Research Progress on Early Diagnosis of Pregnancy in Cattle and Sheep
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摘要:
规模化养殖中，母畜空怀将使养殖场遭受严重的经济损失，应用早期妊娠诊断技术，可有效降低空怀母畜数量，缩短产犊（羔）间隔，提高养殖效益。本文从诊断方法、诊断时间、诊断准确率等方面对牛羊早期妊娠临床诊断法和妊娠相关标志物诊断法进行综述。在临床诊断中，B超妊娠诊断较普遍，妊娠28～35 d超声检查可以获得比较可靠的结果，但其可靠性很大程度上取决于所用设备频率、操作者技能。在妊娠相关标志物诊断中，孕酮（Progesterone, P4）浓度检测法操作繁琐，对检测环境要求高，难以在生产中大面积推广。早期妊娠因子是受精后最早出现的特异性指标，但目前其检测完全依赖于使用玫瑰花环抑制试验，后续需开发简便的检测方法。干扰素刺激基因和外泌体miRNAs可能有助于牛羊早期妊娠诊断，但这些技术尚处于研究开发阶段。商业化的妊娠相关糖蛋白（pregnancy-associated glycoproteins, PAGs）检测试剂盒可作为B超妊娠诊断的替代方法，用于确定牛羊的早期妊娠或晚期胚胎损失。未来需要研发国产的商业化PAG检测试剂盒，以降低检测成本。本文通过总结不同检测方法的优缺点及实际应用效果，为生产者选择早期妊娠诊断方法提供参考，为牛羊早期妊娠诊断方法后期研究方向提供参考。
Abstract:In a commercial livestock or dairy farm, the so-called “empty sows” of those female animals failed to conceive in impregnation means economic loss for the business. Being able to accurately detect a pregnancy early, therefore, is indispensable for a timely determination allowing a short calving interval to enhance operational efficiency and profitability. This article reviewed the availability and advancements in the techniques and markers for the diagnosis on cattle and sheep. Published reports on the methodologies, examination time, and test result accuracy on the diagnosis are summarized with comments. For instance, B-ultrasound is commonly applied in clinical practices for 28–35 d pregnancy in the animals. Although relatively reliable results can be expected from the tests, they depend on the frequency and equipment selected as well as the skill of the operator. As a marker, progesterone (P4) is an indicator whose concentration in the animal is measured for the diagnosis. The method required specific testing environment, and consequently, is not popularly employed in the field. The initial fertilization indicator or physiochemical pregnancy signs in impregnated animals are currently detected by using the complex erythrocyte rosette test. Hence, not until a simple method is developed can a wide application based on the approach be realized. In theory, the interferon-stimulated genes and exosomal miRNAs may be useful for the diagnosis, but no testing technology has yet been established. And the commercially available PAG test kits for early-stage pregnancy or late-stage embryo loss are cost prohibitive for average farmers at present. By briefly describing the basics and presenting the pros and cons of various available devices and methods, this article provides a concise reference for the livestock ranchers in their decision-making and for the animal husbandry scientists in directing their efforts to develop a reliable and affordable means of fertilization detection in cattle and sheep.
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Keywords:
- cattle /
- sheep /
- pregnancy diagnosis /
- miRNAs /
- PAGs
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0. 引言
【研究意义】随着国家绿色发展战略和农药化肥减施政策的提出，充分利用农闲田种植绿肥，还田培肥地力，提高农作物产量和质量，已成为促进化肥减量增效及农业可持续发展的重要手段[1]。红萍，学名满江红（Azolla Lam.），江浙一带又称绿萍，具有固氮[2]、富钾[3]、增磷效[4]、培肥[5]等多重生态功能，繁殖力强[6]，是稻田生态系统中一种重要的水生绿肥。通过倒萍（将红萍埋入土中进行的还田操作）或插秧前耘田翻压，腐解后释放的养分可供后茬作物当季利用，替代部分化肥的同时还能改善土壤养分状况，是实现稻田土壤可持续利用的有效途径，在稻田化肥减施增效方面具有巨大潜力。开展红萍绿肥替代化肥效应的量化研究，对于红萍绿肥利用具有重要的指导意义。【前人研究进展】早在20世纪70～80年代，就有许多学者对稻田红萍的减肥增效作用进行了研究，在不同耕作制度和施肥水平下红萍替代化肥的效果不同，如早稻田用红萍做基肥或套养红萍作追肥，施用70%红萍氮可以相当于70%化肥氮的效果（每667m2总施氮量为12.25 kg），而在萍-稻-稻耕作制下，红萍氮可以替代总施氮量的30%～40%（每667m2施氮量为7～8 kg）[7]。近年来，随着国家高质量发展战略的提出，越来越多的学者关注红萍绿肥利用研究，并将利用从稻田[8-11]扩展到旱地[12-13]，关注点也逐渐从减肥增效[10-11]向固碳减排[9,14]倾斜。而关于稻田红萍绿肥利用的研究更多地集中在红萍与水稻共生或红萍与其他有机物料共同还田方面，如红萍和水稻秸秆联合还田，可以促进土壤碳氮矿化[8]；联合使用红萍和脲酶抑制剂的施肥模式可以减少农田NH3挥发，促进水稻增产[9]；红萍与水稻共作，可以减少稻田15%～30%的氮肥施用，减少氨挥发[10]等。红萍单独还田在稻田化肥减施方面的研究仅见Malyan的报道，认为红萍替代25%尿素氮后，水稻仍有增产[11]。【本研究切入点】近年来，随着耕作制度的变革，单季稻已逐渐发展成为南方稻区水稻（Oryza sativa）生产主要的耕作方式。红萍作为水生绿肥在新型耕作制度下对水稻化肥减量增效有很好的作用，但其减肥量和减肥效果都不甚明确，这些都亟待开展系统的量化研究。此外，前人通常利用冬闲田（12月至次年4月）养殖红萍，通过红萍多次倒萍腐烂起到养地肥田作用，但该法在复种指数高的地区势必影响冬种面积，且冬季红萍的繁殖能力不强，有些寒冷地区甚至需要增加防护措施才能保证萍母越冬。实际上在春季，单季稻插秧前（3～5月），气温达15 ℃以上时，养殖1～2个月红萍的产量就可达30 t·hm−2以上[7]。如果扩繁的红萍就地还田能为当季水稻提供肥效，将为红萍的利用与化肥减施增效提供新的途径。【拟解决的关键问题】为了明确新型耕作制度下，红萍还田对单季稻化肥减施及改良土壤的可行性和效果，在福建省粮食主产区浦城县开展红萍还田减肥试验，以春季培养可获得的30 t·hm−2红萍鲜萍还田，研究不同减肥处理下红萍翻压还田对单季稻生长、产量的影响和改土减肥的效果，旨在为红萍在单季稻中的利用提供数据支持，对于红萍在单季稻耕作制度下的减肥增效技术的应用和推广具有实际意义。
1. 材料与方法
1.1 试验材料
试验于2021年5月～10月在福建省浦城县莲塘镇马西村（经度118°29′50″，纬度27°59′2″，海拔255.75 m）进行，还田红萍为当地冬闲田种植的红萍，鲜萍平均含水率为92.23%，干物质中含全N 1.94%、全P 0.21%、全K 1.46%。种植的水稻品种为中稻嘉丰优2号。试验地土壤为灰泥田，土壤肥力为中上水平，耕层土壤（0～20 cm），有机质26.60 g·kg−1，碱解氮103.83 mg·kg−1，有效磷49.60 mg·kg−1，速效钾68.67 mg·kg−1，pH 4.92。
1.2 试验设计
试验共设6个处理，每个处理3次重复。T1：不施红萍和化肥；T2：常规（配方）施肥；T3：常规（配方）施肥+红萍还田；T4：减少常规施肥氮、钾肥用量20%+红萍还田；T5：减少常规施肥氮、钾肥用量40%+红萍还田；T6：减少常规施肥氮、钾肥用量60%+红萍还田。红萍还田量均为30 t·hm−2鲜萍。小区面积24 m2（6 m×4 m），小区间以地沟隔开，外设保护行。各小区随机区组排列。
1.3 供试肥料及施用方法
试验中常规施肥水平为N 180 kg·hm−2、P2O5 60 kg·hm−2、K2O 90 kg·hm−2。氮、磷和钾肥分别为尿素（46% N）、过磷酸钙（12% P2O5）和氯化钾（60% K2O）。磷肥全部作基肥，氮肥和钾肥按m（基肥）∶m（分蘖肥）∶m（穗肥）=4∶3∶3施用。基肥于水稻移栽前1 d施入，分蘖肥在移栽后5～7 d撒施，穗肥在水稻幼穗分化2期施用。各小区施肥及田间管理措施相同。水稻采用单本插秧，株行距23 cm×23 cm。各处理具体施肥量见表1。
表 1 不同处理小区施肥量Table 1. Amounts of fertilizer applied on plots for treatments处理
Treatment红萍还田量
Plow-down amount/kg基肥
Base fertilizer分蘖肥
Tiller fertilizer孕穗肥
Booting fertilizer尿素
Urea/g过磷酸钙
Superphosphate/g氯化钾
Potassium chloride/g尿素
Urea/g氯化钾
Potassium chloride/g尿素
Urea/g氯化钾
Potassium chloride/gT1 0 0 0 0 0 0 0 0 T2 0 375.65 1200.00 144.00 281.74 108.00 281.74 108.00 T3 72.00 375.65 1200.00 144.00 281.74 108.00 281.74 108.00 T4 72.00 300.52 1200.00 115.20 225.39 86.40 225.39 86.40 T5 72.00 225.39 1200.00 86.40 169.04 64.80 169.04 64.80 T6 72.00 150.26 1200.00 57.60 112.70 43.20 112.70 43.20 表中施肥量为单个小区（24 m2）的施肥量。
The fertilization amount in the table is the fertilization amount of a single plot (24 m2).1.4 田间操作管理
整地耘田后划出小区，小区田埂包膜，各小区设置独立进排水口，插秧前10 d将红萍按处理设置的还田量施入小区，人工翻压均匀。试验水稻5月1日播种育秧，5月5日进行小区隔离，5月25日红萍翻压入田，6月9日施基肥，6月15日水稻插秧移栽，7月1日施分蘖肥，8月4日施孕穗肥。10月13日收割，小区单打单收，实割测产。
1.5 测定项目及方法
水稻成熟后，各小区单收测产。同时每小区采水稻3穴地上部进行室内考种，记录稻谷和稻草鲜重产量，测定样品干重。并取各小区耕层土壤（0～20 cm）测定pH、有机质、碱解氮、有效磷和速效钾。其中土壤pH、有效磷和速效钾的测定分别采用中华人民共和国农业行业标准《NY/T 1377—2007 土壤pH的测定》[15]、《NY/T 1121.7—2014 土壤检测 第7部分：土壤有效磷的测定》[16]和《NY/T 889—2004 土壤速效钾和缓效钾含量的测定》[17]的方法；土壤有机质和碱解氮的测定分别采用鲍士旦的重铬酸钾容量法——外加热法和碱解扩散法[18]。
1.6 数据处理与分析
数据用Excel和SPSS 19.0进行统计和分析，以平均值±标准差的形式表示，显著性分析采用LSD法（P＜0.05）。
文中经济效益相关指标的计算公式如下：
单位面积水稻产值/（万元⋅hm−2）=（单位面积稻谷产量×稻谷单价）/10000 单位面积水稻种植成本/（元·hm−2）=单位面积肥料投入成本+单位面积红萍还田成本
单位面积肥料投入成本/(元⋅hm−2)=∑(小区各类型肥料投入量×肥料单价)/24×10000 单位面积水稻种植收益/（万元·hm−2）=（单位面积水稻产值−单位面积水稻种植成本）/10000
式中，稻谷单价按2.8 元·kg−1计算，10000为换算系数，各处理肥料投入量见表1，其中尿素（46% N）、过磷酸钙（12% P2O5）和氯化钾（60%K2O）的单价分别按浦城当地市场价格3.6、1.9、4.8 元·kg−1计算。
2. 结果与分析
2.1 红萍还田对水稻产量的影响
红萍还田对水稻产量有显著影响（表2）。与无肥处理（T1）相比，不同处理对水稻的增产效应为T4＞T3＞T2＞T5＞T6。其中，T4处理（氮、钾肥减量20%+红萍还田）稻谷产量最高，显著高于除T3外的其他处理（P＜0.05），比常规施肥处理（T2）平均增产11.11%。氮、钾肥减量40%或60%并辅以红萍还田的处理（T5和T6），分别比常规施肥处理（T2）减产0.92%、4.86%，但差异未达显著性水平（P＜0.05）。
表 2 红萍还田对水稻产量的影响Table 2. Effect of Azolla application on rice yield处理
Treatment稻谷产量
Grain yield/(t·hm−2)比T1
Compared to T1比T2
Compared to T2秸秆产量
Straw yield/(t·hm−2)收获指数
Harvest index增产量
Yield increment/(t·hm−2)增产率
Yield increase rate/%增产量
Yield increment/(t·hm−2)增产率
Yield increase rate/%T1 7.32±0.09 d — — — — 6.59±0.11 c 0.53±0.01 a T2 9.07±0.30 bc 1.75 23.86 — — 8.90±1.34 ab 0.51±0.03 a T3 9.68±0.26 ab 2.36 32.28 0.62 6.80 9.82±1.11 a 0.50±0.03 a T4 10.07±0.35 a 2.75 37.63 1.01 11.11 9.51±0.66 ab 0.51±0.02 a T5 8.98±0.86 bc 1.66 22.73 −0.08 −0.92 7.80±1.43 bc 0.54±0.03 a T6 8.63±0.91 c 1.31 17.84 −0.44 −4.86 8.33±0.49 abc 0.51±0.02 a 同列不同字母表示不同处理间差异显著（P ＜0.05）； “―”表示未参与比较。下同。
The different letters in the same column indicate significant difference between different treatments (P ＜ 0.05)." —"means not participating in the comparison. Same as below.地上部秸秆产量以T3最高，与T2、T4、T6无显著差异，但与T1、T5有显著差异（P＜0.05）。收获指数T5最高，但与其他处理无显著性差异。从稻谷产量、秸秆产量和收获指数结果看，T4处理对提高水稻产量效果最好，说明每公顷翻压30 t红萍鲜萍，可以替代常规施肥中20%的氮钾肥，还能显著提高稻谷产量。
2.2 红萍还田对水稻产量构成因素的影响
红萍还田对水稻产量构成因素的影响结果见表3。各处理对水稻的穗粒数、结实率无显著影响，但对株高、有效分蘖数、丛穗数、穗长、千粒重有显著影响。株高以T3最高，与T2、T4无显著差异，显著高于T1、T5、T6（P＜0.05）。有效分蘖数以T4最高，与T2、T3、T5无显著差异，与T1、T6差异显著（P＜0.05）。丛穗数以T3、T4最高，与T5无显著差异，与T1、T2、T6差异显著（P＜0.05）。穗长以T1最低，除T6外，其他处理均显著高于T1（P＜0.05）。千粒重以T6最高，与T3差异显著（P＜0.05），与其他处理无显著性差异。说明减氮钾肥20%，并辅以红萍还田的处理（T4）提高了水稻株高、有效分蘖数和丛穗数，且对水稻丛穗数的影响达到显著性水平（P＜0.05）。而在红萍还田的情况下，减肥至40%以上，水稻的株高、有效分蘖数开始明显降低，并在减氮钾肥60%+红萍还田处理（T6）下对水稻的有效分蘖数产生显著降低的影响（P＜0.05）。
表 3 红萍还田对水稻产量构成因素的影响Table 3. Effect of Azolla application on rice yield components处理
Treatment株高
Plant height/cm有效分蘖数
No. of effective tillers丛穗数
No. of spike per plant穗长
Spike length/cm穗粒数
No. of grains per spike结实率
Seed setting rate/%千粒重
1000 grain weight/gT1 125.03±1.76 d 5.27±0.35 c 5.56±0.39 c 22.50±0.20 b 275.24±24.12 a 91.00±0.04 a 25.43±0.70 a T2 140.91±3.80 abc 7.62±0.39 a 7.07±0.78 bc 24.22±0.47 a 287.12±23.79 a 91.70±0.01 a 25.29±0.64 a T3 145.33±0.71 a 7.84±0.37 a 8.33±0.00 a 23.95±0.46 a 299.13±15.24 a 81.67±0.11 a 23.70±1.22 b T4 144.25±3.43 ab 7.87±0.58 a 8.33±0.34 a 23.70±0.71 a 264.46±24.49 a 87.67±0.04 a 24.47±0.72 ab T5 138.46±4.70 bc 6.87±0.70 ab 8.00±1.20 ab 23.68±1.00 a 283.35±12.03 a 88.33±0.09 a 25.53±0.15 a T6 137.49±3.75 c 6.11±0.84 bc 6.11±0.51 c 23.48±0.59 ab 279.03±52.64 a 88.98±0.04 a 25.60±0.36 a 2.3 红萍还田的经济效益分析
试验根据市场价格对各处理进行经济效益分析比较，结果见表4。红萍还田主要的成本在于还田红萍萍母的养殖成本及红萍萍母的投放人工费，而红萍还田可结合插秧前耘田一起进行，故与常规施肥处理相比不增加还田费用。据报道，越冬萍母田平均每667 m2花工4个[7]，折工费500元，需肥料或农药约20元，合计每667 m2耗本约520元。按冬闲田平均每667 m2产萍7500 kg[7]折算，扩繁每公斤萍母需耗本0.07元。春季单季稻田每667 m2需投放萍母200～250 kg[19]，故每公顷所需母萍的成本约210～262.5元。每公顷投放母萍按1个工计，则稻田红萍绿肥利用每公顷共需耗本约330～380元。经扣除肥料和红萍还田成本后，收益以T4处理最好，显著高于T1、T5和T6处理（P＜0.05），收益比T1处理增加31.17%，比T2处理增加11.07%，增收2680.58元·hm−2，但未达显著性水平。说明实施红萍绿肥还田，不仅能减少后季水稻化肥施用量20%，还能增加经济收益。
表 4 红萍还田对水稻经济效益的影响Table 4. Effect of Azolla application on economic benefits for rice farming处理
Treatment产值
Output value/ (万元·hm−2)肥料成本
Fertilizer cost/（元·hm−2）红萍还田成本
The cost of returning Azolla to the field/（元·hm−2）收益
Income/（万元·hm−2）收入比T1增加
Income increased
over T1/%收入比T2增加
Income increased
over T2/%T1 2.05±0.03 c 0 0 2.05±0.03 c — — T2 2.54±0.08 b 1194.00 0 2.42±0.08 ab 18.09 — T3 2.71±0.07 ab 1194.00 380.00 2.55±0.07 ab 24.75 5.64 T4 2.82±0.10 a 978.00 380.00 2.69±0.10 a 31.17 11.07 T5 2.52±0.24 b 762.00 380.00 2.40±0.24 b 17.84 −0.21 T6 2.42±0.26 b 546.00 380.00 2.32±0.26 bc 13.47 −3.91 2.4 红萍还田对水稻收获后土壤养分含量的影响
红萍还田对水稻收获后土壤养分含量的影响结果见表5，施化肥显著降低了土壤pH（P＜0.05），而红萍还田并减肥的处理（T4、T5、T6）pH比100%施肥的处理（T2）pH均有所提高。可见，利用红萍还田，减少化肥施用，可以缓解土壤酸化。红萍还田处理，土壤有机质平均含量均高于无红萍还田的处理（T2）。其中T3处理有机质含量比T2提高了2.77 g·kg−1，且差异显著（P＜0.05）。此外，红萍还田处理的土壤碱解氮、有效磷和速效钾含量也均比无红萍还田处理高，但差异没有显著性。其中减60%氮钾肥并辅以红萍还田的处理（T6）速效钾含量比100%施肥处理（T2）显著提高（P＜0.05）。说明红萍还田替代化肥施用，可减缓化肥引起的土壤酸化的同时，还可以增加土壤有机质、碱解氮、有效磷和速效钾等养分的积累。
表 5 红萍还田对水稻收获后土壤养分含量的影响Table 5. Effect of Azolla application on nutrients in soil after rice harvest处理
TreatmentpH 有机质
Organic matter/
（g·kg−1）碱解氮
Alkaline hydrolyzed
nitrogen/（mg·kg−1）有效磷
Available phosphorus/
（mg·kg−1）速效钾
Available
potassium/（mg·kg−1）T1 4.97±0.04 a 29.01±3.07 ab 141.56±24.12 a 43.26±5.09 a 42.52±4.38 ab T2 4.89±0.04 b 27.10±3.38 b 138.44±15.88 a 43.75±5.91 a 41.85±6.07 b T3 4.88±0.04 b 29.87±3.72 a 150.50±22.95 a 43.90±6.30 a 47.01±12.98 ab T4 4.93±0.03 ab 28.81±2.14 ab 144.86±11.16 a 45.36±5.13 a 45.68±1.51 ab T5 4.92±0.01 ab 28.59±1.27 ab 144.09±13.37 a 49.46±4.38 a 45.35±1.07 ab T6 4.91±0.03 ab 30.43±2.13 a 157.50±9.26 a 46.60±2.60 a 55.66±9.35 a 3. 讨论
3.1 红萍还田对水稻生长的影响
大量的生产实践和科学研究已证明红萍对水稻生长发育具有促进作用[20]，具体表现为前期抑制，中期赶上，后期促进的效果[21]。王在德等[21]研究认为，红萍还田腐解会释放大量养分，促进水稻生长，推迟有效分蘖的终止期，株高和生物学产量均高于不施萍的对照。本试验结果亦是如此。同样100%施肥，施萍还田处理（T3）的株高，有效分蘖数和生物学产量均高于不施萍的处理（T2）。红萍还田主要是增进水稻中后期的营养，如果水稻在早期受抑不重，后期供肥适时，就会增加穗数，如果后期供肥不过量，就会增进结实率和千粒重取得增产[21]。本试验中，红萍还田显著增加了水稻丛穗数，并在减施氮钾肥后，水稻结实率和千粒重有明显增加。该结果与王在德等[21]的研究结果一致，也反映了本试验常规施肥处理中水稻后期供肥有过剩的可能。
3.2 红萍还田的减肥效应
前人对红萍还田减肥增效作用的研究结果不甚相同，这主要与应用田块的土壤肥力、水稻需肥特性及耕作制度的不同有关。叶国添等[7]报道，早稻田用红萍做基肥或套养红萍作追肥，在每667 m2总施氮量为12.25 kg的情况下，施用70%红萍氮可以相当于70%化肥氮的效果，而在萍-稻-稻耕作制下，每667 m2施氮量为7～8 kg时，用红萍氮替代总施氮量的30%～40%，水稻产量还能增产3.46%～5.21%。Malyan等[11]研究认为红萍替代25%尿素氮后，水稻产量比100%施尿素氮的处理增加14.8%。本试验是在单季稻耕作制下开展的研究，应用的水稻品种——嘉丰优2号属于耐肥能力强的品种，试验地块的土壤肥力属于中上水平（土壤有机质26.60 g·kg−1，碱解氮103.83 mg·kg−1，有效磷49.60 mg·kg−1，速效钾68.67 mg·kg−1），此土壤条件下每公顷翻压30 t红萍不仅可以替代常规施肥中20%的氮钾肥（T4），还能显著增加稻谷产量（比常规施肥增产1.01 t·hm−2，P＜0.05），替代40%的氮钾肥（T5），稻谷产量虽略有减少，但与常规施肥相比没有显著差异（P＞0.05）。可见，调整后期施肥策略（适当减施），可替代更多数量的化肥。此外，前人研究认为红萍钾的效益可以达到甚至略高于同数量化肥钾的效益[22]，本试验也尝试利用红萍替代钾肥，减氮的同时也减同比例钾，试验水稻未出现明显缺钾症状，说明红萍的钾对当季水稻的生产是有效的。
3.3 红萍还田的经济效益
明确红萍还田的经济效益，是推广红萍利用的重要依据。稻田红萍绿肥利用时，通常是利用冬闲田养萍，随插秧前的耘田实现红萍还田，故红萍还田的成本主要在萍母养殖和投放上。叶国添等[7]对萍母田的产萍量、花工和耗本都做了详细的统计，每元可平均产萍442 kg。现如今，人工成本大幅上涨，红萍绿肥利用的成本也随之升高。经本试验统计，现如今每元可平均产萍仅14.3 kg，成本增加了近30倍。尽管如此，红萍还田增产减肥的效益明显，减氮钾肥20%，扣除红萍还田成本后，仍比常规施肥增收2680.58元·hm−2。另一方面，生态种养的价值在现代化农业中越发凸显。据报道，南方稻区冬闲田种植紫云英在一个轮作周期中能够产生1.125万元·hm−2的生态价值[23]，倘若对红萍还田的生态效应进行价值核算，预计这部分的效益将远远超过水稻增产减肥带来的直接经济效益。此外，经多年调查和实践发现，连年生长红萍的田块，每到春季可自然生萍，不需额外投放母萍或作为新增地块替代施肥的萍母地，这将大大降低红萍绿肥利用的投入成本，显著提高红萍还田的增收效果。可见，实施红萍还田，尤其是连年红萍还田，不仅减肥还能增收。
3.4 红萍还田的改土培肥效果
大量研究表明，施用化肥显著降低农田土壤pH[24]。而绿肥还田后土壤有机质含量明显提高，pH值上升[25]。施书莲等[26]报道，100 kg红萍的干物质在一年内就可以转化成39 kg土壤有机质。Singh等[27]的盆栽模拟试验表明，红萍还田后，土壤pH呈上升趋势。本试验也表明，施用化肥会导致土壤酸化，而红萍还田并减少氮钾肥施用可明显提高土壤pH和有机质，可见红萍改土培肥效果显著。研究表明，绿肥还田对土壤肥力的影响及对后茬作物增产节肥效应的发挥随翻压年限的增加而增强[28, 29]，红萍亦是如此[30]。利用红萍还田，减少化肥施用，是改善稻田土壤酸化的有效措施，但红萍翻压还田改土培肥需要长期的施用累积，才能达到显著效果。
4. 结论
红萍还田量达30 t·hm−2时，不仅可以替代常规施肥中20%的氮钾肥，还能显著增加稻谷产量（比常规施肥处理增产11.11%，P＜0.05），并达到培肥地力（比常规施肥处理平均增加土壤有机质1.71 g·kg−1，碱解氮6.42 mg·kg−1，有效磷1.61 mg·kg−1，速效钾3.83 mg·kg−1，P＞0.05）的作用，是实现单季稻减肥增效的有效措施。
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表 1 不同妊娠相关标志物诊断法在牛羊生产中的应用
Table 1 Various methods for pregnancy diagnosis on cattle and sheep based on pregnancy-related markers
妊娠诊断方法
Pregnancy diagnostic生产中应用效果
Application effect in production优点
Advantage局限性
Disadvantage孕酮浓度检测
P4 detectionAI后10、21和30 d，流产奶牛P4显著低于妊娠奶牛[29]；
羊在AI后14～30 d，妊娠组P4显著高于未妊娠组[30]；方便、可批量操作 程序复杂、对检测环境要求严格 早孕因子检测
EPF detection牛配种后1个月，妊娠与空怀差异显著 [32]；
绵羊进行早孕诊断，准确率90.81%[33]；诊断时间早，妊娠24 h后即可检出 肿瘤 EPF可能造成假阳性结果 IFN-τ及ISGs
IFN-τ and ISGsAI后22 d流产奶牛ISG15比妊娠奶牛低[41]；
羊在AI后15 d内 ISG15的表达均上调[41]；诊断时间早 判别标准需要进一步研究 妊娠相关糖蛋白检测
PAG detection牛PAG检测试剂盒准确率可达93.9%[60]；
羊PAG检测试剂盒敏感性100.0%，特异性95.8%[57]；诊断时间早 检测成本和技术要求高 外泌体miRNAs 牛：miR-26a可能是潜在妊娠诊断标志物[65]；
羊：miR-379可能是潜在妊娠诊断标志物[69]；诊断时间早 在试验研究阶段，无相应检测产品 
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