Optimization of ethylene glycol concentrations, freezing rates and holding times in liquid nitrogen vapor for cryopreservation of rhesus macaque (Macaca mulatta) sperm

Ethylene glycol (EG) has been speculated to be the most appropriate penetrating cryoprotectant for cryopreservation of rhesus macaque sperm due to its higher permeability coefficient. The present study aimed to determine the optimal EG concentration, freezing rate and holding time in liquid nitrogen (LN(2)) vapor for rhesus sperm cryopreservation. Among six tested EG concentrations (0, 0.18, 0.35, 0.7, 1.4 and 2.1 M), 0.7 M EG showed the most effective cryoprotection (P<0.05). Sperm frozen with 0.7 M EG at -183°C/min showed higher post-thaw motility than sperm frozen at -10, -67 or -435°C/min (P<0.05). Sperm frozen in LN(2) vapor at -183°C/min with 0.7 M EG and a holding time of 10 min showed higher post-thaw motility compared with a holding time of 5 or 15 min (P<0.05). The function of sperm cryopreserved at the optimized EG concentration, freezing rate and holding time was further evaluated by in vitro fertilization. Of the 36 oocytes collected from gonadotropin-stimulated rhesus macaques, 61.1% were fertilized, and 61.1, 44.4 and 36.1% of the oocytes developed to 2 cells, morulae and blastocysts, respectively. Our findings provide an alternative penetrating cryoprotectant and optimal protocol for genetic preservation purposes in this important species.     

Optimization of a protocol for cryopreservation of mouse spermatozoa using cryotubes

The rapid increase in the number of genetically modified mouse strains has produced a high demand for their frozen spermatozoa from laboratories and mouse banking facilities. Historically, plastic straws have been used preferentially as containers for frozen mammalian spermatozoa because spermatozoa frozen in plastic straws have a high survival rate after thawing. However, plastic straws are more fragile and are used less often than the cryotubes used for conventional cell freezing. In this study, we sought to develop a new protocol for sperm freezing using cryotubes as the container to increase the accessibility of mouse sperm cryopreservation. Epididymal spermatozoa were collected from mature ICR or C57BL/6J (B6) males and were suspended in 18% raffinose and 3% skim milk solution. We then optimized the following conditions using the sperm survival rate as an index: 1) distance of cryotubes from the surface of the liquid nitrogen at freezing, 2) volume of the sperm suspension in the cryotube and 3) temperature of warming sperm during thawing. The best result was obtained when cryotubes containing 10 μl of sperm suspension were immersed 1 cm below the surface of the liquid nitrogen and then thawed at 50 C. The fertilization rates using spermatozoa frozen and thawed using this method were 63.1% in ICR mice and 28.2% in B6 mice. The latter rate was increased to 62.3% by adding reduced glutathione to the fertilization medium. After embryo transfer, 68% and 62% of the fertilized oocytes developed into normal offspring in the ICR and B6 strains, respectively. These results show that cryotubes can be used for cryopreservation of mouse spermatozoa under optimized conditions. This protocol is easy and reproducible, and it may be used in laboratories that do not specialize in sperm cryopreservation.     

Use of Penicillin and Streptomycin to Reduce Spread of Bacterial Coldwater Disease I: Antibiotics in Sperm Extenders

Bacterial coldwater disease caused by Flavobacterium psychrophilum has led to the loss of significant numbers of hatchery-reared salmonids. The bacteria can be spread from parent to progeny within contaminated sperm and ovarian fluid. Methods for disinfecting ovarian fluid and unfertilized eggs are available, but methods for disinfecting sperm have not been described. In this study we determined whether sperm extenders containing a mixture of penicillin and streptomycin can be used to eliminate F. psychrophilum. In vitro trials demonstrated that when Rainbow Trout Oncorhynchus mykiss sperm is mixed with an extender, a 15-min exposure to 0.197 mg penicillin plus 0.313 mg/mL streptomycin is effective at killing the bacteria and has no effect on sperm motility. Small-scale trials showed that egg fertilization rates were not reduced when sperm held in an extender solution containing the same antibiotic mixture for 15 min was used to fertilize eggs. Production-scale trials, however, showed a roughly 18% decrease in egg fertilization rate when sperm stored in an antibiotic containing extender was used. To determine why a reduction in fertilization capacity was observed, a small-scale experiment testing the fertilization of eggs with larger quantities of sperm was performed and showed that increasing the volume of sperm used did not increase fertilization rates. Our results demonstrate that extenders containing penicillin and streptomycin can be used to disinfect sperm, especially when small quantities of eggs are fertilized, but factors negatively affecting egg fertilization and survival on a production scale still need further investigation.

Received May 1, 2014; accepted August 10, 2014     

Effect of cumulus cell removal and sperm pre‐incubation with progesterone on in vitro fertilization of equine gametes in the presence of oviductal fluid or cells

In spite of many attempts to establish an in vitro fertilization (IVF) technique in the equine, no efficient conventional IVF technique is available. The presence of oviductal fluid or oviductal cells during IVF helps to improve embryo production in vitro but is not sufficient to reach high fertilization rates. Thus, our aim was to perform equine IVF either after sperm pre‐incubation with oviductal fluid or in the presence of oviductal cells, and to evaluate the effect of cumulus removal from the oocyte or sperm pre‐incubation with progesterone. In experiments 1 and 2, IVF was performed in the presence of porcine oviduct epithelial cells. The removal of cumulus cells from equine oocytes after in vitro maturation tended to increase the percentage of fertilization when fresh sperm was used (1/33 vs. 4/31, p > 0.05) but had no effect when frozen sperm was used (1/32 vs. 1/32). Equine sperm pre‐incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/14 vs. 2/18 for fresh, 1/29 vs. 1/25 for frozen). In experiments 3 and 4, IVF was performed after pre‐incubation of sperm with porcine oviductal fluid. The removal of cumulus cells tended to increase the percentage of fertilization when fresh sperm was used (1/24 vs. 3/26, p > 0.05). Sperm pre‐incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/39 vs. 2/36 for fresh, 2/37 vs. 1/46 for frozen), but two 3–4 cell stage zygotes were obtained with fresh sperm pre‐incubated with progesterone. This is an encouraging result for the setting up of an efficient IVF procedure in equine.     

Rescue In Vitro Fertilization Method for Legacy Stock of Frozen Mouse Sperm

Sperm cryopreservation has been widely adopted for maintenance of the genetically engineered mouse (GEM). The cryopreserved sperm are being exchanged among many institutes worldwide. However, the recipients are not always able to obtain high fertilization rates with the frozen sperm shipped from senders. In this study, we cryopreserved mouse sperm via various methods and performed in vitro fertilization (IVF) in which the combination of methyl-beta-cyclodextrin for sperm preincubation and reduced glutathione for insemination was used (the MBCD-GSH IVF). In addition, frozen sperm sent from the Jackson Laboratory (USA) were thawed and used for IVF in the same manner. The fertilization rates of both the sperm cryopreserved via the methods applied in some countries and the cryopreserved GEM sperm improved when used with the MBCD-GSH IVF method. Therefore, we strongly believe that the MBCD-GSH IVF method brings about relatively high fertilization rates with any strain of frozen mouse sperm.     

谷氨酰胺替代部分甘油对冻融猪精子获能和体外受精能力的影响

本试验旨在探索用谷氨酰胺（Gln）替代部分甘油对冻融猪精子体外获能和受精能力的影响，试验分为6组：3%甘油对照组和5个处理组（Ⅰ~Ⅴ组：2%甘油＋谷氨酰胺（0、20、40、80和100 mmol/L））。对冻融松辽黑猪精子的精子活力、质膜完整性、顶体完整性、线粒体膜电位、鱼精蛋白水平、获能及体外受精等指标进行了检测。结果显示，用谷氨酰胺替代部分甘油均对冻融精子质量有一定的改善作用，改善的程度受谷氨酰胺浓度的影响。与对照组相比，Ⅰ组精子的质量参数均显著下降（P<0.05）；与Ⅰ组相比，Ⅱ组精子活力、顶体完整性和活率显著提高（P<0.05），Ⅲ组精子线粒体膜电位显著提高（P<0.05），Ⅴ组精子活力、质膜完整性、顶体完整性、活率和线粒体膜电位均显著提高（P<0.05）。说明用谷氨酰胺替代部分甘油对精子质量具有很大的影响，且当谷氨酰胺为100 mmol/L时可得到更高质量的精子，因此，后续试验使用浓度为100 mmol/L的谷氨酰胺进行研究。与对照组相比，2%甘油＋100 mmol/L谷氨酰胺处理组精子鱼精蛋白缺失率显著下降（P<0.05），精子获能无显著差异（P>0.05），但胚胎卵裂率显著提高（P<0.05）。综上所述，谷氨酰胺可作为一种新型冷冻保护剂替代部分甘油来提高猪精液的质量，并降低甘油对猪精液的毒性作用，为猪精液的冷冻保存及商业化生产提供技术支撑。     

多种因素对小鼠卵母细胞体外受精效果影响的分析

为了探讨不同精子获能时间,精卵孵育时间,精子密度以及颗粒细胞对小鼠卵母细胞体外受精的影响,从而达到对卵母细胞体外受精体系优化的目的。比较了精子获能时间分别为40 min、60 min、80 min试验组的受精卵卵裂率。结果表明,带颗粒细胞卵母细胞(COCs)在三个试验组中卵裂率无显著差异,不带颗粒细胞卵母细胞(NO)在精子获能时间为60 min时卵裂率最高;比较了精卵孵育时间分别为2 h、4 h、6 h、8 h试验组的受精卵卵裂率,结果显示COCs精卵孵育时间2 h试验组的效果最好,NO孵育时间为6 h试验组的效果最好;比较了精子密度分别为3×10⁵/mL,3×10⁶/mL,3×10⁷/mL试验组受精卵卵裂率,结果显示COCs和NO均为3×10⁶/mL试验组卵裂效果最好;比较COCs和NO的受精卵卵裂率,结果显示COCs与NO之间存在显著差异(P<0.05),裸卵卵裂效果显著优于颗粒细胞卵裂效果。试验结果表明,在卵母细胞体外受精过程中,精子获能时间60 min,精子密度为3×10⁶/mL,精卵孵育6 h,培养24 h后卵裂率最高。     

Optimization of the in vitro fertilization protocol for frozen epididymal sperm with low fertilization ability in Ban—A native Vietnamese pigs

The aim of the present study was to improve the penetration during in vitro fertilization (IVF) of a frozen lot of epididymal sperm with a notoriously low fertilization ability of a Ban boar which is a native Vietnamese breed by optimizing different parameters of the IVF system. In Experiment 1, we determined that Pig‐fertilization medium was superior medium to Tyrode's albumin lactate pyruvate‐polyvinyl alcohol medium for IVF and defined the optimum the sperm concentration (1 × 10⁶ sperm/ml). In Experiment 2, we clarified that partial removal of cumulus cells from cumulus‐oocyte complexes by hyaluronidase treatment before IVF enhances sperm penetration, whereas complete cumulus removal reduces penetration. Finally, in Experiment 3 the elevation of concentration of caffeine in Pig‐fertilization medium from 2 to 5 mmol/L and the prolongation of the co‐culture of gametes from 3 to 5 hr significantly increased the total penetration rate from 15.2% to over 50%. In conclusion, the combination of partial oocyte denudation, an elevated caffeine concentration in Pig‐fertilization medium and an extended interval of IVF with using an optimized sperm concentration was a potent way to improve the fertilization results for a frozen epididymal Ban sperm lot with low fertility.     

白藜芦醇对奶牛性控冻精品质和体外受精能力的影响

本研究旨在探究不同浓度白藜芦醇处理对奶牛性控冻精品质和体外受精能力的影响。在解冻后的奶牛性控冻精中分别添加0、10~(-3)、10~(-4)、10~(-5) mol/L的白藜芦醇,各组精子在受精液中获能孵育1.5 h后,测定精子质量和体外受精能力。结果表明:添加白藜芦醇均可有效降低性控冻精中活性氧(ROS)含量、提高顶体完整活精子比例(P0.05),其中10~(-3)、10~(-4) mol/L的白藜芦醇处理对降低ROS含量最为显著;10-4 mol/L的白藜芦醇处理可显著降低丙二醛(MDA)含量并提高性控冻精的卵裂率和囊胚率(P0.05)。综上所述,白藜芦醇作为一种外源性天然抗氧化剂,通过降低性控精液中过量的ROS水平、抑制精子脂质过氧化反应、保护顶体完整性,从而提高性控精子质量和体外受精能力。     

Motility and Fertility Evaluation of Thawed Frozen Stallion Semen After 24 Hours of Cooled Storage

Breeding mares with cryopreserved semen requires specialized equipment for storage and thawing and more intensive mare management. The objectives of this study were (1) evaluate the longevity of frozen stallion semen once it had been thawed, extended, and maintained at 5°C for 48 hours in a passive cooling container, and (2) determine fertility potential of frozen semen that had been thawed, extended, and used to inseminate mares after 24 hours of cooled storage. Eight ejaculates were collected and aliquots were cooled in either INRA96 and CryoMax LE minus cryoprotectant at a concentration of 50 million total sperm/mL. The remainder of the ejaculate was frozen in CryoMax LE extender at a concentration of 200 million total sperm/mL. Semen was thawed using 1 of 3 thawing protocols, and diluted to a concentration of 50 million total sperm/mL in either INRA96 or CryoMax LE minus cryoprotectant and cooled to 5°C. Sperm motility was evaluated at 24 and 48 hours. Eight mares were inseminated over two estrous cycles using frozen semen that had been thawed, extended in INRA96, and cooled for 24 hours. There was no difference in progressive motility at 24 or 48 hours of cooled-storage post-thaw between the 3 thawing protocols. An overall per cycle pregnancy rate of 56% (9/16 cycles) was achieved using frozen-thawed semen that had been extended and cooled for 24 hours. In summary, frozen stallion sperm was thawed, extended, and cooled to 5°C for 24 hours and still maintained adequate (>30%) sperm motility and fertility.     

不同激活条件及半胱氨酸处理对猪ICSI胚胎发育影响研究

本试验探讨了不同辅助激活方法(Calciumionophore A23187激活、Calciumionophore A23187+6-DMAP联合激活和电激活)、不同精子预处理方法(液氮冻融处理和0.1%Triton X-100处理)和在添加半胱氨酸的胚胎培养液中培养不同时间(0 h、4 h、12 h和168 h)对猪卵母细胞内单精子注射(ICSI)胚胎体外发育的影响。结果显示:与无辅助激活相比,A23187+6-DMAP联合激活和电激活均能显著提高ICSI卵母细胞的激活率、卵裂率和囊胚率(P0.05),A23187+6-DMAP联合激活能显著提高ICSI卵母细胞的受精率(P0.05)。液氮冻融精子组ICSI卵母细胞的雄原核形成率显著高于活精子组(P0.05)。在添加半胱氨酸的胚胎培养液中培养4 h的ICSI卵母细胞受精率、雄原核形成率和囊胚率显著高于0 h组(P0.05)。以上结果表明,猪卵母细胞在ICSI后需要辅助激活来启动胚胎顺利发育,A23187+6-DMAP激活效果较好。液氮冻融精子可以促进ICSI后雄原核的形成。半胱氨酸处理4 h对猪ICSI卵母细胞受精和发育均有促进作用。     

Fertilizing Ability of Electro-Ejaculated Cryopreserved Semen in the Domestic Cat

Semen collection and AI in the cat are still not routine procedures. The correlation between semen quality and fertility under natural conditions is a relatively unknown field in the cat. In the present study, functional in vitro tests, such as the ability to bind and penetrate the zona pellucida or to fertilize in vitro, were used to determine fertilizing ability of sperm cryopreserved with a practical and efficient freezing protocol previously developed in our laboratory. Semen was collected by electroejaculation, evaluated for motility and diluted with Tris-glucose-citrate egg-yolk extender supplemented with Equex STM paste (0.5% v/v). After equilibration and loading into 0.25 ml straws, semen was frozen at 3.85 degrees C/min. Frozen-thawed semen was co-cultured with in vitro matured cat oocytes. Penetration rate was recorded 30 h after in vitro fertilization and cleaved zygotes were cultured in vitro until day 7. A correlation was found between sperm motility index (SMI) after thawing and semen fertilizing ability (p<0.05). In conclusion, it was demonstrated that the post-thaw motility quality, expressed as SMI, of spermatozoa frozen using the protocol mentioned above can be considered an index of the sperm ability to penetrate in vitro matured oocytes.     

Effect of the Interaction Between Cryoprotectant Concentration and Cryopreservation Method on Frozen/Thawed Chicken Sperm Variables

This work examines the effect of the interaction between different concentrations of two cryoprotectants – glycerol (GLY) and dimethylacetamide (DMA) – and two methods of cryopreservation – pellets produced by plunging into liquid nitrogen and gradual in‐straw freezing – on frozen/thawed chicken sperm variables. Sperm was cryopreserved using: (i) 6% DMA, following the in‐straw and the pellet methods (ii) 11% GLY, following the in‐straw and the pellet methods; and (iii) 8% GLY in the in‐straw method and 3% DMA in the pellet method (i.e. reduced cryoprotectant concentrations). When 6% DMA was used as the cryoprotectant, no differences were seen between the in‐straw and pellet methods in terms of frozen/thawed sperm variables or fertility (10.8% and 12.8%, respectively). The viability and motility variables of the frozen/thawed sperm produced using the in‐straw method with 11% GLY were higher (p < 0.05) than those recorded for the sperm preserved using the same cryoprotectant and concentration in the pellet method. However, fertility was extremely low in both groups (2.1% and 4.2% for the in‐straw and pellet methods, respectively). Finally, the use of 8% GLY in the in‐straw method returned higher sperm viability, intact acrosome and motility values than the use of 3% DMA in the pellet method (p < 0.01). No differences were seen, however, in the fertility results obtained (28.8% and 25.0%, respectively). These results suggest that cryoprotectant concentrations can be reduced and still provide acceptable fertility rates.     

A 32-kDa tyrosine-phosphorylated protein shows a protease-dependent increase in dead boar spermatozoa

Boar sperm TyrP32 is a 32-kDa tyrosine-phosphorylated protein that increases during the capacitation and acrosome reaction and during cryocapacitation. However, it is still unclear whether the increase in TyrP32 is an event that is limited to the process of sperm fertilization, including cryocapacitation. The aims of the present study were to demonstrate that TyrP32 is increased in dead spermatozoa after freeze-thawing without a cryoprotectant and to find the causal factors for this increase. Washed spermatozoa were resuspended in a salt solution and then frozen. The frozen samples were rapidly thawed in a warm water bath and then used for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)/Western blotting to detect TyrP32, SDS-PAGE/silver staining of sperm proteins and staining of acrosomal contents with fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA). In the samples before freezing, TyrP32 was barely detectable, and the distribution of the acrosomal contents was normal in most spermatozoa. One cycle of freeze-thawing induced an increase in TyrP32, a decrease in major sperm proteins and disorder in the acrosomal contents. However, the addition of a protease inhibitor (APMSF, 1 mM) suppressed the increase in TyrP32 and the decrease in the major sperm proteins, although it did not have any influence on the disorder in the acrosomal contents. Additionally, the spermatozoa did not exhibit any flagellar movement after freeze-thawing, which showed that almost all of them were dead. These results indicate that TyrP32 can show a protease-dependent increase in dead spermatozoa after freeze-thawing without a cryoprotectant even though the dead spermatozoa do not undergo cryocapacitation.     

牛卵母细胞体外受精技术研究

本研究从3方面进行了牛卵母细胞的体外受精试验,即用不同离心法、不同温度上浮法、不同精子浓度等方法对COCs进行体外受精,测定其体外受精率。结果表明,两种离心法中,以上清液二次离心法的精子获能效果最好,受精率最高,该法首次采用低速离心技术,突破了离心法的传统方法,离心效果明显提高;不同温度条件下的精子上浮效果以CO2培养箱恒温（38.5℃）上浮法最好,受精率最高;对于选择受精时获能精子的浓度,宜控制在1.0×106～1.5×106个/ml,不仅能提高受精率,还可避免或降低多精受精现象。     

Sperm of the giant grouper: cryopreservation,physiological and morphological analysis and application in hybridizations with red-spotted grouper

In order to develop excellent germplasm resources for giant grouper (Epinephelus lanceolatus), cryopreservation of giant grouper sperm was examined in the present study. Firstly, 13 kinds of sperm dilution (ELS1-3, EM1-2, TS-2, MPRS, ELRS0-6) were prepared with physiological salt, sucrose, glucose and fetal bovine serum. The physiological parameters of ELRS3 (ratio of fast motion, ratio of slow motion, time of fast motion, time of slow motion, lifespan and motility) and ELS3 (sperm ratio of slow motion, time of slow motion and motility) were significantly higher than those of the other dilutions (P < 0.05). Secondly, after adding 15% DMSO and 10% FBS to ELRS3 and ELS3, most physiological parameters of frozen sperm were also significantly higher than the other gradients (P < 0.05), and sperm motility was as high as 63.68 ± 4.16% to74.75 ± 12.71% (fresh sperm motility, 80.70 ± 1.37% to 80.71 ± 1.49%). Mixed with the above dilutions, a final volume of 105 ml semen was cryopreserved. Finally, the sperm of giant grouper cryopreserved with cryoprotectants (ELRS3 + 15% DMSO + 10% FBS) was used for electron-microscopic observation and crossbreeding with red-spotted groupers (Epinephelus akaara). The electron-microscopic observation revealed that part of the frozen-thawed sperm was cryodamaged, e.g., flagellum fracturing and mitochondria falling out, while the ultrastructure of sperm membrane, mitochondria and flagellum remained intact. Also, the fertilization and hatchability rates of giant grouper frozen sperm and red-spotted grouper eggs were as high as 94.56% and 75.56%, respectively. Thus, a technique for cryopreservation of giant grouper sperm was successfully developed and applied to crossbreeding with red-spotted grouper eggs.     

猪精子顶体素水平与精液质量参数和体外受精间的联系

本研究目的是评估不同处理对精子顶体素活性的影响以及顶体素活性、精液参数和体外受精间的联系.鲜精、稀释精液(1:1)和液相精液(17℃)的精子顶体素活性分别是5.27,4.28和4.41 mIU/106.冻精或液相精液保存4 d后顶体素活性显著下降(P＜0.05).公猪间液相精液顶体素活性和低渗膨胀精液百分数有显著的差异(P＜0.05).冷、热应激对精子顶体素活性没有影响.鲜精和冻精的顶体素活性与低渗膨胀精子百分数和顶体完整率呈正相关.不同种猪液相精液精子顶体素活性与体外受精率和随后的囊胚发育率相关性不大.因此建议顶体素活性与其它精子功能参数相结合才能精确预测精子受精力.     

小鼠肾细胞冻存过程中甘油浓度的优化

目的:探讨在冻存小鼠肾细胞过程中,通过优化冷冻保护剂中甘油浓度的加入量,以达到小鼠肾细胞的最佳冻存效果。方法:将小鼠肾细胞制备成单细胞悬液,然后加入不同浓度的冷冻保护剂甘油,冻存细胞,比较细胞冻存效果,计算细胞成活率。四个组的甘油加入量分别为5%、10%、15%、20%。结果:在小鼠肾细胞冻存过程中,当冷冻保护剂中甘油的浓度达到10%时,冻存效果最佳,细胞成活率达到89.57%。     

红腹锦鸡精液冷冻稀释液配方的筛选

为加强濒危珍稀动物种质资源的保护,开展了濒危珍稀禽类—红腹锦鸡的人工授精研究。2005年5月26日~6月8日,对8只红腹锦鸡用手按摩其背、尾部采精12次。初测其精液品质,结果表明,采精量平均(0.114±0.016)mL(0.01~0.2 mL),每毫升精液中精子平均3.2亿个(3.0~3.3亿),活力9级以上,pH值6.5,偏酸性,淡乳白色(半透明似冲熟的藕粉),微腥。鲜精分别加11%蔗糖—卵黄稀释液(3号液);11%蔗糖—0.1%柠檬酸三钠—卵黄稀释液(5号液);5%葡萄糖—卵黄稀释液(8号液)。精子活力达8~9级。用3%柠檬酸三钠—卵黄稀释液(2号液)稀释,精子活力2级。冷冻时,用11%蔗糖溶液100 mL中加16 mL鲜卵黄,加5 mL甘油,配制的3号冷冻稀释液稀释,解冻后,精子活力达4级。优于试验中选拟的其它配方(加入5 mL甘油的5号冷冻液,解冻后精子活力为3级;加入5 mL甘油的8号冷冻液,解冻后未见活的精子)。如果冷冻稀释液中甘油改为6 mL,则解冻后精子全部死亡。     

利用性控精液体外生产奶牛早期胚胎

实验进行了精子处理方法、洗精液中添加抗生素种类及浓度、常规培养小滴(50μL)中入胚数的筛选,按照不同荷斯坦公牛个体分选的性控精液对体外生产的牛早期胚胎发育潜力的影响。结果表明:离心法处理性控精液效果最优。利用经过分选的性控精液进行体外受精(IVF)后都较易导致污染,且受精后的体外胚胎发育效果都较差。在洗精液中添加10μL/mL头孢哌酮钠的IVF胚胎体外发育较好,囊胚率达到16.9%;且在常规培养小滴中适当减少入胚数,能有效抑制利用性控精液制备的奶牛IVF胚胎自身对培养微滴的污染。利用直接离心法处理精液,在洗精液中添加头孢哌酮钠10μL/mL,可以成功进行牛性控精子的体外受精,但目前的受精效果低于用普通精子的体外受精效果。