副珠蜡蚧阔柄跳小蜂的寄生影响因子

副珠蜡蚧阔柄跳小蜂Metaphycus parasaissetiae是橡副珠蜡蚧Parasaissetia nigra成虫的重要内寄生蜂。寄生蜂的寄生效果受多种因素影响,就温度、光周期、寄主成虫日龄、雌蜂日龄及交配等因子对副珠蜡蚧阔柄跳小蜂的寄生率、每头寄主的出蜂量及后代雌性率的影响进行研究,以期探明几种因子对该蜂寄生效果的影响。结果表明:温度为27—30℃时寄生率最高为91.7%,出蜂量以27℃最高为7.5头,雌性率以24℃条件最高为72.1%,其次27℃为62.6%,说明24—30℃是寄生效果较好的适温范围;光周期对副珠蜡蚧阔柄跳小蜂的影响明显,光暗比为12 h∶12 h条件下,其寄生率、出蜂量、后代雌性率均最大,分别为70.6%、5.3头、68.3%;副珠蜡蚧阔柄跳小蜂寄生用20—21日龄橡副珠蜡蚧成虫的寄生率、出蜂量及后代雌性率均为最高,分别为87.0%、5.6头、51.2%,说明20—21日龄的橡副珠蜡蚧成虫是副珠蜡蚧阔柄跳小蜂最适合寄生的阶段;雌蜂日龄对寄生效果的影响明显,3日龄的出蜂量最多为5.6头,4日龄的小蜂寄生率最高为81.7%,5日龄的雌性率最大为55.2%;交配是另一影响寄生效果及性比的重要因子,已交配的寄生率及出蜂量显著高于未交配的,分别为91.7%、7.5头,交配的雌性率为62.1%,而未交配所育后代均为雄蜂。综合以上结果分析,可考虑选用20—21日龄橡副珠蜡蚧成虫、充分交配的3—6日龄小蜂、温度27℃及光周期L∶D=12∶12作为室内扩繁副珠蜡蚧阔柄跳小蜂的理想条件组合。     

低温对副珠蜡蚧阔柄跳小蜂发育及繁殖的影响

副珠蜡蚧阔柄跳小蜂是橡副珠蜡蚧的重要寄生蜂。为了探明副珠蜡蚧阔柄跳小蜂对低温的适应性,本文在15℃-24℃范围内对该蜂的产卵、发育、出蜂量及性比进行观察。结果表明:副珠蜡蚧阔本跳小蜂在15℃-24℃范围内,随温度的升高,产卵量逐渐增加;该蜂在21℃、24℃时蜂能完成世代发育,在15℃、18℃时少量跳小蜂能发育到蛹,但均不能羽化为成蜂;温度对性比的影响明显,24℃的雌性比例高于21℃。综上所述,副珠蜡蚧阔柄跳小蜂对18℃以下的低温适应性较差,室内繁殖或野外放蜂时注意选择较高的温度条件。     

温度对副珠蜡蚧阔柄跳小蜂发育和繁殖的影响

通过观察副珠蜡蚧阔柄跳小蜂Metaphycus parasaissetiae Zhang and Huang在18,21,24,27,30,33和36℃恒温条件下的生长发育情况,求得其世代发育起点温度和世代有效积温分别为13.10℃和215.00日.度,建立世代发育历期预测式为N=[215.00±44.32]/[T-(13.10±3.24)],世代发育速率与温度之间的关系拟合为S形曲线V=-e(1.33+37.73/T),推算出该蜂在海南1年可发生18代。所设试验温度范围内,该蜂对橡副珠蜡蚧Parasaissetia nigra(Nietner)的寄生率在13.33%～20.00%范围内波动,30℃时寄生率最高;雌性比率在27℃时最大为70.03%。随着试验温度的升高,成蜂羽化相对集中,羽化持续时间逐渐缩短,羽化高峰提前。30℃条件下雌蜂终生产卵量最多,平均25.77粒。雌蜂寿命随试验温度的升高而逐渐缩短,同一恒温条件下补充20%蜂蜜水可显著延长雌蜂寿命。27～30℃可视为有利于副珠蜡蚧阔柄跳小蜂发育繁殖的适温范围,最适温度为30℃。     

日本食蚧蚜小蜂对橡副珠蜡蚧的控制作用研究

【目的】为了明确日本食蚧蚜小蜂Coccophagus japonicus Compere对橡副珠蜡蚧Parasaissetia nigra Nietner的控害潜能。【方法】在18、21、24、27、30、33℃条件下研究了日本食蚧蚜小蜂对橡副珠蜡蚧3龄若虫的寄生功能反应及27℃条件下该蜂自身的干扰效应。【结果】该蜂的寄生功能反应符合Holling-Ⅱ和Holling-Ⅲ模型,但各温度间功能反应的参数存在差异,其中以瞬时攻击率/处理时间(a'/T_h)为评价指标,在18-33℃范围内,在24℃时寄生效能最大,为53.86;日本食蚧蚜小蜂个体间存在干扰效应,随着自身密度增大,相互干扰作用增强,在27℃时,Hassell模型E=QP~(-m)能够很好的反映出不同寄生蜂密度对橡副珠蜡蚧的寄生情况。【结论】日本食蚧蚜小蜂对橡副珠蜡蚧3龄若虫有较好的控害能力。     

蓝色长盾金小蜂对橡副珠蜡蚧的控制作用

蓝色长盾金小蜂Scutellista caerulea Fonscolombe是橡胶树重要害虫橡副珠蜡蚧Parasaissetia nigra Nietner的一种外寄生性天敌。为了明确蓝色长盾金小蜂对橡副珠蜡蚧的控害潜能,为该寄生蜂的进一步利用提供数据支撑。在室内通过体视镜下解剖观察寄生蜂在寄主腹下是否产卵测定了不同温度、寄主发育阶段下蓝色长盾金小蜂的寄生功能反应、寻找效应和自身密度干扰效应。温度设置有21、24、27、30、33、36℃共6个处理,寄主发育阶段设初期成虫（1-2 d成虫）、褐色期成虫（体色褐色,产卵前3-4 d成虫）、黑色期成虫（体色黑色,产卵1-2 d成虫）三个处理。结果显示：蓝色长盾金小蜂对橡副珠蜡蚧的寄生功能反应符合Holling-Ⅱ型和Holling-Ⅲ型模型,在21-36℃范围内,33℃时该蜂寄生效能最大,为44.4201,21℃时最小,为9.2458;在橡副珠蜡蚧为初期成虫-黑色期成虫范围内,寄生效能由大到小为黑色期成虫>褐色期成虫>初期成虫,分别为18.9044、13.7410、7.2002。采用Hassell-Varley干扰模型对不同温度下蓝色长盾金小蜂受自身密度干扰的寄生作用率进行拟合,表明蓝色长盾金小蜂在寄生时存在种群内个体间自我干扰情况。温度会影响蓝色长盾金小蜂的搜寻和自我干扰,在21-33℃范围内,33℃搜寻常数和干扰常数达到最大值,分别为0.6116、0.7535。当温度为33℃,寄主发育阶段为黑色期成虫时,蓝色长盾金小蜂对橡副珠蜡蚧有较强的控制能力,自身干扰作用最强。     

寄主大小对副珠蜡蚧阔柄跳小蜂产卵选择及繁殖的影响

为明确寄主体型大小对副珠蜡蚧阔柄跳小蜂产卵选择及繁殖的影响,在室内观察了副珠蜡蚧阔柄跳小蜂在不同大小的橡副珠蜡蚧上的产卵量、寄生率、发育历期及性比。结果表明:在选择性和非选择性的产卵试验条件下副珠蜡蚧阔柄跳小蜂对中型个体寄主(长4.0 mm,宽3.4 mm)的寄生率最高;小蜂的产卵量在选择性试验时,以大型寄主(长4.8 mm,宽4.0 mm)体内的最高(67.0粒),非选择性试验时,以中型寄主(长4.0 mm,宽3.4 mm)体内的最高(131.0粒),小型寄主(长3.2 mm,宽2.9 mm)体内产卵量最少(61.5粒);副珠蜡蚧阔柄跳小蜂后代出蜂数与寄主体型的大小呈显著正相关,雌性比则随寄主体型的增大而增减小,发育历期则受寄主体型的影响不明显。综上所述,副珠蜡蚧阔柄跳小蜂雌蜂能够根据寄主质量来调整产卵量及后代性比,以使后代适应度最大化。     

温度和光周期对斑翅食蚧蚜小蜂发育与繁殖的影响

斑翅食蚧蚜小蜂是橡副珠蜡蚧的重要寄生性天敌之一。研究了温度和光周期对斑翅食蚧蚜小蜂发育、寄生及繁殖等方面的生态学特性。研究表明:温度和光周期对斑翅食蚧蚜小蜂种群增长影响明显。在32℃下不能完成世代发育,在温度为18-27℃范围内随温度升高发育加快,18℃发育历期最长(54.0d),27℃最短(22.8d);世代发育起点温度和有效积温分别为12.76℃和307.62日度。高温和低温均不利于斑翅食蚧蚜小蜂寄生,21℃时寄生率最高为(36.0%),30℃时寄生率最低(4.5%)。结合发育历期、体长、产卵、抱卵、寄生率等参数,该蜂发育繁殖的适宜温度为21-27℃度范围内,30℃以上不利于小蜂的发育及存活。斑翅食蚧蚜小蜂对长光的刺激比较敏感,随光照时间增长,发育加快、产卵量明显增加,长日照条件(LD16∶8h)的发育历期最短(24.8d),短日照条件(LD10∶14h)的最长(27.8d),产卵量在长日照条件(LD16∶8h)时最多(119.6粒),短日照条件(LD10∶14h)时最少(86.2粒)。结合发育、产卵、寄生等参数,长日照条件(LD14∶10-LD16∶8h)有利于种群增长。     

斑翅食蚧蚜小蜂的生物学特性

斑翅食蚧蚜小蜂Coccophagus ceroplastae是橡副珠蜡蚧Parasaissetia nigra的重要内寄生蜂。对斑翅食蚧蚜小蜂的形态特征进行了描述,在实验室条件下观察了斑翅食蚧蚜小蜂的行为、发育、存活、繁殖等生物学特性。结果表明:斑翅食蚧蚜小蜂幼虫分3龄,完成一个世代需21-26d。卵巢左右成对,各由3条卵巢管组成,为发育成熟型,成虫期补充营养和发育时间对抱卵量的影响不明显。羽化后即可进行交配,交配时间2-3s,雄蜂有竞争交配行为,交配对雌蜂产卵有显著刺激作用。产卵行为有寻找、寄主检查、产卵器刺探、产卵、产卵针拔出等步骤,存在过寄生现象。羽化主要是在08:00-10:00,温度、相对湿度、光照对其羽化节律均有影响,温度升高羽化提前,且相对集中;相对湿度在20%-90%范围内,随湿度的升高成虫羽化高蜂延后;而光照时间过长或过短均可使斑翅食蚧蚜小蜂的羽化相对分散。随着温度的升高,雌成蜂的寿命逐渐缩短;补充营养能延长成虫寿命,取食10%蔗糖的寿命最长,清水和无补充营养寿命短。     

班氏跳小蜂对扶桑绵粉蚧的寄生功能反应

为评估班氏跳小蜂Aenasius bambawalei Hayat对其寄主扶桑绵粉蚧Phenacoccus solenopsis Tinsley的控制作用, 在实验室条件下（温度25±1℃, 相对湿度70%±5%, 光周期14L∶10D）, 研究了班氏跳小蜂对扶桑绵粉蚧3龄若虫、 雌成虫的寄生功能反应及其自身密度效应。结果表明: 寄生功能反应均符合Holling Ⅱ型方程, 且受寄主密度和寄生物密度的影响。当扶桑绵粉蚧 3龄若虫和雌成虫的密度分别大于15头/容器和10头/容器时, 班氏跳小蜂的寄生量增加幅度开始减小; 以瞬间攻击率/处理时间（α/Th)为评价指标, 班氏跳小蜂雌蜂寄生扶桑绵粉蚧3龄若虫时, α/Th为21.1307, 且大于雌蜂寄生雌成虫的情况（α/Th为6.2506）; 班氏跳小蜂寄生功能反应有较强的种内干扰作用, 随着自身密度的增加, 寄生效能逐渐降低; 通过方程E=QP^-m能很好地模拟班氏跳小蜂的寻找效应（E）与其自身密度（P）之间的关系, 对于3龄若虫和雌成虫的模拟结果分别为E=0.2931P^-0.6240和E=0.0944P^-0.4840。本研究为利用班氏跳小蜂开展扶桑绵粉蚧生物防治提供了基础数据和方法。     

葡萄绵蜡蚧的重要寄生蜂——多孔阔柄跳小蜂(膜翅目:跳小蜂科)

记述了西藏昌都地区葡萄绵蜡蚧Pulvinaria vitis （Linnaeus）的重要寄生蜂多孔阔柄跳小蜂Metaphycus annasor Guerrieri ＆ Noyes,该种为中国新记录种。研究标本保存在中国科学院动物研究所。     

Examination of heat shock protein mRNA accumulation in early Xenopus laevis embryos

Elevation of the incubation temperature of Xenopus laevis neurulae from 22 to 33-35 degrees C induced the accumulation of heat shock protein (hsp) 70 mRNA (2.7 kilobases (kb)) and a putative hsp 87 mRNA (3.2 kb). While constitutive levels of both hsp mRNAs were detectable in unfertilized eggs and cleavage-stage embryos, heat-induced accumulation was not observed until after the mid-blastula stage. Exposure of Xenopus laevis embryos to other stressors, such as sodium arsenite or ethanol, also induced a developmental stage-dependent accumulation of hsp 70 mRNA. To characterize the effect of temperature on hsp 70 mRNA induction, neurulae were exposed to a range of temperatures (27-37 degrees C) for 1 h. Heat-induced hsp 70 mRNA accumulation was first detectable at 27 degrees C, with relatively greater levels at 30-35 degrees C and lower levels at 37 degrees C. A more complex effect of temperature on hsp 70 mRNA accumulation was observed in a series of time course experiments. While continuous exposure of neurulae to heat shock (27-35 degrees C) induced a transient accumulation of hsp 70 mRNA, the temporal pattern of hsp 70 mRNA accumulation was temperature dependent. Exposure of embryos to 33-35 degrees C induced maximum relative levels of hsp 70 mRNA within 1-1.5 h, while at 30 and 27 degrees C peak hsp 70 mRNA accumulation occurred at 3 and 12 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biology and thermal requirements of Trichogramma atopovirilia Oatman & Platner (Hymenoptera: Trichogrammatidae) parasitizing eggs of Diaphania hyalinata L. (Lepidoptera: Pyralidae)

The development and parasitism of Diaphania hyalinata L. eggs by Trichogramma atopovirilia Oatman & Platner and its thermal requirements were studied at the temperatures of 18, 21, 24, 27, 30 and 33 degrees C. Thirty eggs of D. hyalinata were exposed to three females of T. atopovirilia for 5h at 25 degrees C and incubated at the different temperatures. The developmental time from egg exposure to adult, parasitism viability, number of adults per parasitized host egg and progeny sex ratio were monitored. The developmental time from egg to adult emergence of the parasitoid exhibited inverse relationship to the temperature, lasting 24.12 days at 18 degrees C and 7.36 days at 33 degrees C. Parasitism viability at 24, 27 and 30 degrees C was higher than 90%. The ratio of T. atapovirilia adult produced per egg and its sex ratio were not affected when using D. hialynata as host. The lowest threshold temperature (Tb) and estimated degree-days over Tb required by T. atopovirilia to develop on eggs of D. hyalinata was 11.99 degrees C and 130.42 masculine C, respectively. Considering the temperature regimes of two areas where cucurbitaces are cultivated in Bahia State (Rio Real and Inhambupe County) it was estimated that T. atopovirilia can achieve more than 32 generation per year. The results suggest that T. atopovirilia has potential to control D. hyalinata eggs with better chance of success under temperature regimes ranging from 24 to 27 degrees C that meets the suitable field conditions for cropping cucurbitaces.     

Life history of the mealybug, Maconellicoccus hirsutus (Hemiptera: Pseudococcidae), at constant temperatures

Important life history parameters of the mealybug, Maconellicoccus hirsutus (Green), were characterized on hibiscus (Hibiscus rosa-sinensis L.) cuttings at six constant temperatures between 15 and 35 degrees C. The development of M. hirsutus was the fastest at 27 degrees C, where the mealybugs completed development in approximately 29 d. The lower (T(min)) and upper (T(max)) developmental thresholds and the optimal developmental temperature (T(opt)) for the development of female mealybugs were estimated as 14.5, 35, and 29 degrees C, respectively. The thermal constant (K), which is the number of temperature-day or degree-day units required for development, of the females was 347 DD. The original distribution range prediction (based on T(min) = 17.5 degrees C and K = 300 DD) indicated that M. hirsutus could complete at least one generation in all of the continental United States. However, results of this study suggested that the distribution range of M. hirsutus may expand northward because of the lower T(min), and the predicted number of generations in a year may be lower because of the higher K required to complete each generation. The average cumulative survival rate of M. hirsutus at 25 and 27 degrees C was 72%, which was significantly higher than 51 and 62% at 20 and 30 degrees C, respectively. M. hirsutus reproduced sexually, with each mated female producing 260-300 eggs between 20 and 27 degrees C but only approximately 100 eggs at 30 degrees C. Female longevity was reduced from 28 d at 20 degrees C to 19-21 d at 25-30 degrees C. At 27 degrees C, the net reproductive rate (R(o)) was estimated at 165 female symbol/female symbol, the intrinsic rate of population increase (r(m)) was 0.119 (female symbol/female symbol/d), the generation time (T(G)) was 43 d, and the doubling time (DT) was 5.8 d. The life table statistics suggested that the currently released biological control agents, which have higher r(m) than M. hirsutus, will be able to complete more generations than the mealybug within the tested temperature range; thus, they are effective against M. hirsutus.     

Effects of temperature and host age upon the encapsulation of Metaphycus stanleyi and Metaphycus helvolus eggs by brown soft scale Coccus hesperidum

Encapsulation of eggs inserted by Metaphycus stanleyi (Hymenoptera: Encyrtidae) into the brown soft scale Coccus hesperidum (Homoptera: Coccidae) became more frequent as the host matured. This occurred with both laboratory reared and field-collected parasites. After parasitism for 24 hr at 27°C, encapsulation frequency did not differ in hosts reared at 20° or at 27°C, but significantly increased in hosts reared at 33°C. When parasitism and rearing were carried out at the same temperature, the percentage of eggs encapsulated increased from 48.7% at 27°C to 94.1% at 33°C. With M. helvolus, the percentage of eggs encapsulated was considerably higher than with M. stanleyi; e.g., 99.3 vs 48.7%, respectively, at 27°C. At 20° and 27°C, some M. helvolus development occurred in the larvae of brown soft scale but none at 33°C; the adult stages of the host encapsulated all the parasite eggs at these temperatures.     

Parasitism capacity of Trichogramma pratissolii Querino & Zucchi (Hymenoptera: Trichogrammatidae) on alternative hosts, under different temperatures

The successful use of Trichogramma as biocontrol agent depends on its mass production in laboratory, a fundamental step for any biological control program among other factors. This work investigated the parasitism capacity of Trichogramma pratissolii Querino & Zuchi (Hymenoptera: Trichogrammatidae), a new recorded Trichogramma species, parasitizing eggs of Anagasta kuehniella (Zeller) and Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) under the temperatures of 15, 18, 21, 24, 27, 30 and 33 degree Celsius. Eggs of these hosts were offered to newly emerged females during 24h. This procedure was repeated daily for each female and each temperature up to female death, in order to estimate daily and accumulated parasitism, and female longevity. On both hosts, the daily parasitism decreased as function of the female age. Under all temperatures studied and both hosts the highest rate of parasitism was observed during the first 24h of host exposure, and reached 80% of total parasitism in the 4th and 3rd days when parasitizing A. kuehniella and C. cephalonica, respectively. On both hosts, the highest parasitism rate was observed under temperatures from 21 degree Celsius to 27 degree Celsius. Average longevities of T. pratissolii females deprived of food emerging from A. kuehniella and C. cephalonica lived for 1.0 and 8.9 days when reared at 15 degree Celsius e 33 degree Celsius, respectively. The results indicate that eggs of A. kuehniella and C. cephalonica and temperatures from 21 degree Celsius to 27 degree Celsius were appropriate to rear T. pratissolii.     

Effect of temperature on nucleotide pools and monoclonal antibody production in a mouse hybridoma

The specific monoclonal antibody productivity (q(Mab)) of a murine hybridoma (CC9C10) increased with incubation temperature in the range 33 degrees C to 39 degrees C. q(Mab) was constant at each temperature and was independent of the phase of culture. The q(Mab) increased 97% at 39 degrees C and decreased by 21% at 33 degrees C compared with controls at 37 degrees C. Specific rates of substrate (glucose and glutamine) utilization and byproduct (lactate and ammonia) formation also increased with temperature but the yield coefficient, Y(Lac/Llc') was constant for 33 degrees C to 39 degrees C and Y(Amm/Gin) was constant for 37 degrees C to 39 degrees C. Y(Amm/Gin) at 33 degrees C was lower than the control. Changes in specific nucleotide concentrations and ratios were monitored by analysis of intracellular nucleotide pools. The NTP ratio, (ATP + GTP)/(UTP + CTP), increased and the U-ratio (UTP/UDP-GNac) decreased during the course of each culture, whereas the adenylate energy charge, (ATP + 0.5ADP)/(ATP + ADP + AMP), remained relatively constant at a value 0.8. The relative content of UDP-/N acetyl galactosamine, UDP-N acetyl glucosamine, and NAD increased with incubation temperature, whereas the relative ATP content, SA(ATP + ADP + AMP)/SU (UTP + UDP-sugars) ratio, purine/pyrimidine, ATP/GTP, and U-ratio decreased at higher incubation temperatures. It is possible that these nucleotide parameters may have a regulatory role in the changes of q(Mab) observed at the higher temperatures. (c) 1994 John Wiley & Sons, Inc.     

Influence of incubation temperature on hatching success,energy expenditure for embryonic development,and size and morphology of hatchlings in the oriental garden lizard,Calotes versicolor (Agamidae)

We incubated eggs of Calotes versicolor at four constant temperatures ranging from 24 degrees C to 33 degrees C to assess the effects of incubation temperature on hatching success, embryonic use of energy, and hatchling phenotypes that are likely to affect fitness. All viable eggs increased in mass throughout incubation due to absorption of water, and mass gain during incubation was dependent on initial egg mass and incubation temperature. The average duration of incubation at 24 degrees C, 27 degrees C, 30 degrees C, and 33 degrees C was 82.1 days, 60.5 days, 51.4 days, and 50.3 days, respectively. Incubation temperature affected hatching success, energy expenditure for embryonic development, and several hatchling traits examined, but it did not affect the sex ratio of hatchlings. Hatching success was lowest (3.4%) at 33 degrees C, but a higher incidence of deformed embryos was recorded from eggs incubated at this temperature compared to eggs incubated at lower temperatures. Most of the deformed embryos died at the last stage of incubation. Energy expenditure for embryonic development was, however, higher in eggs incubated at 33 degrees C than those similarly incubated at lower temperatures. A prolonged exposure of eggs of C. versicolor at 33 degrees C appears to have an adverse and presumably lethal effect on embryonic development. Hatching success at 24 degrees C was also low (43.3%), but hatchlings incubated at 24 degrees C did not differ in any of the examined traits from those incubated at two intermediate temperatures (27 degrees C and 30 degrees C). Hatchlings incubated at 33 degrees C were smaller (snout-vent length, SVL) than those incubated at lower incubation temperatures and had larger mass residuals (from the regression on SVL) as well as shorter head length, hindlimb length, tympanum diameter, and eye diameter relative to SVL. Hatchlings from 33 degrees C had significantly lower scores on the first axis of a principal component analysis representing mainly SVL-free head size (length and width) and fore- and hindlimb lengths, but they had significantly higher scores on the second axis mainly representing SVL-free wet body mass. Variation in the level of fluctuating asymmetry in eye diameter associated with incubation temperatures was quite high, and it was clearly consistent with the prediction that environmental stress associated with the highest incubation temperatures might produce the highest level of asymmetry. Newly emerged hatchlings exhibited sexual dimorphism in head width, with male hatchlings having larger head width than females.     

Cryopreservation of mouse oocytes using a medium with low sodium content: effect of plunge temperature

The effect of various combinations of plunge temperature and thawing protocol on the survival and viability of mouse oocytes was examined. The oocytes were frozen either in a standard freezing medium (ETFM, embryo transfer freezing medium) or in a low-sodium, choline-based freezing medium (CJ2), with 1.5 M 1,2-propanediol and 0.1 M sucrose, and using a conventional slow cooling method. The criteria used to assess survival were morphological state after thawing (intact or lysed), ability to become fertilized, and ability to develop to the two-cell, morula, and blastocyst stage in vitro. Oocytes frozen in CJ2 and plunged into liquid nitrogen (LN(2)) from -10, -20, or -33 degrees C remained intact and developed to the blastocyst stage at significantly higher rates than oocytes frozen in ETFM. For oocytes plunged into LN(2) from -33 degrees C, very rapid thawing (10 s in 30 degrees C water) was more detrimental than rapid or slow thawing (holding in air at room temperature for 10 or 30 s, respectively, prior to submersion in water at 30 degrees C for 10 s). By contrast, oocytes plunged into LN(2) from -10 or -20 degrees C survived better when thawing was very rapid or rapid than when thawing was slow. With the current protocol CJ2 was very effective over a wide range of plunge temperatures (-20 to -33 degrees C), although the optimal thawing protocol depended on the particular plunge temperature. Over 90% of oocytes surviving after slow cooling in CJ2 to -33 degrees C could be plunged to -196 degrees C with little or no further damage.