Modification of maturation condition improves oocyte maturation and in vitro development of somatic cell nuclear transfer pig embryos

This study examined effects on the developmental competence of pig oocytes after somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) of : 1) co-culturing of oocytes with follicular shell pieces (FSP) during in vitro maturation (IVM); 2) different durations of maturation; and 3) defined maturation medium supplemented with polyvinyl alcohol (PVA; control), pig follicular fluid (pFF), cysteamine (CYS), or β-mercaptoethanol (β-ME). The proportion of metaphase II oocytes was increased (p < 0.05) by co-culturing with FSP compared to control oocytes (98% vs. 94%). However, blastocyst formation after SCNT was not improved by FSP coculture (9% vs. 12%). Nuclear maturation of oocytes matured for 39 or 42 h was higher (p < 0.05) than that of oocytes matured for 36 h (95-96% vs. 79%). Cleavage (83%) and blastocyst formation (26%) were significantly higher (p < 0.05) in oocytes matured for 42 h than in other groups. Supplementation of a defined maturation medium with 100 µM CYS or 100 µM β-ME showed no stimulatory effect on oocyte maturation, embryo cleavage, or blastocyst formation after PA. β-ME treatment during IVM decreased embryo cleavage after SCNT compared to pFF or PVA treatments, but no significant difference was found in blastocyst formation (7-16%) among the four treatment groups. The results indicated that maturation of oocytes for 42 h was beneficial for the development of SCNT embryos. Furthermore, the defined maturation system used in this study could support in vitro development of PA or SCNT embryos.     

The role of forskolin as a lipolytic stimulator during in vitro oocyte maturation and the in vitro embryo production of livestock

Cryopreservation is a modern technique which assists in the preservation of genetic material from oocytes and embryos for a long time. However, elevated vulnerability to cryopreservation due to the large accumulation of intracellular lipids within oocytes or embryos avoids success of this method. These lipids remain the main crucial factor limiting survival rates of oocytes and embryos after thawing. Lipid ingathering in the oocyte cytoplasm augments lipid peroxidation (LPO) and oxidative stress increases the apoptosis process, declines the viability after thawing, declines cytoskeleton actin filament injuries, lowers the blastocyst rates and reduces cryotolerance in the early stages of embryo development. There have been several attempts to reduce the ingathering of intracellular lipids in oocytes or embryos during the cryopreservation process, in that way enhancing the competence of cryopreserved oocytes or embryos and increasing their viability. One of the most applied agents for chemical delipidation is forskolin. Forskolin exhibited a possible part in improving the oocytes cryopreservation through stimulating cyclic adenosine monophosphate (cAMP) production. The main purpose of cAMP modulation is to provide energy to sustain the mammalian oocytes´ meiotic arrest. The purpose of the existing article is to assess and offer more evidence concerning the forskolin utilization as a modulator of cAMP during the cryopreservation of oocytes and its influence on meiosis completion and the reorganization of cytoplasm, which are prerequisites for the development of oocytes in addition to the contribution to fertilization and subsequently, the development of embryos.     

白藜芦醇对猪卵母细胞体外老化的抑制作用

旨在研究白藜芦醇(RES)对猪卵母细胞体外老化的影响及机制。添加不同浓度RES(0、1、2、4μmol/L),体外培养猪卵母细胞44 h后,通过卵丘扩散和第一极体排出率筛选出最佳有效浓度为2μmol/L。卵丘-卵母细胞复合体(cumulus-oocyte complexes, COCs)成熟培养44 h作为对照组,连续培养68 h作为体外老化组,置于含2μmol/L白藜芦醇成熟培养液中培养68 h,为白藜芦醇处理组。激光扫描共焦显微镜检测染色体和纺锤体,Western blot检测凋亡相关蛋白Caspase-3、Bcl-2和Bax表达水平。结果表明:RES处理组和对照组的猪卵母细胞染色体与纺锤体异常率均较老化组显著降低(P<0.01)。用2μmol/L RES处理后,猪卵母细胞Caspase-3和Bax蛋白表达量均有下降,显著低于老化组(P<0.001);Bcl-2蛋白表达量有所升高,但差异不显著。试验表明,RES可明显抑制猪卵母细胞的老化,这一作用可能主要通过抑制线粒体凋亡途径来实现的。     

Production of Ban miniature pig embryos by in vitro fertilization: A comparative study with Landrace

Ban is an endangered miniature pig breed in Vietnam. This study aimed to set up an in vitro embryo production (IVP) system for this breed. Ban's epididymal sperm concentration (1240 ± 35 × 10⁶/mL) was lower (P < 0.01) compared with Landrace (4160 ± 42 × 10⁶/mL). However, sperm characteristics before and after freezing in Ban and Landrace were similar. The numbers of follicles with diameter larger than 2 mm per ovary in Ban females treated with equine chorionic gonadotropin and human chorionic gonadotropin (27.1 ± 1.3) were higher (P < 0.05) than those in Landrace (12.9 ± 2.0) and in non‐hormone stimulated Ban (no > 2 mm follicles). After in vitro maturation, the percentages of oocytes with expanded cumulus cells and the first polar body (matured oocytes) were not different among Ban, hormone‐stimulated Ban and Landrace. The percentages of two‐cell embryos and morulae derived from oocytes collected from three sources did not differ. However, the rate of blastocysts derived from oocytes in non‐stimulated Ban (4.0 ± 3.8%) was lower (P < 0.05) than that in Landrace (15.3 ± 1.8%). In conclusion, an effective IVP system for good quality embryos in Ban, that is essential for genetic conservation of this breed, was established.     

Theca cell-conditioned medium added to in vitro maturation enhances embryo developmental competence of buffalo (Bubalus bubalis) oocytes after parthenogenic activation

Theca cells (TCs) play a key role in follicular growth and atresia. TCs synthesize androgens that act as substrate for granulosa cells (GCs) aromatization to estrogens needed for oocyte maturation. However, the effects of TCs in the form of conditioned medium on in vitro maturation (IVM) and developmental competence of buffalo oocytes remain unclear. In the present study, we examined the impacts of TC-conditioned medium (TCCM) on maturation efficiency and embryo development of buffalo oocytes after parthenogenic activation (PA). Our results showed that TCCM that was collected on day 2 and added to IVM medium at a 20% proportional level (2 days & 20%) exerted no significant effect on IVM rate (43.06% vs. 44.71%), but significantly (p  < .05) enhanced embryo development (oocyte cleavage, 80.93% vs. 69.66%; blastocyst formation, 39.85% vs. 32.84%) of buffalo oocytes after PA compared with the control group. However, monolayer TC significantly (p < .05) promoted both maturation efficiency (48.84% vs. 44.53%) and embryo development (oocyte cleavage, 80.39% vs. 69.32%; blastocyst formation, 35.38% vs. 29.25%) of buffalo oocytes after PA compared to that in the control group. Furthermore, TCs secreted some testosterone into the conditioned medium, which significantly (p < .05) promoted the expression levels of oestrogen synthesis-related genes (CYP11A1, CYP19A1 and 17β-HSD) in buffalo cumulus–oocyte complexes (COCs). Our study indicated that TCCM (2 days & 20%) did not significantly affect IVM efficiency, but enhanced embryo developmental competence of oocytes after PA principally by stimulating the secretion of testosterone and facilitating estradiol synthesis of buffalo COCs.     

水牛卵泡颗粒细胞对卵母细胞体外成熟的影响

为了系统研究颗粒细胞对水牛卵母细胞体外成熟的影响,使用颗粒细胞条件液处理或单层颗粒细胞和卵母细胞共培养的方法,探讨颗粒细胞共培养对水牛卵母细胞体外成熟和早期胚胎发育的影响.结果显示,添加颗粒细胞传代接种第2天收集的20％颗粒细胞条件液到水牛卵母细胞成熟液中能显著提高水牛卵母细胞体外成熟率和囊胚发育率(P＜0.05);然...     

树鼩卵母细胞的体外成熟培养及体外受精

利用树鼩卵母细胞的成熟培养、体外受精等方法,生产树鼩的试管婴儿,期望通过此路径来实现生产转基因树鼩动物模型。使用促性腺激素进行树鼩超数排卵、TCM199完全培基对卵母细胞进行体外成熟培养、卵母细胞与体外获能的附睾精子进行体外受精、受精卵发育至桑椹胚、囊胚的实验。结果表明,卵母细胞成熟率A级76.7%、B级55.01%、C级17.39%。受精率52%,受精卵采用体细胞共培养与非共培养的分裂率分别为37.14%和9.6%。桑椹胚/囊胚发育率分别为13.57%和0,(P<0.05)。树鼩卵母细胞的成熟培养、体外受精实验方法可行,受精卵在体外能发育到囊胚阶段。     

高浓度葡萄糖对猪卵母细胞体外成熟和早期胚胎发育的影响

试验旨在探究高浓度葡萄糖对猪卵母细胞体外成熟及早期胚胎发育能力的影响。取体外分离处于生发泡期的猪卵丘卵母细胞复合体(COCs),分为3个处理组。分别用含葡萄糖浓度为5.6 mmol/L(C组)、10 mmol/L(G-1组)、15 mmol/L(G-2组)的培养液,进行体外成熟(IVM)处理,42 h后观察,并统计卵丘细胞扩散情况和第一极体排出率;对体外成熟42 h后的卵母细胞孤雌激活,统计2-细胞、4-细胞和第7天囊胚发育。结果发现,G-1组和G-2组卵丘细胞扩散度显著低于C组(P<0.05);G-1组和G-2组的MII期卵母细胞死亡率和存活率与C组相比无显著差异(P>0.05),但G-1组极体率显著降低(P<0.05),G-2组极体率极显著低于C组(P<0.01)。孤雌激活后,与C组相比,G-1组和G-2组的2-细胞分裂率显著降低(P<0.05),4-细胞分裂率以及囊胚发育率均极显著降低(P<0.01),但G-1、G-2组囊胚细胞数量与C组相比无显著性差异(P>0.05)。进一步线粒体染色发现,G-1组和G-2组的线粒体与C组相比分布不均。...     

Comparison of production systems for efficient use of indigenous pig breeds in developing countries

Conserving pig genetic resources and improving their productivity is important to increase returns over investment in developing countries. The purebred, first‐cross, rotational cross and backcross matings representing production systems based on pig breeds indigenous to the country and exotic pig breeds were investigated. The number of pigs in the nucleus and commercial herds necessary to produce a defined quantity of pork was considered. The amount of heterosis between the indigenous and exotic breeds, superiority in meat production, and degree of inferiority in reproductive performance of the exotic breed compared with that of the indigenous breed were investigated. The number of breeding pigs in the whole system was in the following order: pure breeding (PB) > first‐cross (F1) > rotational cross (RC) > backcross (BC) systems. The number of breeding pigs in the nucleus herds of the RC and BC systems was smaller than that in the nucleus herds of the PB and F1 systems. The degree of inferiority in reproductive performance of the exotic breed compared with that of the indigenous breed affected the efficiency of the production system.     

Effects of electromagnetic waves on oocyte maturation and embryonic development in pigs

Our living environment has been full of electromagnetic radiation (EMR) due to the prevailing electronic devices and equipment. Intermediate frequency electromagnetic field (IF-EMF) or waves constitute a significant part of EMR; therefore, an increasing number of household electrical appliances have become a source of IF-EMF, and concerns about IF-EMF on health are gaining more attention. However, little information is available about its impact on female reproductive traits, such as germ cell viability and early embryonic development, particularly at the cellular and molecular levels. In this study, we used porcine oocytes as a model system to explore the effect of IF-EMF at various intensities on the in vitro maturation (IVM) of oocytes and their subsequent embryonic development. Our results showed that no difference in oocyte maturation rates was detected among groups, but the cleavage and blastocyst rates of parthenotes derived from EMF-treated oocytes decreased with the weaker IF-EMF intensity (25 and 50 Gauss) groups compared to the control group (P < 0.05). For cytoplasmic maturation, the weaker IF-EMF intensity groups also showed a peripheral pattern of mitochondrial distribution resembling that of immature oocytes and increased autophagy activity. No obvious differences in cytoskeletal distribution and total cell numbers of blastocysts were investigated in the four IF-EMF treatments compared to those in the control group. Although the underlying mechanism associated with EMF effects on oocytes and embryos is still elusive, we have demonstrated that low intensity IF-EMF exerts harmful effects on porcine oocytes during the maturation stage, carrying over such effects to their subsequent embryonic development.     

Supplementation with exogenous coenzyme Q10 to media for in vitro maturation and embryo culture fails to promote the developmental competence of porcine embryos

The coenzyme Q10 (CoQ10) is a potent antioxidant with critical protection role against cell oxidative stress, caused by the mitochondrial dysfunction. This study evaluated the effects of CoQ10 supplementation to in vitro maturation (IVM) or embryo culture media on the maturation, fertilization and subsequent embryonic development of pig oocytes and embryos. Maturation (Experiment 1) or embryo culture (Experiment 2) media were supplemented with 0 (control), 10, 25, 50 and 100 μM CoQ10. The addition of 10–50 μM CoQ10 to the IVM medium did not affect the percentage of MII oocytes nor the fertilization or the parameters of subsequent embryonic development. Exogenous CoQ10 in the culture medium neither did affect the development to the 2–4‐cell stage nor rates of blastocyst formation. Moreover, the highest concentration of CoQ10 (100 μM) in the maturation medium negatively affected blastocyst rates. In conclusion, exogenous CoQ10 supplementation of maturation or embryo culture media failed to improve the outcomes of our in vitro embryo production system and its use as an exogenous antioxidant should not be encouraged.     

激素和血清对牛卵母细胞体外成熟的影响

卵母细胞的体外成熟直接关系到体外受精胚胎的数量和质量,从而影响到胚胎移植的成功率。本文就促性腺激素、类固醇激素和血清对牛卵母细胞体外成熟的影响,进行了较为详尽的阐述。     

Supplementation with sunflower seeds in beef cattle did not impact on oocyte and in vitro embryo production

Supplementation with compounds rich in linoleic acid, including sunflower seed supplementation, promotes increase in conception rates in cows. We aimed to evaluate whether the sunflower seed (linoleic acid source) supplementation in beef donor females alters the plasma concentrations of cholesterol, triglycerides, HDL and LDL, increases the number and quality of oocytes, increases the cleavage rates and determines an improvement in number and quality of in vitro produced blastocysts. Thus, Nelore females were divided into two groups of 15 animals to receive supplementation with or without sunflower seed for 57 days. Females underwent follicular aspiration and the oocytes were subjected to in vitro embryo production. There was no difference (p > .1) between control group and group supplemented with sunflower seed on the number of displayed follicles; number of aspired oocytes; recovery rate; cleavage rate; number of embryos; number of blastocysts; embryos number of grades I and II; plasma concentrations of total cholesterol, triglycerides; HDL and LDL. Therefore, sunflower seed supplementation in oocyte donors did not increase the number and quality of oocytes, cleavage rates and the number and quality of blastocysts produced in vitro.     

季节对延边黄牛卵母细胞质量及体细胞核移植胚胎发育的影响

研究季节对延边黄牛卵巢质量和卵母细胞的数量、质量、体外成熟以及核移植重组胚发育的影响。持续1年采集延边黄牛卵巢,分级卵巢,抽取卵巢表面直径2～8mm的卵泡内的卵母细胞,收集后进行体外成熟和重组胚的体外培养。结果表明,1级卵巢的比例在春冬季时明显高于秋季（P〈0．05）,2级卵巢的比例在春夏季高于冬季（P〈0．05）,秋季的3级卵巢比例最高,与其他季节有显著差异（P〈0．05）;在秋冬季,平均每个卵巢拣出卵母细胞数高于春夏季（P〈0．05）,成熟率上,秋季显著高于春夏季（P〈0．05）,但与冬季并无差异且其他各组间无显著差异（P〉0．05）;卵裂率上,秋季与春季有差异（P〈0．05）,但与其他季节无显著差异（P〉0．05）,囊胚率上,各组之间均无显著差异（P〉0．05）。通过研究发现,季节对延边黄牛卵母细胞的可用回收数量及其质量都会产生影响且秋季为延边黄牛体细胞核移植实验最适合季节。     

Optimization of recovery and maturation methods for cumulus-oocyte complexes in jennies

Embryo production in donkeys is inefficient compared with that in other livestock. Obtaining a sufficient number of MII oocytes is the first step to solving this problem. In this study, the number, morphology and maturation rates of cumulus-oocyte complexes (COCs) obtained from abattoir-derived ovaries or live jennies were compared. The diameter of follicles from abattoir-derived ovaries was measured and divided into group 1 (2–6 mm), group 2 (6–10 mm), group 3 (10–20 mm), group 4 (20–28 mm) and group 5 (>28 mm). The results showed that the number of follicles per ovary in group 2 (3.6 ± 0.28) and 3 (4.2 ± 0.90) was higher than that in the other groups (p < .05). The recovery rate in group 3 was higher than group 1 (48.8% vs. 26.8%, p = .00), but lower than group 5 (48.8% vs. 76.5%, p = .025). The percentage of grade A COCs in group 3 was higher than group 2 (59.3% vs. 39.5%, p = .00) and group 1 (59.3% vs. 26.7%, p = .00). Moreover, the percentage of grade A COCs in group 4 (55.0%, p = .710) and group 5 (46.2%, p = .351) was reduced compared with that in group 3. From the above results, the developing follicles (group ovum pick-up [OPU], 10–20 mm) and preovulation follicles (group OPU-Preov, >35 mm) were aspirated from live jennies using OPU. Although there was no difference in the recovery rates of COCs between group 3 and OPU (48.8% vs. 43.0%, p = .184), the percentage of grades A COCs in group OPU was higher than group 3 (72.5% vs. 59.3%, p = .036). There was no difference in the maturation rate between group 3 and OPU (60.3% vs. 69.3%, p = .171) after the COCs matured in vitro. The rates of recovery (72.2%) and maturation (92.3%) in group OPU-Preov were higher than those in other groups (p < .05). Moreover, the effects of maturation time and serum type on maturation rates were evaluated in groups B44 (44 h, FBS), B36 (36 h, FBS) and D44 (44 h, foetal donkey serum, FDS). These results indicated that the maturation rate in group B36 was lower than group B44 (13.1% vs. 47.0%, p = .00) and group D44 (13.1% vs. 53.3%, p = .00). In conclusion, the quality of donkey COCs from OPU was higher than that from abattoir-derived ovaries, the suitable time of donkey in vitro maturation (IVM) was 44 h, and FBS could be replaced with FDS in donkey IVM medium.     

密闭气袋在牛卵母细胞体外成熟培养中的应用

通过简易避光密闭气袋在人体呼出的气相环境下（CO2浓度约5%,O2浓度约15%）,利用生化培养箱进行牛卵母细胞的体外成熟培养。结果显示,牛卵母细胞在密闭气袋中培养与对照组（正常培养）相比成熟率（74.79%比71.69%,P〉0.05）、孤雌激活后卵裂率（83.07%比81.39%,P〉0.05）和囊胚发育率上（26.75%比22.87%,P〉0.05）均无显著差异,但是密闭气袋培养各项数据均略高于对照组。本试验验证了该技术的方便性和有效性,为此技术在生产实践中的推广应用提供依据。     

The use of human and bovine commercial media for oocyte maturation and embryo development in the domestic cat (Felis catus)

The aim of this study was to examine the suitability of commercial media designed for humans and cattle for oocyte maturation and embryo culture in the domestic cat. In Exp. I, feline oocytes collected ex vivo were subjected to in vitro maturation in a laboratory‐made culture medium (based on M199) or a commercial medium designed for cattle cells (BO‐IVM^®). In Exp. II, ICSI‐derived feline embryos were cultured for 7 days in a commercial human (Continuous Single Culture^®) or bovine (BO‐EC^®) cell medium. The rates of cleavage, morula and blastocyst formation were evaluated at 24 hr, 6 days and 7 days after ICSI, respectively, and compared between experimental groups. At the end of culture, embryos were assessed for viability and apoptotic changes. In Exp. I, no statistically significant difference in oocyte maturation outcome between laboratory‐made (52.7%) and commercial media (58.9%) was observed. However, the use of a commercial medium prepared for use with bovine cells resulted in a significantly lower variance of the maturation rate. In Exp. II, no statistically significant differences between two commercial media were observed for cleavage (67.5% and 64.5%), morula (39.3% and 47.1%) and blastocyst rates (25.0% and 19.6%), as well as for the percentage of late apoptotic blastomeres. Morulae cultured in medium marketed for humans exhibited significantly more early apoptotic (43.2 ± 31.2% vs. 23.4 ± 23.2%) and necrotic (60.6 ± 47.6% vs. 29.4 ± 22.6%) blastomeres. In conclusion, both commercial media tested are suitable for in vitro oocyte maturation and embryo culture procedures in cats. It is remarkable that a culture medium designed for use in cattle for in vitro maturation of cat oocytes provides more reproducible results.     

Evaluation of smallholder pig production systems in North Vietnam: Pig production management and pig performances

This study investigated the productive adaptability of pig breeds under different smallholder production conditions in Vietnam, comparing an indigenous with a Vietnamese improved breed. Fieldwork was conducted in four villages with different remoteness in North Vietnam from 2001 to 2002, in 64 households keeping the improved Mong Cai or indigenous Ban as sow breeds and progeny for fattening. Four visits per farm yielded 234 structured interviews. Reproductive performances were derived from 135 litters. Individual weights of pigs (n = 755) were obtained. Data were analysed by regression, linear and generalised linear, especially loglinear models.

Two distinct pig production systems were identified, that differed in remoteness, market access, resource availability, distribution of pig breeds and pig production intensity. Higher performances of 1.4 and 1.5 litters year^− 1, 8.4 and 8.4 piglets weaned litter^− 1, 66.6 and 93.0 kg piglets weaned sow^− 1 year^− 1, and 136 and 177 g day^− 1 ADG were found in two villages near town with semi-intensive production conditions and a high rate of improved Mong Cai sows and MC and LW × MC offspring in the observed population. Lower performances of 1.1 and 1.1 litters year^− 1, 4.6 and 5.5 piglets weaned litter^− 1, 20.5 and 30.9 kg piglets weaned sow^− 1 year^− 1, and 66 and 85 g day^− 1 ADG were observed in parallel to higher incidences of indigenous Ban pigs away from town under extensive conditions. Total live weight offtake per household per year was higher near town and in one village distant to town. MC and LW × MC remained even under demand-driven conditions below the performance potential reported for improved genotypes. Under resource-driven conditions with a saving-oriented production pattern, the higher-yielding but more demanding Mong Cai might not be an efficient production alternative as a further performance reduction can be assumed. A considerable live weight output was observed under resource-driven conditions and with a higher percentage of crossbred LW × Ban offspring in the population, possibly representing a promising production alternative. Factors influencing the performance expression of pigs, and the suitability of different methods for the assessment of performance parameters on-farm are discussed.     

Effects of complement component 3 derivatives on pig oocyte maturation, fertilization and early embryo development in vitro

Complement component 3 (C3) has well-established roles within immune system, but its roles outside of immune system are less characterized. The extensive presence of C3 throughout the female reproductive tract, and its temporal, and gamete-specific regulation of expression suggest a potential role for C3 in reproduction. In the present investigation, the effects of C3, C3b and iC3b on porcine oocyte maturation, fertilization and embryonic development were examined. We identified the ability of iC3b to positively influence oocyte maturation. No effects on fertilization efficiency, penetration rates, polyspermy and blastocyst formation were observed. However, C3, C3b and iC3b presence in embryo culture medium resulted in fewer total cells in test blastocysts compared to control blastocysts. The results of this study indicate a potential function for iC3b in oocyte maturation. Furthermore, it was demonstrated that the presence of either C3, C3b or iC3b has a negative influence on early embryonic development in the porcine species.