诱导滞育的温度和光照节律对家蚕滞育激素基因Dh表达的影响

滞育激素(DH)是导致家蚕滞育生理现象出现的关键因素。研究卵期不同温度和光照节律引起家蚕滞育激素基因Dh表达的变化,探讨温度和光照对家蚕滞育调控的机制。催青期25℃持续光照(25LL)条件下,蚕卵中Dh基因mRNA高水平稳定转录;而在15℃持续黑暗(15DD)条件下,蚕卵中只有痕迹量Dh基因mRNA存在;25LL或者20℃、光照与黑暗12h交替(20LD)条件下,胚胎发育至单眼着色后(有效积温2160℃.h以后)Dh基因mRNA转录水平显著上调,与此时期胚胎对诱导滞育的温度和光照节律更加敏感一致。从催青期开始整个世代的保护温度和光照节律,呈现高温25℃显著上调整个胚后时期Dh基因mRNA转录水平,光照上调蛹期与蛾期Dh基因mRNA转录水平,且高温的作用强于光照。5龄第3天幼虫的基因芯片表达谱显示,Dh基因mRNA转录水平存在显著的性别与组织差异,雌蚕多个组织中的Dh基因mRNA转录水平都显著低于雄蚕,卵巢中的Dh基因mRNA只有痕迹量。亲代卵的催青温度与光照节律决定子代卵的滞育性,也影响到Dh基因mRNA的转录水平;但子代卵内Dh基因mRNA转录水平高低不是蚕卵滞育与否的关键。推测家蚕Dh基因表达的性别差异及在早期蚕卵中表达特征与滞育性不一致的现象,可能与Dh基因的前体多肽翻译产物剪切有关。     

滞育诱导温度与光照对家蚕近日节律基因per表达的影响

温度和光照是昆虫近日节律基因表达的授时因子,也是影响家蚕滞育的决定因素。为了解家蚕滞育与近日节律调控的关系,研究了滞育诱导发生温度与光照对家蚕近日节律基因per表达的影响。家蚕整个世代在25℃高温环境下,或幼虫期与蛹期在20℃的较高温度下,per基因在黑暗环境中的转录水平高于光照环境。家蚕幼虫及蛹期在相同光照条件下,per基因转录水平在高温环境中高于低温环境,黑暗环境下又呈现20℃25℃15℃的变化趋势,且温度的影响大于黑暗环境;在25℃持续光照条件(25LL)下,胚后时期per基因的表达水平高于15℃持续黑暗的条件(15DD),而20℃及光照与黑暗各12 h条件(20LD)下变化更加敏感,这与温度和光照对家蚕滞育的诱导作用一致。在处女蛾和卵产出后0.5 h的未受精蚕卵中都检测到了高水平的母源per基因mRNA。亲代卵催青期的单因子25℃高温或黑暗、以及20LD,都会引起子代卵内per基因mRNA水平的上调,这种上调只出现在滞育出现或活化发育的转折时间点(卵龄72 h)之前。家蚕滞育诱导温度和光照条件直接影响per基因在整个世代的差异表达,该基因在卵期的表达与胚胎滞育性差异密切关联。     

滞育诱导发生温度与光周期对家蚕羽化激素基因表达的影响

羽化激素(EH)是调控生长发育的重要神经肽激素。本文研究了家蚕滞育发生诱导温度和光周期条件对羽化激素基因(Eh)表达的影响。结果显示,在15DD非滞育卵诱发环境下,催青期胚胎的Eh基因mRNA一直处于很低水平;而在25LL滞育卵诱发环境或20LD滞育卵与非滞育卵同时出现的诱发条件下,能够检测到Eh的高水平表达,提示胚胎期可能已经开始合成EH。其中25LL蚕卵Eh基因的表达水平高于20LD区,并在积温高于1 440℃.h(反转期)后,Eh基因表达水平显著上调,这与在此阶段的蚕卵胚胎对滞育诱导环境温度和光照节律更加敏感相一致。在胚后幼虫至成虫期维持催青期的15DD或25LL环境中,1-4龄幼虫在食桑期Eh基因继续了胚胎后期25LL中高表达的现象,但5龄幼虫至成虫期出现15DD环境Eh基因表达水平显著高于25LL区的现象。胚后改用25℃自然光照环境保护,25LL区的子代滞育性卵和15DD区的子代非滞育性卵中,表现出15DD环境Eh基因表达水平高于25LL区的现象,在卵龄48h后更加明显。结果表明亲代卵的孵化温度与光周期在决定子代卵的滞育性同时,显著影响整个世代Eh基因的表达。     

滞育诱导发生温度与光周期对家蚕垂体同源框激素基因Pitx表达的影响

家蚕垂体同源框激素PITX对滞育激素DH的分泌与作用有直接影响。为了探讨家蚕滞育的信号传导机制,研究了3种典型的滞育发生诱导温度和光周期条件对Pitx基因表达的影响。结果显示,亲代催青期滞育卵诱导发生条件25LL显著上调了蚕卵中Pitx基因表达水平。整个世代持续25LL环境中,3龄前幼虫和成虫体内,Pitx基因的表达水平高于15DD环境中的蚕体;胚后蚕体Pitx基因的表达水平有显著组织差异,幼虫眠中表达水平有下调趋势。子代卵内Pitx基因mRNA的水平变化与亲代诱导所致的蚕卵滞育性差异密切相关,滞育性卵中未检测到母源Pitx基因的mRNA,卵龄12～24h的胚胎发生期则有较高水平的表达,卵龄72h后的滞育发生阶段后只有痕迹量;非滞育性卵中有较高水平的母源Pitx基因mRNA,胚胎发生及发育期,Pitx基因转录水平显著高于滞育性卵;亲代20LD催青条件下所产卵滞育性不稳定,Pitx基因的mRNA水平显著高于滞育性卵,也高于非滞育性卵。亲代卵的孵化温度与光周期决定子代卵的滞育性,也影响到Pitx基因表达水平,子代卵内Pitx基因表达水平高低不是蚕卵滞育与否的关键。     

滞育诱导发生温度与光周期对家蚕垂体同源框激素基因Pitx表达的影响

<正>家蚕垂体同源框激素PITX对滞育激素DH的分泌与作用有直接影响。为了探讨家蚕滞育的信号传导机制,研究了3种典型的滞育发生诱导温度和光周期条件对Pitx基因表达的影响。结果显示,亲代催青期滞育卵诱导发生条件25LL显著上调了蚕卵中Pitx基因表达水平。整个世代持续25LL环境中,3龄前幼虫和成虫体内,Pitx基因的表达水平高于15DD环境中的蚕体;胚后蚕体Pitx基     

催青温度对家蚕二化性品种滞育激素受体基因表达的影响及基因的结构特征

家蚕二化性品种的卵滞育与否受上代环境影响,25℃高温明催青将产滞育卵,而15℃低温暗催青将产非滞育卵。为探究催青温度调控二化性家蚕滞育的分子机制,对从家蚕卵巢细胞中克隆的家蚕滞育激素受体基因(Bmdhr)的5种cDNA进行生物信息学分析,结果表明Bmdhr基因的5种cDNA由相同的mRNA转录本通过不同的剪接方式而来,其中Bmdhr mR-NA-1与Bmdhr mRNA-2编码的氨基酸序列相同,Bmdhr mRNA-4编码的氨基酸序列与家蚕滞育激素BmDHR-1的序列相似度达99.2%。将家蚕二化性品种秋丰的蚕卵用蛾区半分法分成2组,分别以15℃暗催青和25℃明催青,利用实时荧光定量PCR分析催青温度对家蚕不同发育时期及蚕体组织中Bmdhr基因mRNA转录的影响。结果显示:Bmdhr mRNA-1主要在蛹期卵巢中表达,在对滞育激素最敏感的化蛹后4 d时,其转录水平急速上升至峰值,并且高温催青的转录水平高于低温催青;Bm-dhr mRNA-4主要在各发育时期的蚕体血液中表达,特别是在高温明催青条件下,其在蛹期血液中的转录水平是低温暗催青的7.7倍,说明BmDHR-4可能是决定家蚕二化性品种卵滞育与否的关键因子之一;Bmdhr mRNA-5在化蛹后2～3 d的卵巢中转录水平高,且低温催青的转录水平高于高温催青,化蛹后3 d其转录水平开始下降,至化蛹后4～5 d 2种催青处理间的转录水平无显著差异。研究结果为阐明家蚕滞育的分子机制积累了实验数据。     

家蚕山梨醇脱氢酶基因的转录水平特性分析

为了探究家蚕山梨醇脱氢酶基因（BmSDH）在家蚕各发育时期和组织的转录水平,根据家蚕二化性品种子代滞育性受亲代胚胎期催青条件调控的原理,利用半定量RT-PCR分析25.0℃常温明催青和17.5℃低温暗催青对家蚕品种“秋丰”各发育时期蚕体和组织中BmSDH转录水平的影响.结果显示,BmSDH-1、BmSDH-2a和BmS-DH-2b在家蚕的各个发育阶段均有表达：在幼虫期5龄3d,BmSDH-1在常温明催青组脂肪体中转录水平较高;BmSDH-2a和BmSDH-2b在常温明催青条件下的血液、脂肪体和卵巢中转录水平均高于低温暗催青;在蛹期3d,BmSDH-2a和BmSDH-2b在常温明催青条件下血液和卵巢中的转录水平均低于低温暗催青;在次代卵产下后6～24h期间,BmSDH-2a和BmSDH-2b的转录水平逐渐升高.因此,BmSDH-1和蚕卵滞育与否没有直接关系,而BmSDH-2a和BmSDH-2b与家蚕二化性品种卵滞育与否有重要的平行关系,为从分子水平阐明家蚕滞育的分子机制积累了试验数据.     

二化性家蚕滞育过程中谷胱甘肽S-转移酶活性的变化

二化性家蚕卵以25℃和15℃催青,可分别诱导成虫产下子代滞育和非滞育卵,而即时浸酸和5℃左右的低温处理可以分别阻止和解除胚胎滞育。利用1-氯-2,4-二硝基苯(CDNB)显色法测定了经上述处理后的胚胎滞育过程中谷胱甘肽S-转移酶(glutathione S-transferase,GST)的活性,结果表明:胚胎发育后期的蚕卵经25℃催青,卵中的GST活性显著高于15℃催青蚕卵;蚕卵即时浸酸未显著改变胚胎滞育发动阶段蚕卵中的GST活性;5℃低温处理显著提高了滞育卵中的GST活性,但是未能显著改变即时浸酸卵的GST活性。上述结果表明,蚕卵中的GST活性变化与二化性家蚕胚胎滞育诱导和解除密切相关。     

诱发滞育的温度和光照条件对家蚕胚胎生物钟信号主要基因表达的影响

为了深入研究温度和光照等昼夜节律授时因子对家蚕滞育的调控机制,以常规诱导滞育发生的温度与光照条件处理胚胎发育后期蚕卵,调查家蚕胚胎生物钟信号环路主要基因Cry1、Cry2、Per和Tim的表达对授时因子振荡变化的响应。结果显示:光照能够上调Cry1基因在家蚕胚胎发育后期的表达,温度则可改变该基因表达的振荡周期相位;温度在改变Cry2基因振荡表达相位的同时,低温(15℃)还能上调该基因在家蚕胚胎发育后期的表达;温度升高可缩短Per基因表达的振荡周期;黑暗诱导Tim基因振荡表达,温度则能够改变Tim基因表达的振荡周期相位。结果提示:家蚕胚胎中可能存在与其他昆虫类似的以CRY1为光感受器因子的生物钟信号环路,Cry1、Cry2、Per和Tim是家蚕胚胎期能够响应昼夜节律授时因子光照和温度而诱导滞育发生的生物钟基因。     

家蚕二化性品种(品系)转基因用蚕卵的预处理技术研究

家蚕为卵滞育昆虫,二化性家蚕品种(品系)的化性由上代卵期温度与光照等调控。将若干个二化性家蚕品种(品系)以17~18℃温度催青诱导子代蚕卵非滞育,筛选出1个经济性状较优的实用品种"秋丰"、1个突变品系P33,二者非滞育卵圈比例分别达到96.4%和89.6%,正常催青的良卵实用孵化率分别达到96.11%和98.58%,解决了采用显微注射法以家蚕实用品种构建转基因系统过程中因蚕卵滞育不能及时孵化的难题。另对利用20%盐酸-2%甲醛混合液刺激解除蚕卵滞育的方法以及非滞育蚕卵表面用70%乙醇消毒的方法进行了尝试,前者可以获得96%以上的孵化率,后者可以缩短蚕卵预处理时间。     

Artificial long-day photoperiod in the subtropics increases milk production in goats giving birth in late autumn

Two experiments were conducted to determine whether exposure to a photoperiod of artificial long days in autumn increased milk yield in subtropical goats milked once (Exp. I) or twice daily (Exp. II). In Exp. I, starting at d 10 of lactation, 1 group of does was kept under naturally decreasing photoperiod (DD1X; n = 8), whereas the other group was submitted to an artificial photoperiod of long days (LD1X; n = 8; 16 h light:8 h darkness). The kids were weaned 28 d after parturition, and dams were manually milked once daily. Milk yield and milk components (fat, protein, and lactose) were assessed up to 140 d of lactation. From d 0 to 28 of lactation (suckling phase), mean daily milk yield did not differ between DD1X and LD1X goats (2.3 ± 0.2 kg vs. 2.4 ± 0.2 kg; P = 0.717). However, between d 29 and 84 (early milking phase), mean daily milk yield was greater in LD1X does than in DD1X does (2.6 ± 0.1 kg vs. 2.1 ± 0.1 kg; P = 0.001). Finally, between d 85 and 140 (late milking phase), mean daily milk yield was greater in LD1X goats than in DD1X goats (P ≤ 0.05) only during the first 2 wk. In Exp. II, one group of goats was exposed to a photoperiod of naturally decreasing days (DD2X; n = 8) and another group was submitted to an artificial photoperiod of long days (LD2X; n = 7). In both groups, kids were weaned on d 28 of lactation and the dams were manually milked twice daily. During the nursing phase, mean daily milk yield did not differ between the DD2X and LD2X groups (2.5 ± 0.3 kg vs. 2.6 ± 0.2 kg; P = 0.767). In the early milking phase, mean daily milk yield was greater in LD2X than in DD2X goats (3.3 ± 0.2 kg vs. 2.8 ± 0.2 kg; P = 0.022), whereas during the late milking phase, milk yield did not differ between the 2 groups (P = 0.946). In both experiments, milk composition was not significantly influenced by exposure to long-day photoperiod. We conclude that, in subtropical female goats that start lactation in late autumn, exposure to an artificial long-day photoperiod stimulates milk yield, even if goats are milked once daily. In addition, combining exposure to long days with twice-daily milking will increase further milk yield in such goats without affecting milk components.     

催青温度对家蚕二化性品种丙酮酸脱氢酶激酶基因(BmPDK)表达的影响

丙酮酸脱氢酶激酶(PDK)在生物体的新陈代谢中具有重要的生理功能。为了探讨家蚕(Bombyx mori)PDK基因的表达特性,用蛾区半分法将二化性家蚕品种的活化越年卵(丙2期)分别以常温(25℃)光照和低温(15℃)黑暗催青,通过实时荧光定量PCR技术检测分析2种温度催青处理后家蚕不同发育阶段和不同组织的BmPDK表达水平。常温光照催青区BmP-DK的表达水平表现出发育时期和组织差异:胚胎发育阶段BmPDK在己4期的表达水平最高,其次是戊2、己3和己5期;幼虫发育阶段BmPDK的表达水平在蚁蚕期极显著高于其它龄期,5龄期在卵巢和精巢的表达水平高于其它组织;蛹期BmPDK的表达水平比其它发育时期低;成虫期的表达水平与胚胎发育末期相当,其中雌蛾的脂肪体和卵巢中BmPDK的表达水平较高。推测BmPDK在家蚕胚胎发育末期及产卵过程中有重要作用。催青温度影响BmPDK在家蚕各发育时期及卵巢组织中的表达水平:胚胎发育阶段戊2期BmPDK的表达水平为常温催青区显著高于低温催青区,己3～己5期常温催青区BmPDK的表达呈低-高-低的趋势,而低温催青区则呈上升趋势;蚁蚕期BmPDK表达水平为低温催青区极显著低于常温催青区;蛹期和成虫期卵巢中,常温催青区BmPDK的表达水平极显著高于低温催青区。催青温度对二化性家蚕BmPDK的表达的影响主要出现在胚胎戊2期、胚胎己3期～蚁蚕期、蛹期、成虫期。     

Effect of moving dawn, dusk, or both on oviposition time in domestic laying hens

1. Lohmann Brown pullets were reared on 8-h photoperiods and transferred to 12 h at 15 weeks by either advancing dawn or delaying dusk by 4 h. At 25 weeks, half of each group was transferred to 16 h by advancing dawn or delaying dusk and, 10 d later, each photoperiod-group was advanced or delayed 4 h by moving both dawn and dusk. Individual oviposition times were recorded over 48 h at 25 weeks and 8-10 d after each lighting change. 2. At 25 weeks, there were no differences in mean time of oviposition, eggs laid in the modal 8 h, or in the proportion of eggs laid before dawn between pullets that had been given a 4-h increase in daylength at 15 weeks by advancing dawn and those photostimulated by delaying dusk. 3. Extensions of the daylength from 12 to 16 h at 25 weeks, whether by advancing dawn or by delaying dusk, delayed mean oviposition time by 2 h and virtually eliminated egg-laying before dawn. All groups laid > or = 94% of eggs in the modal 8 h. 4. Moving the complete 12- or 16-h photoperiod forwards by 4 h delayed egg-laying by 0.5 h (relative to dawn), whilst moving them backwards advanced it by 0.7 h. Pullets given 16 h of light laid very few eggs before lights-on, but the divergent movements in oviposition time, relative to dawn, resulted in more eggs being laid before lights-on when the 12-h photoperiod was moved backwards and fewer eggs when it was moved forwards. However, a slower adjustment to the new times of dawn and dusk by the pullets whose day had been moved forward, as indicated by fewer eggs being laid in the modal 8 h and poorer rates of lay compared with pullets that had their photoperiod moved backwards, may explain these differences. It is doubtful that the differences would have persisted after all birds had adjusted their ovulatory cycle, and so any reduction in pre-dawn egg-laying is likely to have been transitory. 5. The only permanent way to minimise pre-dawn egg-laying in brown-egg hybrids is to provide a photoperiod of at least 16 h, though 14-15 h may be long enough for white-egg hybrids and 12-13 h sufficient for broiler breeders.     

Free-running rhythms of adrenocorticotropic hormone (ACTH), cortisol and melatonin in pigs.

In the domestic pig, a circadian rhythm of plasma cortisol occurs, with greatest concentrations in the morning and lowest concentrations in the afternoon. However, photic entrainment of the rhythms of ACTH and melatonin in pigs have not been defined clearly. This experiment was designed to evaluate free-running rhythms of ACTH, cortisol and melatonin in pigs housed in constant light (LL) and constant darkness (DD). Twelve crossbred barrows, maintained under ambient photoperiod, were catheterized and tethered individually in two environmentally controlled rooms, one with LL and the other with DD. For animals in LL, fluorescent lights provided 202 +/- 15 (mean +/- standard deviation) lux of light at 65 cm above the floors. Incandescent nightlights equipped with 7 watt red bulbs provided 7 +/- 2 lux and were illuminated continuously in both rooms. Pigs were given at least 14 d exposure to LL and DD, then samples of plasma and serum were obtained at hourly intervals for 48 hr. Plasma was assayed for ACTH, and serum for cortisol and melatonin. Periodograms were constructed to analyze the data. For this type of analysis, a statistic, Qp, is calculated, and circadian periodicity is suggested if maximum Qp (Qp max) occurs at or near 24 hr. The period of the free-running rhythms (tau) at Qp max for ACTH, cortisol and melatonin for pigs in LL (23.80 +/- .01, 23.78 +/- .01, and 23.21 +/- .02 hr, respectively) did not differ significantly from those for pigs in DD (23.39 +/- .01, 23.20 +/- .01, and 22.55 +/- .02 hr, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of temperature on transovarial transmission of Babesia bigemina (Smith and Kilborne, 1893) in Boophilus annulatus (Say, 1821)

The effect of the temperature on the transovarial passage of Babesia bigemina has been studied in female Boophilus annulatus during their oviposition. Kinetes of Babesia were present in eggs laid at the temperatures of 16, 20, 26, 28, 30 and 35 degrees C. The rate of infection was temperature dependent. It reached at least 50%, even at low temperatures. The average infection level at 26 and 30 degrees C was 270 kinetes per egg. However, between 20 and 35 degrees C the eggs laid during the first 3 days were parasite free. At 16 degrees C, no kinetes were detected during the first 13 days of oviposition.     

The control of lymphoid leukosis in a flock White Plymouth Rock chickens

Summary Lymphoid leukosis (LL) was successfully controlled in a commercial basic breeding line of White Plymouth Rock chickens. The control method has been developed for breeder flocks and consists of three elements: - In the flock under study, homogenates of embryos from all eggs collected during a number of I4-day periods are tested for the presence of LL viruses. - Only eggs from hens that have been shown not to shed virus in their eggs are used for the production of progeny. The offspring are reared in isolation during the first two months of life, at which time the age-related resistance against tumour formation by LL viruses appears to be sufficiently developed. - The chickens are subsequently inoculated intramuscularly with LL viruses of subgroups A and B transferred to a conventional chicken house. The vaccination raises a solid immunity to horizontal LL virus exposure and, due to the age-related resistance, tumour formation does not follow. No excretion of LL viruses could be detected in three generations of White Plymouth Rock chickens to which the three elements of the control procedure were applied. Clinical disease was not observed in any of the chickens under notice.     

Removal of nocturnal secretion of melatonin fails to reduce antibody synthesis and interleukin-2 production of lambs.

Crossbred ewe and wether lambs were used to evaluate the effects of a normal, nocturnal elevation in the concentration of melatonin in the serum on immunological functions. The nocturnal elevation in melatonin was eliminated by exposing half the lambs to constant light (LL), whereas the remainder received a 12-h light, 12-h dark cycle (LD). Immune function was challenged by treating half the lambs in LL and half of the lambs in LD with dexamethasone (DEX; .04 mg/kg); the remainder of the lambs received only a saline vehicle (SAL). The resulting treatment combinations were designated LD+SAL (n = 5), LD+DEX (n = 5), LL+SAL (n = 5), and LL+DEX (n = 5). Lambs were stanchioned individually in environmental rooms; photoperiod treatments commenced on that day (d -14). Also on d -14, lambs were given 1 mg ovalbumin/lamb in adjuvant. Lambs were given a booster injection of .5 mg ovalbumin/lamb on d 0. Treatments with DEX and SAL also began on d 0 and were repeated every 48 h through d 14. Catheters were placed in the jugular vein of all lambs on d 12; samples of plasma and serum were collected hourly from 0800 on d 14 to 0800 on d 15; plasma was assayed for adrenocorticotropic hormone (ACTH) and serum was assayed for cortisol and melatonin. In addition, samples of serum obtained at 0800 on d 15 were used to evaluate antibody titers to ovalbumin. Samples of whole blood also were obtained at 0800 on d 15, and total and differential leukocyte numbers and production of interleukin-2 (IL-2) by lymphocytes were determined.(ABSTRACT TRUNCATED AT 250 WORDS)