肪酸乙酯在各自的质量浓度范围内线性关系良好,相关系数R 2均大于0.99;方法检出限和定量限分别为9.1~42.9μg/L和5.2~200.0μg/L;回收率为79.6%~99.3%,相对标准偏差(n=3)为8.0%~10.7%。应用此方法,并结合同位素内标法对24种不同香型和品牌的白酒样品中8种高级脂肪酸乙酯进行精准定量,发现它们的种类和质量浓度存在显著差异。其中,在酱香型白酒中,高级脂肪酸乙酯的检出率最高,达到100%。该方法简便快捷,样品萃取只需10 min,具有高灵敏度和准确性,能够有效避免传统液-液萃取技术的背景干扰问题,可为实现白酒中高级脂肪酸乙酯的精准检测提供一种新的方法。

长链脂肪酸钠/芳基酰胺脂肪酸钠复配成核剂对聚丙烯性能影响研究

使用长链脂肪酸钠盐与芳基酰胺脂肪酸钠(SCAB)复配作为聚丙烯(PP)成核剂,研究了复配前后SCAB对PP结晶及力学性能的影响。结果表明,长碳链脂肪酸钠盐在复配过程和PP加工温度下均未改变SCAB的分子结构且复配可以明显改善SCAB分子间聚集状态;PP的结晶速率、缺口冲击强度和拉伸强度随着长链脂肪酸钠盐的碳链长度延长大幅提高且复配物未影响PP的晶体生长方式;硬脂酸钠复配的SCAB由于在PP基体中优异的分散性,能够更有效细化PP球晶,完善晶体结构,使其结晶速率提高6.3倍,缺口冲击强度提升27.3%,拉伸强度增长8.2 MPa。

蒜头果种子萌发过程中油脂的变化

为了解富含神经酸等超长链脂肪酸的种子在萌发过程中油脂的变化,检测了蒜头果种子萌发6个阶段的不同器官、组织中的含油量以及脂肪酸组成,通过分析其变化趋势和差异性,为蒜头果成熟种子中积累大量超长链脂肪酸提供合理解释。结果表明,蒜头果萌发过程中对脂质的利用集中在幼苗形态建成阶段,此时胚乳中的脂质含量迅速下降,靠近胚根的胚乳脂质消耗早于其他部分。在胚乳中检测出11种脂肪酸,其中神经酸(C24∶1)含量最为丰富,萌发时降幅最大。而营养器官根、茎、叶未表现出对神经酸、芥酸的积累偏好。综合表明,超长链脂肪酸是蒜头果种子萌发的能量和营养来源。

人体二十二碳六烯酸检测方法的研究进展

二十二碳六烯酸(DHA)作为人体必需的长链多不饱和脂肪酸之一,对儿童尤其是婴幼儿大脑发育、成人脑功能维持及心血管疾病预防等均有重要作用。DHA的人体来源主要依靠膳食补充,其缺乏人群较为普遍。因此,监测人体内DHA水平是预防因DHA不足或缺乏引起疾病的关键。目前人体DHA水平检测方法较多,但标准化的检测方法尚少见文献报道。该文通过综述近几年已发表的人体DHA水平评估或检测方法,主要包括膳食调查法、色谱法、核磁共振技术和近红外光谱技术,并对各种方法的优势和局限性进行分析总结,以期为人体DHA水平检测提供新思路及科学指导,并助力健康人群DHA水平参考区间的建立。

Case Report: Carnitine Palmitoyl Transferase II (CPT II) Deficiency

Carnitine Palmitoyl Transferase II (CPTII) is a very important enzyme that helps with the oxidation of long-chain fatty acid to produce energy. Deficiency in CPTII will lead to energy deficiency in the case of fasting and the accumulation of the long chain fatty in the body. There are three types of CPT II deficiency, the myopathic form, the severe infantile hepatocardiomuscular form and the lethal neonatal form. They are all inherited as an autosomal recessive. Diagnosis of the CPTII are 1) tandem mass spectrometry (MS/MS) in adult form and 2) CPTII polymorphism (F352C), which is linked to reducing the activity of CPTII in infantile form [1]. Glucose is the primary management and medium-chain fatty acid is an alternative due to the bypass of the CPTII enzyme in the pathway. For the prevention of CPTII deficiency are to avoid long chain fatty acid (C12-fatty acid), fasting, prolonged exercise, known triggers, and certain medications such as anti-epileptics and general anesthesia. During the rhabdomyolysis and myoglobinuria attack, it is very important to maintain hydration to avoid acute renal failure. If, however, renal failure occurs, dialysis is recommended. We present a case of a 27-year-old African American woman with the significant past medical history of CPT II deficiency leading to recurrent rhabdomyolysis and myoglobinuria. Together with all the research studies from diagnosis to treatment of CPTII deficiency will help in clinical management of patients. And this case report will add to the existing case reports of patients who have CPTII deficiency in terms of how we diagnose, how we treat, and how we prevent symptoms from re-occurring.

油脂厌氧消化中LCFAs的危害及调控策略研究进展

重点阐述了油脂厌氧代谢途径及微生物作用机理,概述了LCFAs积累对厌氧消化的抑制作用,并从联合消化、添加微量元素或缓冲剂、微生物驯化和反应器开发等角度总结了调控油脂厌氧消化的措施和方法,并结合存在的问题对今后的发展方向和应用原则进行了概括。

Synthesis of Long Chain Fatty Acids Acylated Coumarin Glycoside Esters with Lipase as Catalyst

A series of novel coumarin glycoside esters（1-9） was synthesized through the acylation reaction of 4-methylcoumarin-7-O-β-D-glucoside（11） with different long chain fatty acids catalyzed by lipase in organic medium. The acylation occurred regioselectively at the 6＇-OH of glycosyl moiety. The enzymatic synthesis was optimized to achieve 54%-70% yield using immobilized lipase（Novozym 435, 10 mg/mL） as catalyst and acetone and pyri- dine（9：1, volume ratio, water content〈1%） as solvent with an acyl donor/coumarin glycoside molar ratio of 10：1 at a temperature of 40--50 ℃ for 96 h. All the synthesized compounds were confirmed.

Oligomerized Amyloid-<i>β</i>_1-40Peptide Favors Cholesterol, Oxysterol, and Fatty Acid Accumulation in Human Neuronal SK-N-BE Cells

Amyloid peptide, the main component of senile plaques, is a major biological characteristic of Alzheimer’s disease (AD). The aim of the present study conducted on human neuronal SK-N-BE cells was to evaluate whether oligomerized Aβ1-40-induced cell damages was associated with lipid modifications. Under treatment with Aβ1-40 (10 - 100 μM;24 - 48 h), cell viability was recorded with the MTT test and by measuring LDH activity. Mitochondrial transmembrane potential and ATP production were assessed using flow cytometry and a luciferase-based ATP bioluminescence assay, respectively. Annexin V-CF647 staining assay for cell apoptosis detection was performed using flow cytometry. Potentially intracellular cytotoxic lipids (oxysterols: 7α-hydroxycholesterol (7α-OHC), 7β-hydroxycholesterol (7β-OHC), and 7-ketocholesterol (7KC), 24(S)-hydroxycholesterol;arachidonic acid (C20:4 n-6);VLCFAs (C22:0, C24:0, C24:6 and C26:0)) were measured using gas chromatography coupled with mass spectrometry. The cellular level of docosahexaenoic acid (C22:6 n-3), often altered in AD, was also quantified. In the presence of Aβ1-40, the percentage of MTT-positive cells decreased and was associated with an increase in LDH activity. In addition, treatment with oligomerized Aβ1-40 induced a decrease of mitochondrial transmembrane potential as well as an apoptotic cell death. Sterol analysis revealed a higher cholesterol level and a significant increase of cytotoxic oxysterols per cell (7KC + 7β-OHC), and of the [(7β-OHC + 7KC)/cholesterol] ratio, considered as a lipid peroxidation index, in Aβ1-40-treated cells. An enhancement of C20:4 n-6, C22:6 n-3 and saturated VLCFAs was also observed. Therefore, Aβ1-40-induced side effects are associated with intracellular accumulation of lipids, especially cholesterol, oxysterols (7β-OHC, 7KC), C20:4 n-6, and saturated VLCFAs, which could in turn contribute to neurotoxicity.

不同日龄北京油鸡胸肌中长链脂肪酸的变化规律研究

为研究相同饲养条件下的北京油鸡胸肌中长链脂肪酸随日龄的变化规律,试验运用气相色谱-质谱联用的靶向代谢组学方法,分别在150、300和450日龄对北京油鸡胸肌中长链脂肪酸含量进行检测。结果显示:北京油鸡胸肌共鉴定到33种中长链脂肪酸,其组成不随日龄而变化;北京油鸡胸肌各脂肪酸分类在450日龄的绝对含量极显著高于150和300日龄(P<0.01),但其相对比例在不同日龄间未发现显著差异(P>0.05);北京油鸡胸肌n-6/n-3PUFAs及PUFAs/SFAs等指标分别在(17.06~17.99)∶1和77.20%~80.95%范围内,其不受日龄影响,表现出相对的稳定性。结果表明日龄对北京油鸡胸肌脂肪酸分类的绝对含量影响程度高于其对相对比例的影响,且主要体现在300~450日龄饲养阶段上。