Effect of orally administrated chlortetracycline microcapsule on microflora quantity in sheep rumen
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摘要:目的
评估口服自主研制的金霉素微囊化颗粒对绵羊瘤胃微生物菌群数量的影响。
方法选取体质量（29.0±2.3） kg、6月龄左右、体况良好的18只绵羊，随机分为6组，包括1个空白对照组和5个给药组，每组3只。空白对照组只饲喂基础饲粮；5个给药组分别为试验I组：基础饲粮＋普通金霉素预混剂颗粒，试验II组：基础饲粮＋氢化油处方组金霉素微囊化颗粒，试验III组：基础饲粮＋PEG4000/单甘脂(质量比为5∶5)处方组金霉素微囊化颗粒，试验IV组：基础饲粮＋氢化油/单甘脂(质量比为9∶1)处方组金霉素微囊化颗粒，试验V组：基础饲粮＋氢化油/单甘脂(质量比为8∶2)处方组金霉素微囊化颗粒。给药组绵羊金霉素最终给药剂量均为25 mg/kg，每天给药1次且均在晨饲前灌胃给药，连续给药5 d，通过瘤胃液采样器采集瘤胃内容物。采集的瘤胃内容物经4层纱布过滤后，收集滤液装于冻存管内，所有样品经液氮速冻后置于−80 ℃保存，运用实时荧光定量PCR(RT-PCR)技术对绵羊瘤胃液相瘤胃微生物进行定量检测。
结果与普通金霉素预混剂颗粒相比，氢化油/单甘脂(质量比为9∶1)处方组金霉素微囊化颗粒有较好效果，只显著增加了产琥珀酸拟杆菌的数量，对绵羊瘤胃其他微生物菌群数量影响均不显著；与空白对照组相比，氢化油/单甘脂(质量比为9∶1)处方组金霉素微囊化颗粒及PEG4000/单甘脂(质量比为5∶5)处方组金霉素微囊化颗粒有相对较好效果，分别只对牛链球菌及产琥珀酸拟杆菌的数量有显著降低趋势，对绵羊瘤胃其他微生物菌群数量影响均不显著。
结论氢化油/单甘脂(质量比为9︰1)处方组金霉素微囊化颗粒对绵羊瘤胃微生物数量抑制作用较小，符合临床应用要求。
Abstract:ObjectiveTo evaluate the effect of orally administrated self-developed chlortetracycline microcapsule on microflora quantity in sheep rumen.
MethodEighteen sheep of (29.0±2.3) kg of 6-month-old with good health condition were randomly assigned to six groups with three replicates in eash grcup, including one control group and five medicated groups. The sheep in the control group were only fed with basal diet. The treatments in five medicated groups were as follows. Group I: Basal diet supplemented with normal chlortetracycline premix granule; Group II: Basal diet supplemented with hydrogenated oil prescription chlortetracycline microcapsule; Group III: Basal diet supplemented with PEG4000/glycerol stearate (mass ratio of 5︰5) prescription chlortetracycline microcapsule; Group IV: Basal diet supplemented with hydrogenated oil/glycerol stearate (mass ratio of 9︰1) prescription chlortetracycline microcapsule; Group V: Basal diet supplemented with hydrogenated oil/glycerol stearate (mass ratio of 8︰2) prescription chlortetracycline microcapsule. The chlortetracycline administration dosages of sheep in the five medicated groups were all 25 mg/kg. The sheep were administrated once a day before morning feeding for five days. The rumen contents were collected by rumen fluid collector and quickly filtered through four layers of gauze after continuous administration. The filtrate were collected in the cryopreserved tube and quickly frozened by liquid nitrogen then stored at −80 ℃. The rumen microorganisms of liquid phase were quantitatively detected by real-time fluorescence quantitative PCR (RT-PCR).
ResultCompared with the normal chlortetracycline premix granule,hydrogenated oil/glycerol stearate (mass ratio of 9︰1) prescription chlortetracycline microcapsule had better effect, only significantly increased the quantity of Fibrobacter succinogens, and had no significant influence on the quantities of other microbial flora in sheep rumen. Compared with the control group, hydrogenated oil/glycerol stearate (mass ratio of 9︰1) prescription chlortetracycline microcapsule and PEG4000/glycerol stearate (mass ratio of 5︰5) prescription chlortetracycline microcapsule had relatively better effects, significantly decreased the quantities of Streptococcus bovis and F. succinogens respectively, and had no significant influence on the quantities of other microbial flora in sheep rumen.
ConclusionThe hydrogenated oil/glycerol stearate (mass ratio of 9︰1) prescription chlortetracycline microcapsule has less inhibition effect on rumen microflora quantity and meets clinical application requirements.
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Keywords:
- sheep /
- rumen /
- microbe /
- chlortetracycline premix /
- chlortetracycline microcapsule
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图 1 瘤胃液基因组DNA凝胶图
1、14：DL2000 marker；2~13：瘤胃液粗提总DNA
Figure 1. Electrophoretogram of rumen fluid genomic DNA
1, 14: DL2000 marker; 2−13: Total DNA of rumen fluid crude extraction
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图 2 8种瘤胃微生物的普通PCR扩增结果
1、10：DL2000 marker，2：白色瘤胃球菌，3：牛链球菌，4：黄化瘤胃球菌，5：瘤胃厌氧真菌，6：普雷沃氏杆菌，7：溶纤维丁酸弧菌，8：瘤胃总细菌，9：产琥珀酸拟杆菌
Figure 2. Normal PCR amplification results of eight kinds of ruminal microbes
1, 10: DL2000 marker; 2: Ruminococcus albus; 3: Streptococcus bovis; 4: Ruminococcus flavefaciens; 5: Rumen anaerobic fungi; 6: Prevotella spp.; 7: Butyrivibrio fibrisolvens; 8: Rumen general bacteria; 9: Fibrobacter succinogens
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图 4 8种瘤胃微生物菌液PCR扩增
图A中，1：DL2000 marker，2、3：普雷沃氏杆菌，5：白色瘤胃球菌，6、7：溶纤维丁酸弧菌，8、9：黄化瘤胃球菌，10：牛链球菌，12、13：产琥珀酸拟杆菌；图B中，1、2、3：瘤胃总细菌，4、5、6：瘤胃厌氧真菌，7：DL2000 marker
Figure 4. Bacterial fluid PCR amplification of eight kinds of ruminal microbes
In figure A, 1: DL2000 marker; 2, 3: Prevotella spp.; 5: Ruminococcus albus; 6, 7: Butyrivibrio fibrisolvens; 8, 9: Ruminococcus flavefaciens; 10: Streptococcus bovis; 12, 13: Fibrobacter succinogens. In figure B, 1, 2, 3: Rumen general bacteria; 4, 5, 6: Rumen anaerobic fungi; 7: DL2000 marker
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图 5 瘤胃厌氧真菌和瘤胃总细菌标准曲线
Figure 5. Standard curves of rumen anaerobic fungi and rumen general bacteria
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图 6 瘤胃厌氧真菌和瘤胃总细菌扩增曲线
Figure 6. Amplification curves of rumen anaerobic fungi and rumen general bacteria
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图 7 瘤胃厌氧真菌和瘤胃总细菌熔解曲线
Figure 7. Melt curves of rumen anaerobic fungi and rumen general bacteria
表 1 瘤胃微生物PCR扩增引物
Table 1 PCR amplification primers of ruminal microbes
引物名称
Primer name序列(5′→3′)
Primer sequenceθ退火/℃
Annealing temperature产物长度/bp
Product length参考文献
Reference瘤胃总细菌
Rumen general bacteriaF: CGGCAACGAGCGCAACCC
R: CCATTGTAGCACGTGTGTAGCC60 130 [7] 瘤胃厌氧真菌
Rumen anaerobic fungiF:GAGGAAGTAAAAGTCGTAACAAGGTTTC
R: CAAATTCACAAAGGGTAGGATGATT60 120 [7] 白色瘤胃球菌
Ruminococcus albusF: CCCTAAAAGCAGTCTTAGTTCG
R: CCTCCTTGCGGTTAGAACA55 175 [8] 黄化瘤胃球菌
Ruminococcus flavefaciensF: TCTGGAAACGGATGGTA
R: CCTTTAAGACAGGAGTTTACAA55 295 [8] 产琥珀酸拟杆菌
Fibrobacter succinogensF: GTTCGGAATTACTGGGCGTAAA
R: CGCCTGCCCCTGAACTATC60 121 [7] 牛链球菌
Streptococcus bovisF: ATTCTTAGAGATAGGGTTTCTCTT
R: ACCTTATGATGGCAACTAACAATA60 134 [9] 普雷沃氏菌属
Prevotella spp.F: GAA GGT CCC CCA CAT TG
R: CAA TCG GAG TTC TTC GTG56 418 [10] 溶纤维丁酸弧菌
Butyrivibrio fibrisolvensF: CGCATGATGCAGTGTGAAAAGCTC
R: CCTCCCGACACCTATTATTCATCG56 625 [9] 
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表 2 普通金霉素预混剂颗粒及4种金霉素微囊化颗粒对绵羊瘤胃微生物数量的影响
Table 2 Effects of normal chlortetracycline premix granule and four kinds of chlortetracycline microcapsules on the quantity of ruminal microbe of sheep
微生物种类
Microbe species微生物数量1） Microbe quantity I II III IV V CK 瘤胃总细菌 Rumen general bacteria 12.65Aa 12.24Aa 12.56Aa 12.43Aa 12.33Aa 12.35a 产琥珀酸拟杆菌 Fibrobacter succinogens 8.75BCcd 9.19Bbc 8.29Cd 10.14Aa 9.40Bb 10.21a 白色瘤胃球菌 Ruminococcus albus 8.78Aa 8.26Bb 9.03Aa 8.70Aa 8.77Aa 8.76a 黄化瘤胃球菌 Ruminococcus flavefaciens 9.79Aa 9.20Bb 9.82Aa 9.61Aa 9.61Aa 9.63a 普雷沃氏杆菌属 Prevotella spp. 11.53Aa 11.22Aa 11.40Aa 11.09Aa 11.46Aa 11.22a 溶纤维丁酸弧菌 Butyrivibrio fibrisolvens 9.00Bb 8.90Bb 10.50Aa 9.34Bb 8.92Bb 9.28b 牛链球菌 Streptococcus bovis 7.32Ab 7.41Ab 7.66Aab 7.21Ab 7.48Ab 8.04a 瘤胃厌氧真菌 Rumen anaerobic fungi 8.77Aa 8.35Aa 8.72Aa 8.98Aa 8.37Aa 8.82a 1) I组：基础饲粮+普通金霉素预混剂颗粒，II组：基础饲粮+氢化油处方组金霉素微囊化颗粒，III组：基础饲粮+PEG4000/单甘脂(质量比为5∶5)处方组金霉素微囊化颗粒，IV组：基础饲粮＋氢化油/单甘脂(质量比为9∶1)处方组金霉素微囊化颗粒，V组：基础饲粮＋氢化油/单甘脂(质量比为8∶2)处方组金霉素微囊化颗粒，CK：基础饲粮；相同微生物种类数据后的不同大写字母表示5个给药组间差异显著，不同小写字母表示5个给药组和空白对照组间差异显著(P<0.05，Duncan’s法)
1) Group I : Basal diet supplemented with normal chlortetracycline premix granule, group II : Basal diet supplemented with hydrogenated oil prescription chlortetracycline microcapsule, group III : Basal diet supplemented with PEG4000/glycerol stearate (mass ratio of 5∶5) prescription chlortetracycline microcapsule, group IV : Basal diet supplemented with hydrogenated oil/glycerol stearate (mass ratio of 9∶1) prescription chlortetracycline microcapsule, group V : Basal diet supplemented with hydrogenated oil/glycerol stearate (mass ratio of 8∶2) prescription chlortetracycline microcapsule, CK: Basal diet; Different capital letters after the same microbe species data indicate significant differences among five medicated groups, different lowercase letters after the same microbe species data indicate significant differences among five medicated groups and the blank control group (P<0.05, Duncan’s method)
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