Abstracts/Zusammenfassungen

Der Embryotransfer im Rahmen einer Sterilitätsbehandlung des Menschen The Commercial Application of Embryo Splitting in Friesian-Holstein Cattle at the Osnabrücker Herdbook Association (OHG) Experimental Studies on the Assessment of Bovine Embryo Quality Based on Morphological and Metabolic Criteria Investigations to Improve the Selection of Donor Animals for Embryo Transfer in Dependance of the Follicular Growth Aknowledgements to the Problem of Unfertilized Eggs in Commercial Embryo Transfer from a Practicioner's Point of View Energy and Mineral Needs of a High Yielding Dairy Cow Under the Aspect of Fertility in Embryo Transfer Programmes Superovulation and Embryo Transfer (ET) in Industrial-Sized Farms in ?SFR Influence of the Qualitative Peripartal Feeding Supply on the Superovulatory Response in Cattle Superovulation and Embryo Transfer in Cattle: Endocrinological Studies During Recognition and Termination of Single and Multiple Pregnancies Einfluß des FSH/LH-Verhätnisses auf den Superovulationserfolg bei Ziegen Einsatzmöglichkeiten der Sonographie im Rahmen des Embryotransfers beim Rind Selection of Bovine Recipients for Embryo Transfer, Comparison of Rectal Palpation and Ultrasonic Corpus Luteum Assessment with Progesterone Data Presentation of a Simple Freezer for Embryos A Vitrification Experiment in Cattle Embryos Cryopreservation of Bovine Oocates at Different Stages of Maturation Pregnancies Following Transfer of Frozen-Thawed in vitro Matured, Fertilized and Cultured Bovine Embryos to Recipients Competence of Bovine Oocytes Derived from Different Size Follicles Development of Follicles and Oocytes After Superovulation Treatment with Different FSH/LH-Ratio In vitro Development of 1-Cell Mouse Embryos in CZB Medium In vitro Fertilization in Cattle with Fresh and Frozen Semen Tested by Computerized Videomicrogaphy Pregnancy and Twinning Rates After Unilateral and Bilateral Transfer of In vitro Produced Bovine Embryos to Recipients In vitro Maturation and In vitro Fertilization of Calf Oocytes In vitro Fertilization and Preconceptional Sexing in Cattle In vitro Culture of Cloned Cattle Embryos Diagnosis of Sex and Milk Protein Genotypes in Preimplantational Bovine Embryos Influence of Cloning on Breeding and Production Kultivierung und Transfer von zonalosen geteilten Ziegenembryonen Dilatation of the Uterine Cervix by Hormones as an Assumption for Transcervical Collection of Ovine and Caprine Embryos     

The effect of co-culture on the development of in vitro matured equine oocytes after intracytoplastic sperm injection

It is clear that, in the horse, there are many weak links in the process of in vitro embryo production; an optimal culture system for equine oocytes does not exist, and related data are conflicting. Therefore, the ability of 3 different culture systems to support embryonic development of ICSI horse oocytes was examined. Oocytes (n = 261) suitable for culture were collected from 55 ovaries and divided, according to cumulus morphology, into 2 categories: expanded cumulus and compacted cumulus. Oocytes with expanded and compacted cumulus were cultured for in vitro maturation in TCM 199 + 10% FCS + 0.1 iu/ml FSH/LH at 38.5 degrees C under 5% CO2 in air for 24 and 40 h, respectively. Oocytes (n = 149) reached metaphase II and were subjected to ICSI with frozen semen and then incubated in 3 different culture systems: A) TCM 199 + 10% FCS alone or B) on granulosa cell monolayer, C) SOF + MEM amino acids + 0.8% BSA. Cultural conditions were 39 degrees C and 5% CO2 in air for A and B, while a gas mixture (5% CO2, 5% O2, 90% N2) was used for C. The fertilisation rate was 32%. The cleavage rate in Group A was 74.4% (32/43); 18 embryos reached 2-6 cell stage, eight 8-16 cell, four 16-32 cell and two >32 cell. In Group B, the cleavage rate was 73.5% (36/49) with better results in embryonic development; 14 reached 2-6 cell stage, eighteen 8-16 cell, twelve 16-32 cell and five >32 cell. In Group C, the cleavage rate was significantly lower then in A and B; only 15 of 47 ICSI oocytes (39.1%) cleaved with maximum development to 2-6 cell stage. The remaining oocytes (68.1%) degenerated during culture. In conclusion, IVM horse oocytes can be fertilised in vitro with high efficiency with ICSI and co-culture systems showed to be superior in supporting in vitro embryo culture compared to simple ones. The identification of the factors beneficial to in vitro embryo development provided by the somatic cells could be important to optimise the embryo culture systems for equine embryos.     

Feeding fodder beet (Beta vulgaris L.) with either barley straw or pasture silage to non-lactating dairy cows

AIMS: To determine the suitability of diets containing either approximately 85% fodder beet (Beta vulgaris L.) with barley straw or 65% fodder beet with pasture silage when fed to non-lactating dairy cows, by measuring intakes, digestibility, rumen function including microbial growth, and N excretion.

METHODS: Holstein-Friesian cows fitted with permanent rumen fistulae were fed either 65% fodder beet with pasture silage (Silage; n=8) or 85% fodder beet with barley (Hordeum vulgare L.) straw (Straw; n=8) in an indoor facility over a 9-day period, for measurement of intakes, digestibility, rumen function and urine production. The cows were adapted to the diets over 2 weeks before the indoor measurements. Feed was available for about 6 hours/day, as practiced commercially for wintering non-lactating cows.

RESULTS: Five cows fed the Straw diet had to be removed from the trial because of acute acidosis; four on Day 1 of the measurement period and one on Day 7. One cow allocated to the Silage diet refused to eat fodder beet bulbs and was also removed from the trial. Two cows fed the Silage diet were also treated for acidosis. DM intakes were lower with the Straw than Silage diets (6.4 (SE 0.4) vs. 8.3 (SE 0.5) kg/day) and organic matter (OM) digestibility was lower with the Straw than Silage diets (77 (SE 1) vs. 83 (SE 1) g/100g). The N content of the two diets was 1.14 and 1.75?g/100?g DM and there was a net loss of N by cows fed the Straw diet (?22.7 (SE 7) g/day). Rumen microbial N production was much lower in cows fed the Straw than the Silage diet (6.6 (SE 1.3) vs. 15.8 (SE 0.7) g microbial N/kg digestible OM intake). Concentrations of ammonia in rumen liquid collected on Days 5–6 were below detection limits (<0.1?mmol/L) in 36/48 (75%) samples collected from cows fed the Straw diet and in 27/48 (56%) cows fed the Silage diet. Mean urinary N excretion was lower in cows fed the Straw than the Silage diet (52.0 (SE 5.8) vs. 87.7 (SE 5.9) g/day).

CONCLUSION AND CLINICAL RELEVENCE: An over-wintering diet for dry cows comprising about 65% fodder beet with 35% pasture silage provided adequate nutrition, although there was some risk of acidosis. In contrast, the diet containing about 85% fodder beet with barley straw resulted in lower DM intakes, poor rumen function, negative N balance so that both nutrition and welfare were compromised.     

海狸鼠卵母细胞体外成熟培养初探

实验用ＰＭＳＧ或ＰＭＳＧ＋ＨＣＧ处理或未经激素处理的海狸鼠８只，共获卵巢卵母细胞１３８枚。激素处理对获取卵巢卵母细胞的数量没有影响，而对体外成熟发育至卵丘扩展和半成熟阶段有促进作用。三种不同培养液（Ｗｈｉｔｅｎ＋ＦＣＳ；ＴＣＭ１９９＋ＰＭＳＧ＋ＦＣＳ；ＴＣＭ１９９＋ＨＣＧ＋ＦＣＳ）共培养１２５枚卵母细胞，培养后卵丘扩展率及半成熟率分别为５６．５％，４５．７％，４７．６％和２１．７％，１２．３％，９．５％，以Ｗｈｉｔｅｎ液较高（分别为５６．５％和２１．７％），但只有ＴＣＭ１９９＋ＰＭＳＧ＋ＦＣＳ组有２枚卵母细胞出现第一极体。结果表明海狸鼠卵母细胞与其它啮齿动物的卵母细胞一样，能够在体外培养成熟，完成第一次减数分裂，排出第一极体     

Effect of Different Combinations of Cryoprotectants on In Vitro Maturation of Immature Buffalo (Bubalus bubalis) Oocytes Vitrified by Straw and Open‐Pulled Straw Methods

This study was designed to evaluate effects of different combinations of cryoprotectants on the ability of vitrified immature buffalo oocytes to undergo in vitro maturation. Straw and open‐pulled straw (OPS) methods for vitrification of oocytes at the germinal vesicle stage also were compared. The immature oocytes were harvested from ovaries of slaughtered animals and were divided into three groups: (i) untreated (control); (ii) exposed to cryoprotectant agents (CPAs); or (iii) cryopreserved by straw and OPS vitrification methods. The vitrification solution (VS) consisted of 6 m ethylene glycol (EG) as the standard, control vitrification treatment, and this was compared with 3 m EG + 3 m dimethyl sulfoxide (DMSO), 3 m EG + 3 m glycerol, and 3 m DMSO + 3 m glycerol. Cryoprotectants were added in two steps, with the first step concentration half that of the second (and final) step concentration. After warming, oocyte samples were matured by standard methods and then fixed and stained for nuclear evaluation. Rates of MII oocytes exposed to CPAs without vitrification were lower (54.3 ± 1.9% in EG, 47.5 ± 3.4% in EG + DMSO, 36.8 ± 1.2% in EG + glycerol and 29.9 ± 1.0% in DMSO + glycerol; p < 0.05) than for the control group (79.8 ± 1.3%). For all treatments in each vitrification experiment, results were nearly identical for straws and OPS, so all results presented are the average of these two containers. The percentages of oocytes reaching telophase‐I or metaphase‐II stages were lower in oocytes cryopreserved using all treatments when compared with control. However, among the vitrified oocytes, the highest maturation rate was seen in oocytes vitrified in EG + DMSO (41.5 ± 0.6%). Oocytes cryopreserved in all groups with glycerol had an overall low maturation rate 19.0 ± 0.6% for EG + glycerol and 17.0 ± 1.1% for DMSO + glycerol. We conclude that the function of oocytes was severely affected by both vitrification and exposure to cryoprotectants without vitrification; the best combination of cryoprotectants was EG + DMSO for vitrification of immature buffalo oocytes using either straw or OPS methods.     

对C57BL／6小鼠超排效果的研究

本文以近交系C57BL／6小鼠为实验对象．研究注射不同剂量的PMSG和hCG对小鼠超排效果的影响。取C57BL／6小鼠各30只,按照注射剂量不同分为A、B、C三组,每组10只,A组注射PMSG2．5IU,HCG2．5IU,B组注射PMSG5．0IU,HCG5．0IU,C组注射PMSG7．5IU,HCG7．5IU。每只小鼠腹腔注射PMSG,间隔48h后分别注射HCG进行超数排卵,再与性成熟同系公鼠合笼,次日早上检查阴道栓．有栓雌鼠用颈椎脱臼法处死。在实体显微镜下由输卵管膨大部冲卵．收集卵母细胞置于盛有M2培养液的表面皿中检查计数．分析超排效果。结果表明。C57BU6小鼠B组的平均取卵数极显著高于A组的平均取卵数（P〈0．05）;B组的平均取卵数显著高于C组的平均取卵数（P〈0．05）;C组与A组的平均取卵数差异不显著（P〉0．05）。     

Aspectos Cronologicos en la Maduracion Nuclear de Oocitos Bovinos Cultivados in vitro

In this study, the chronological changes in the meiotic progress of in vitro maturation of bovine oocytes was analysed. Oocytes (n = 1044) were obtained from a local abbatoir and were classified into two groups according to the presence or absence of cumulus cells. They were incubated in microdrops (5 per drop) in TCM-199 (control), TCM-199 + 10% fetal calf serum (treatment 2) and TCM-199 + 10% estrous cow serum (treatment 3). Oocytes were fixed and stained at the end of 0, 6, 12, 18, 24 and 30h of the beginning of in vitro culture, were evaluated according to the nuclear stage of maturation. These results demonstrated that the immature oocytes at the time of collection (0 h) were in the germinal vesicle stage (GV), and that the highest maturation rate was at 24 h of culture in all treatments. Serum treatments enhanced the maturation rates obtained (52.1 and 55.7%) compared to control (serum-free) medium (42.7%; P<0.05) in cumulus-cell-enclosed oocytes. In denuded oocytes, the maturation rates were lower compared to cumulus-cells-enclosed oocytes in all treatments. In conclusion, meiotic progression of bovine oocytes can be influenced by the inclusion of sera in the maturation media and by the presence of the cumulus cells.     

牛GV期卵母细胞冷冻保存后发育潜力的研究

二甲基亚砜(DMSO)、丙二醇(PROH)、乙二醇(EG)和甘油(GL)4种冷冻保护剂程序化冷冻牛GV期卵母细胞的结果表明,EG和PROH的保护效果比GL和DMSO好。4种不同冷冻方法冷冻保存牛GV期卵母细胞,比较解冻后卵母细胞的体外成熟率、受精后卵裂率。结果表明,在程序化冷冻法与细管玻璃化法(Straw)之间的差异不显著(P>0.05),在开放式拉管法(OPS)与毛细玻管法(GMP)之间的差异不显著(P>0.05);但OPS和GMP与程序化冷冻法和Straw之间的差异极显著(P<0.01)。玻璃化冷冻效果优于程序化冷冻。说明GMP和OPS玻璃化冷冻优于Straw玻璃化冷冻。说明可以采用GMP方法冷冻保存牛GV期卵母细胞。     

亮甲酚蓝染色对牛卵母细胞体外成熟的影响

为了探讨不同浓度亮甲酚蓝对牛卵母细胞染色选择后对其体外成熟（IVM）的影响,本研究在牛卵母细胞成熟培养前直接用不同浓度梯度的亮甲酚蓝进行不同时间的染色。结果表明：①经过不同浓度的亮甲酚蓝染色后,所选择卵母细胞染色后着色的百分率及其成熟率是不同的。用26、39、52 μmol/L的亮甲酚蓝染色后所得到的胞质蓝色组（27.7%、27.6%、31.9%）较13 μmol/L的亮甲酚蓝染色后所得到的胞质蓝色组（2.1%）差异显著（P＜0.05）;②经过不同浓度的亮甲酚蓝染色后,胞质蓝色组（77.4%）较胞质无色组（51.3%）和对照组（60.2%）成熟率差异显著（P＜0.05）;③不同浓度亮甲酚蓝染色的胞质蓝色组间成熟率差异不显著（P＞0.05）;④不同浓度亮甲酚蓝染色组与对照组卵母细胞的成熟率差异不显著（P＞0.05）;⑤经过不同时间的亮甲酚蓝染色后,所选择卵母细胞着色的百分率是不同的。用26 μmol/L的亮甲酚蓝染色90 min后所得到的胞质蓝色组（75%）较亮甲酚蓝染色60、30 min后所得到的胞质蓝色组（57.14%,40%）差异显著（P＜0.05）。以亮甲酚蓝染色为基础的牛卵丘卵母细胞复合体的区分可以用来有效的选择更具发育活力的牛卵母细胞。     

马鹿冻精解冻后精子超微结构观察

实验利用透射电镜技术和扫描电镜技术对冷冻/解冻后的塔里木马鹿精子的超微结构进行了观察。结果表明:电镜下观察,塔里木马鹿精子头部呈扁平的卵圆形,长约5.95μm。颈部很短且不明显,长约0.45μm,宽为0.62μm。尾部中段横切面近圆形,直径约为0.58μm,轴丝为9×2+2型;线粒体鞘螺旋段为64～70转。尾部末段仅见质膜包围,9束微管和1对中央微管,形成9+2微管结构。冷冻/解冻后塔里木马鹿部分精子结构发生了变化,一些精子肿胀、质膜皱褶、扭曲,部分质膜损坏或溃散;顶体轻度肿胀,顶体外膜凸凹不平,形成突起。冷冻对头部破坏程度较小。     

High survival rate of bovine oocytes matured in vitro following vitrification

Improving pregnancy rates associated with the use of cryopreserved human oocytes would be an important advance in human assisted reproductive technology (ART). Vitrification allows glasslike solidification of a solution without ice crystal formation in the living cells. We have attempted to improve the survival rates of oocytes by a vitrification technique using bovine models. In vitro matured oocytes with or without cumulus cells were vitrified with either 15.0% (v/v) ethylene glycol (EG) + 15% (v/v) dimethylsulfoxide (DMSO) + 0.5 M sucrose or 15% (v/v) EG + 15% (v/v) 1,2-propanediol (PROH) + 0.5 M sucrose, using 'Cryotop' or 'thin plastic sticker', respectively. The oocyte survival rates after vitrifying-warming, and the capacity for fertilization and embryonic development were examined in vitro. The rate of embryonic development to blastocyst was significantly higher (P<0.05) in the oocytes vitrified with 15% (v/v) EG + 15% (v/v) PROH + 0.5 M sucrose than in the oocytes vitrified with 15% (v/v) EG + 15% (v/v) DMSO + 0.5 M sucrose (7.4% +/- 4.1 vs. 1.7% +/- 3.0, respectively). Oocytes vitrified without cumulus cells had a higher survival rate after thawing and a superior embryonic developmental capacity compared with oocytes vitrified with cumulus cells. Prolonged pre-incubation time after thawing adversely affected the rates of embryonic cleavage and development. These results indicate that in vitro matured bovine oocytes can be vitrified successfully with the mixture of the cryoprotectants, EG + PROH, the absence of cumulus cells for vitrification does not affect oocyte survival rate after warming, and vitrified and warmed oocytes do not require pre-incubation before in vitro fertilization.     

Characterisation of movement pattern and velocities of stallion spermatozoa depending on donor, season and cryopreservation

The aim of the study was to compare different types of movement pattern and velocities of stallion spermatozoa depending on cryopreservation during breeding and non-breeding season. Ejaculates were collected from four stallions during May (n = 24) and December (n = 24). Parameters of sperm movement were evaluated by computer-aided sperm analysis (CASA) system, and included percentages of motile spermatozoa, different patterns of motility, the velocity, linearity (LIN), amplitude of lateral head displacement (ALH) and beat-cross frequency (BCF). In winter the average percentages of motility were slightly higher compared to the breeding season in May (70.8 +/- 12.7% vs. 66.8 +/- 12.2%, respectively). Cryopreservation and thawing led to a significant decrease in the number of motile sperm to 11.3 +/- 5.8% in May and 15.6 +/- 7.0% in December. The pattern of motility was also changed. Detailed analysis by CASA demonstrated that cryopreservation resulted in a shift from the proportions of linear to more non-linear motile spermatozoa and to a significant increase of local motile and hyperactivated spermatozoa. Mean velocity of fresh motile spermatozoa differed between May and December (119.1 +/- 43.9 vs. 164.4 +/- 66.4 microm/sec, respectively; P < 0.05). Cryopreservation and thawing led to a slight increase of curvilinear velocity (VCL) and straight line velocity (VSL). The motility analysis has shown that the parameters BCF and ALH were highly correlated in stallion spermatozoa (r = -0.67; P < 0.001). The BCF of stallion spermatozoa was slightly reduced in the non-breeding season. Altogether, the influence of factors on the motility of stallion spermatozoa has the following rank order: cryopreservation (P < 0.0001) > stallion (P < 0.001) > season (P < 0.05).     

血清、促性腺素、EGF和水对山羊卵母细胞体外成熟的影响

为了通过比较培养筛选出能改进和提高山羊卵母细胞体外成熟效率的培养体系 ,培养的山羊卵母细胞以 TCM-199为基础培养液 ,添加 :(1) 10 %血清 (胎牛血清 (FBS)或发情山羊血清 (EGS) ) 2 0 m g/ L促黄体素 (L H) 10 mg/ L促卵泡素 (FSH) 1m g/ L 雌二醇 (E2 ) ;(2 ) 10 % EGS 促性腺素 (L H∶ FSH=5 mg/ L∶ 0 .5 m g/ L 或 2 0 mg/ L∶ 10mg/ L )或者 0 .0 75 IU / m L人绝经期促性腺素 (HMG) 1mg/ L estradiol 17β;(3) 10 % EGS 0 .0 75 mg/ L HMG 10～ 2 0 μg/ L EGF。此外 ,以 M199 10 % EGS 0 .0 75 mg/ L HMG 10～ 2 0 μg/ L EGF为培养基 ,溶解于自制超纯水或储存的商品化超纯水来培养卵母细胞。培养条件为 38℃ ,5 % CO2 。培养 2 4 h后 ,在体式显微镜下统计处于 M 期的卵母细胞比例。结果显示 :在卵母细胞成熟液中添加 10 % EGS比 10 % FBS的成熟培养效果好 ;添加 HMG能够促进卵母细胞的成熟 ,其效果比添加不同比例的 L H/ FSH好 ;成熟液中添加 10～ 2 0μg/ L EGF能促进卵母细胞的成熟 ,但成熟率没有明显提高 ;新鲜的超纯水对于卵母细胞的培养是必要的。结论 :新鲜超纯水配制的 M199 10 % EGS 0 .0 75 IU / m L HMG 10～ 2 0μg/ L EGF培养液可以获得最佳卵母细胞培养效果     

秸秆饲草的几种调制贮存技术探讨

秸秆饲草经过青贮、半干黄贮（添加酶或生物制剂）、黄贮（氨化）调制处理后,可以最大限度地保持原秸秆饲草的营养物质,改善适口性,净化乡村环境卫生,氨化饲草还能提高饲草营养价值。本文就几种调制方法的特点、过程及注意事项等做了探讨。     

猪精子冷冻损伤与保护剂的研究进展

猪精液冷冻保存技术主要是利用液氮(-196℃)作为冷源,在超低温的环境下使精子生理代谢完全暂停,实现长期保存的目的.猪精子在低温环境主要损伤是细胞内结冰晶造成的机械损伤、游离的活性氧造成的化学损伤,对这两种损伤机制研究是国内外科研工作者们的研究热点.文章主要简述猪精液冷冻技术发展简况,精子在冷冻过程中受损伤机制和现阶段...     

Meiosis in bovine oocytes matured in vitro and in vivo

Meiosis in bovine oocytes has; been studied after maturation in vitro or in vivo. Oocytes for in vitro maturation were collected from the ovaries of slaughtered cattle without regard to the phase of the estrous cycle while in vivo maturation was studied in oocytes from gonadotrophin-stimulated heifers at times varying between 6 and 36 h after the beginning of behavioural estrus. Oocytes from slaughtered cattle were classified according to their cumulus complex and ooplasm and were cultured for 6, 12, 18, 24, 36 or 48 h in modified Krebs-Ringer bicarbonate buffer before fixation) for cytogenetic analysis. Oocytes from stimulated heifers were aspirated from follicles or flushed from the oviducts, classified according to cumulus and ooplasm, and fixed within 6 h of collection. Nuclear maturation was more rapid in vitro than in vivo. The largest proportion of oocytes reached maturity (Mil) after 12 to 18 h in culture or 30 to 36 h after the onset of behavioural estrus. Oocytes devoid of cumulus cells or showing signs of vacuolation or degeneration had virtually no capacity for nuclear maturation.     

Effect of Temporary Meiotic Attenuation of Oocytes with Butyrolactone I and Roscovitine in Resistance to Bovine Embryos on Vitrification

This study aimed to produce in vitro bovine embryos by the addition of two drugs, which is responsible for oocyte meiosis inhibition: roscovitine (ROS) and butyrolactone I (BL‐I). Oocytes were recovered from slaughtered cows and matured in a commercial medium and maintained in a 5% CO₂ atmosphere. Oocytes were maintained for 6 h in an in vitro maturation (IVM) medium containing ROS (12.5 μm ), BL‐I (50 μm ) and association of drugs (ROS 6.25 μm and BL‐I 25 μm ). Oocytes were cultured for 18 h in an agent‐free medium for the resumption of meiosis. After 24 h of maturation, oocytes were inseminated in the commercial in vitro fertilization (IVF) medium. Presumptive zygotes were cultured in SOFaa medium in a 5% CO₂ atmosphere. On day 3, rate of cleavage was evaluated and on days 6 and 7, rate of blastocyst formation. BL‐I and its association with the ROS increased the rates of cleavage and blastocyst formation (p < 0.05). The ROS alone was inefficient, impairing embryonic development, with low rates of blastocyst formation when compared to the control group and other treatments (p < 0.05). The embryos from BL‐I and ROS+BL‐I groups presented higher number of cells and lower rates of cellular apoptosis compared to other groups, either for the fresh or for post‐thawing embryos. Embryos from ROS+BL‐I group showed to be more resistant to the vitrification process, presenting a higher rate of embryonic re‐expansion (p < 0.05). In conclusion, block of meiosis using BL‐I or its association with ROS increased the rate of blastocyst formation, and the association of ROS+BL‐I resulted in a better resistance to the embryo cryopreservation process.     

Presence of nitric oxide synthase immunoreactivity and mRNA in buffalo (Bubalus bubalis) oocytes and embryos

This study examined the presence of immunoreactivity and mRNA for different nitric oxide synthase (NOS) isoforms in immature and in vitro matured oocytes and in embryos at two‐, four‐ and eight‐cell, and morula and blastocyst stages in buffalo. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to in vitro maturation in TCM‐199 + 10% FBS + 5 μg/ml pFSH + 1 μg/ml estradiol‐17β + 0.81 mm sodium pyruvate + 10% buffalo follicular fluid + 50 μg/ml gentamycin sulphate for 24 h in a CO₂ incubator (5% CO₂ in air) at 38.5°C. Following in vitro fertilization carried out by incubating them with 2–4 million spermatozoa/ml for 18 h, the presumed zygotes were cultured in mCR2aa medium containing 0.6% BSA and 10% FBS for up to 8 days post insemination. Immunofluorescence staining of NOS using antibodies that cross‐reacted either with all the NOS isoforms i.e., universal (uNOS) or specifically with inducible (iNOS) or endothelial (eNOS) isoforms revealed that NOS was present in oocytes and embryos at all the stages examined. Examination of the semi‐quantitative expression of NOS genes by RT‐PCR revealed that the iNOS, eNOS and nNOS mRNA was present in the immature and mature oocytes and in all the embryonic stages examined. In conclusion, it was demonstrated in the present study that immunoreactivity and mRNA for different NOS isoforms was present in buffalo oocytes and pre‐implantation stage embryos.     

Effects of activation methods on DNA synthesis and development of parthenogenetic porcine embryos

This study investigated the timing of DNA synthesis and patterns of pronuclear (PN) formation during the first cell cycle, and its influence on developmental competence, velocity and proliferation index of porcine parthenote blastocysts produced by different activation treatments. Oocytes were activated as follows: electrical stimulation (EST), EST combined with 7.5 μg/ml cytochalasin B (EST + CCB), 10 μg/ml cycloheximide (EST + CHX) and 1.9 mm 6-dimethylaminopurine (EST + 6-DMAP) for 3 h. DNA synthesis and PN formation were evaluated using 1 mm 5'bromo-2'deoxy-uridne (BrdU) at 2 h intervals from 1 to 13 h or 5 to 13 h of post-activation (hpa), respectively. In EST, DNA synthesis started at 3 hpa, reached the peak at 11 hpa and decreased at 13 hpa. Treatment with 6-DMAP resulted in an early increase of DNA synthesis at 3 hpa, whereas CCB delayed DNA synthesis for 2 h. In EST and EST + 6-DMAP, most of the eggs showed 1PN, whereas, incidence of 2PN in EST + CCB was higher than 1PN. EST + CHX was observed with 1PN, 2PN and multiple PN. Blastocyst rate in EST + CCB and EST + 6-DMAP were significantly (p<0.05) higher than EST + CHX. But, the developmental velocity was not different among groups. Proliferation index of blastocysts, as indicated the number of blastomere at S-phase of the cell cycle was low in all groups. In conclusion, CCB, CHX and 6-DMAP used for producing porcine parthenogenetic embryos induced different onset of DNA synthesis and PN, but they did not affect the subsequent embryo development.