Effects of Partial and Complete Replacement of Synthetic Cryoprotectant with Carrot (Daucus carota) Concentrated Protein on Stability of Frozen Surimi

ABSTRACT

The study aimed to evaluate the effects of carrot concentrated protein (CCP) as additive on the functional and textural properties of surimi from striped catfish (Pangasianodon hypophthalmus) during six months of frozen storage (?20°C). The CCP (82.22% crude protein) was used as an additive either a lone or with a synthetic cryoprotectant (sucrose-sorbitol-sodium tri-polyphosphate). Control was made with synthetic cryoprotectant only. Molecular weight of CCP was found to be 36 kDa. After six months, the results revealed that up to 50% of synthetic cryoprotectants could be replaced by CCP during frozen storage of surimi. Biochemical parameters such as protein solubility, Ca²⁺ATPase activity, and gel strength decreased significantly (p < .05) during storage. Treatment T-3 (CCP 0.5% + 50% of synthetic cryoprotectant) maintained quality of protein significantly superior (p < .05) in respect to denaturation and other functional and sensory attributes compared to all the treatments. The microstructure images of surimi confirmed that addition of CCP modified the ice crystal growth during frozen storage. This study suggests that CCP can be a potential additive to protect protein from denaturation along with partial replacement of chemical cryoprotectants.     

长期冻存对昆虫细胞系SL2和NIH-SaPe-4活性的影响

对2种双翅目昆虫细胞系(黑腹果蝇胚细胞系SL2以及麻蝇胚细胞系NIH-SaPe-4)38个月长期液氮深低温保存效果进行了研究,通过对冻存时间和保护剂对2种细胞系冻后活力、圆度以及恢复时间影响的测定,比较了3种不同配方冻存保护液的效果。结果表明:2种昆虫细胞系冻后活性均随着冻存时间的增长而逐渐降低。长期冻存期中,使用90%胎牛血清(FBS)+10%二甲基亚砜(DMSO)冻存SL2效果较好,NIH-SaPe-4使用10%DMSO即可满足冻存的需要,而无需额外添加高浓度FBS。甘油不适用于这2种昆虫细胞系的长期保存。     

达氏鲟精巢细胞消化分离和超低温冷冻保存

通过研究两种酶对精巢细胞的消化效果,探究抗冻剂、降温程序、糖类和卵磷脂对达氏鲟(Acipenser dabryanus)精巢细胞冻存效果的影响,获得消化冻存后高存活率的细胞。实验使用0.25%胰蛋白酶和2 mg/m L胶原酶H+500 U/m L中性酶II的组合酶对达氏鲟精巢细胞进行消化,获得不同消化时间内的活细胞数量。另外,还研究了冷冻稀释液中分别添加10%二甲基亚砜(DMSO)、乙二醇(EG)、甲醇(MET)作为抗冻剂,采用-1℃/min慢速降温以及直接投入液氮中的快速降温方法冻存,冷冻稀释液采用D-海藻糖或同浓度D-蔗糖,以及添加5%、8%、11%卵磷脂对冻存效果的影响。结果显示:两种消化酶在同一时间消化所得的活细胞数和活细胞率无显著差异,并且都在3 h获得最多活细胞。慢速降温的冻存效果极显著地好于快速降温(P=0.01)。不同抗冻剂的保存效果差异显著,复苏后细胞相对存活率EG(51.70%±5.24%)MET(45.09%±3.15%)DMSO(40.18%±3.90%)。不同糖对达氏鲟精巢细胞冻存效果无显著影响;不同浓度的卵磷脂冻存效果有极显著差异。含8%卵磷脂的冻存液对细胞的冻存效果最好,细胞存活率可达(93.55±2.56)%,培养10 d后细胞数目为0 d时的3.19倍。     

Spermatophore cryopreservation and artificial insemination of black tiger shrimp, Penaeus monodon (Fabricius)

To develop an appropriate cryopreservation protocol for spermatophores of black tiger shrimp, Penaeus monodon, three cryoprotectants (dimethyl sulphoxide (DMSO), methanol (MeOH) and ethylene glycol (EG)) at two concentrations (5% and 10%) were examined. Artificial implantation of spermatophores was also carried out to assess the fertilizing ability of fresh and post‐thaw spermatophores. Spermatophores were collected during consecutive regenerations (15‐day intervals) and assessed for qualitative and quantitative changes and also for fertilizing ability by implantation. The mean fertilization rate for artificial insemination using post‐thaw spermatophore was 79.9±3.7%, lower than the fertilization rates observed for artificial implantation using fresh spermatophore and natural mating. Mean hatch rates for fresh spermatophore, frozen‐thawed spermatophore and natural mating were 88.8±0.6%, 87.8±0.4% and 88.3±0.5%, respectively; and there was no difference among the three groups. The mean fertilization rate of spermatophores collected during the first stripping was higher (90.6±0.6) than during the second stripping (85.7±2.6), but the mean hatch rate was not different between the two strippings. The highest mean sperm viability (79.7±0.4%) was obtained from DMSO (5%), with no survival observed in the 10% MeOH treatment. Spermatophore weight, total sperm count and percentage of abnormal sperm were not different between spermatophores collected at the first and second stripping. This is the first study to report high fertilization and hatch rates from cryopreserved spermatophore using artificial implantation of spermatophore before spawning.     

脱防冻剂方法对牛体外受精胚胎冷冻后成活率的影响

为了观察脱防冻剂方法对牛体外受精冷冻胚胎在体内、外发育率的影响,应用常规冷冻法在０．７５ｍｏｌ／Ｌ甘油＋０．５ｍｏｌ／Ｌ丙二醇溶液中冷冻受精后７、８ｄ的囊胚,解冻后在０．２５、０．５ｍｏｌ／Ｌ蔗糖液中二步或在０．２５ｍｏｌ／Ｌ蔗糖液中三步脱防冻剂的胚胎孵化率（分别为６８．６％、６２．２％、６８．７％）与用ＰＢＳ／ＦＣＳ六步脱防冻剂的差异不显著（７０．４％,Ｐ＞０．０５）,但在０．５ｍｏｌ／Ｌ蔗糖液中三步脱防冻剂后,孵化率显著降低（４７．６％,Ｐ＜０．０１）。用０．２５ｍｏｌ／Ｌ或０．５ｍｏｌ／Ｌ蔗糖或海藻糖预先使胚胎脱水后冷冻,解冻后可使胚胎直接在ＰＢＳ中一步脱掉防冻剂,胚胎孵化率（４５．０％～４９．５％）与在０．２５ｍｏｌ／Ｌ蔗糖液中二步脱防冻剂的相似（５１．５％,Ｐ＞０．０５）。移植试验表明,在０．２５ｍｏｌ／Ｌ蔗糖液中二步脱防冻剂获得的妊娠率与六步脱防冻剂相似,与在ＰＢＳ中一步脱防冻剂的亦无明显差异,但妊娠率均偏低（２１．４％～３７．５％）。     

犬精液冷冻保存技术的研究进展

国内犬精液冷冻保存技术起步较晚,尚处在研究探索阶段,犬精液冷冻保存技术还不成熟,冷冻精液受胎率、窝产仔数与自然交配相比还有一定的差距,而且品种、地域、采精时间的不同,精液冷冻效果也不一致。从犬精液的冷冻保存方法、冷冻保护剂、解冻方法三个方面,综述了近年来国内外的犬精液冷冻研究成果,为探索更加有效的犬精液冷冻技术,提高犬精液冷冻效果提供参考。     

Is sperm cryopreservation in absence of permeable cryoprotectants suitable for subfertile donkeys?

Sperm from fertile donkeys have been successfully frozen in absence of permeable cryoprotectants. The aim of this study was to determine whether this cryopreservation method is suitable for subfertile donkeys in comparison to conventional sperm freezing with glycerol. Ejaculates were collected from four Andalusian Donkeys: three fertile and one subfertile. Semen was frozen with an extender containing glycerol (GLY), or adding instead sucrose 0.25 molar and 1% bovine serum albumin (SUC) as non‐permeable cryoprotectants. After thawing, samples were assessed for total (TM, %) and progressive (PM, %) sperm motility by CASA, plasma membrane integrity (PMI, %) by epifluorescence microscopy and DNA integrity (DFI, %) by SCSA. Results (mean ± SD) were compared between extenders in fertile and subfertile donkeys using the Student's t test. No differences between GLY and SUC treatments were found in the fertile group for the sperm parameters assessed. In subfertile donkey ejaculates, GLY resulted in significantly higher values than SUC for TM (25.5 ± 3.1 vs. 19.6 ± 1.9) and PM (13.3 ± 5.1 vs. 4.0 ± 1.2), respectively. In conclusion, considering all the sperm parameters assessed, sperm freezing in absence of permeable cryoprotectants may not be still an option for cryopreservation of subfertile donkey sperm.     

成年小鼠雄性生殖细胞的冷冻保存

在含10％小牛血清（NBS）的DMEM培养基中，分别各添加5％，10％，15％，20％和25％的二甲基亚砜（DMSO）,丙二醇（PG），乙二醇（EG）和甘油（G），对成年小鼠睾丸生殖细胞冷冻保存；复苏后台盼蓝染色测定细胞复苏率。结果显示，5％－25％DMSO冻存液组的细胞复苏率分别为88.5%,88.0%,65.6%及51.3%;5%-25%PG冻存液组的细胞复苏率分别为87.2%,86.4%,79.0%,73.4%及40.1%.;5%-25%EG冻存液组的细胞复苏率分别为6.6%,80.9%,60.8%,51.3%及30.0%;5%-25%G冻存液组的细胞复苏率分别为86．5％，86．3％，65．3％，36．0％及31．4％。其中各抗冻剂5％和10％组的细胞复苏率最高，与15％组相比均存在显著或极显著差异。4种抗冻剂的最高细胞复苏率之间无显著差异。DMSO，PG，EG和G分别冷冻保存成年小鼠睾丸生殖细胞对的最小损失率分别为4．8％，6．1％，6．7％，6．8％。结果表明，采用慢速冷冻时，DMSO，PG，EG及G均适宜用作成年小鼠睾丸生殖细胞的抗冷冻剂，最佳使用含量均为5％－10％。成年小鼠睾丸生殖细胞分别在含5％－10％的DMSO，PG，EG和G的DMEM（含10％NBS）冻存液中，2步慢速降温，液氮储存，37℃水浴复苏，是一种具有较高复苏率的冷冻保存方法。     

牛精液冷冻保护剂研究进展

牛的人工授精技术是目前应用最为广泛的动物繁殖技术之一,对牛的遗传改良做出了巨大贡献。但是,在冷冻一解冻过程中仍然有大约40％～50％的精子失去活性,限制良种公牛种用性能的发挥。论文在分析精子冷冻保存原理的基础上,阐述了渗透性、非渗透性冷冻保护剂以及低密度脂蛋白对牛精子的冷冻保护作用,以期为开发新型冷冻保护剂的研究提供一定参考,进一步提升牛冷冻精液的质量。     

猪精液冷冻保护剂研究进展

 冷冻保护剂作为精液冷冻稀释液中的添加剂,能防止和减少细胞在冷冻及复苏过程中造成的损伤,确保细胞复苏后有较高存活率。论文就国内外猪精液渗透性和非渗透性精液冷冻保护剂的作用机理、应用进展等研究进行了综述。