Laboratory Production of Equine Embryos

Assisted reproduction technologies (ART) are well developed in humans and cattle and are gaining momentum also in the equine industry because of the fact that the mare does not respond to superovulation but can donate large numbers of oocytes through ovum pick up (OPU). After collection, the oocytes can be fertilized by intracytoplasmic sperm injection (ICSI) using a variety of stallion semen samples, even of poor quality, and the resulting embryos can establish high pregnancy rates after cryopreservation and transfer. The discoveries that equine oocytes can be held at room temperature without loss of viability and that an increase in vitro maturation time can double the number of embryos produced are fueling the uptake of the OPU technique by several clinics that are shipping oocytes of their client’s mares to specialized ICSI laboratories for embryo production and freezing. In this article, we present a retrospective analysis of 10 years of work at Avantea with a special focus on the last 3 years. Based on our data, an average production of 1.7 to 2 embryos per OPU-ICSI procedure can be obtained from warmblood donor mares with a pregnancy rate of 70% and a foaling rate in excess of 50%. OPU-ICSI offers the added value of freezing embryos that allows the development of embryo commercialization worldwide to the benefit of top horse breeders who are endorsing this technology as never before.     

In Vitro Production of Equine Embryos

The development of methods to produce embryos in vitro in the horse has been delayed compared with other domestic species. Oocytes can be collected from excised ovaries or from the small or preovulatory follicles of live mares. Intracytoplasmic sperm injection is the only reliable method to fertilize equine oocytes in vitro. Intracytoplasmic sperm injection-produced embryos can be transferred into the oviducts of recipient mares or cultured to the morula or blastocyst stage of development for nonsurgical embryo transfers into recipients' uteri. Embryos cultured in vitro have some morphological differences compared with embryos collected from the mares' uteri. Most notably, the embryonic capsule does not form in culture, and the zona pellucida fails to expand completely. However, embryo produced in vitro can result in viable pregnancies and healthy offspring.     

Improved embryo development in Japanese black cattle by in vitro fertilization using ovum pick-up plus intracytoplasmic sperm injection with dithiothreitol

The purpose of this study was to determine whether dithiothreitol (DTT) treatment of sperm and ethanol activation improve embryo production by intracytoplasmic sperm injection (ICSI). Further, we compared ICSI with standard in vitro fertilization (IVF) in oocytes obtained from cattle. We demonstrated that DTT reduced the disulfide bond in the bovine sperm head. Using oocytes obtained from a slaughterhouse, ICSI-DTT treatment without ethanol showed the highest rate of blastocyst formation. We applied these results to fertilization using ovum pick-up (OPU). Eleven Japanese black cattle served as donors for OPU plus standard IVF (OPU-IVF). Of them, four donors with low embryo development rates were selected to determine whether embryo development was enhanced by OPU plus ICSI (OPU-ICSI). We assessed effects on embryo development following IVF and ICSI in oocytes obtained using OPU. Blastocyst rates were significantly higher for OPU-ICSI than for OPU-IVF. Our results suggest that OPU-ICSI improves the blastocyst development rate in donors with low embryo production compared with the standard OPU-IVF.     

Current Reproductive Technologies Impacting Equine Embryo Production

Numerous reproductive technologies have been developed in the past several decades, which have dramatically changed the way mares are bred. This review will focus on embryo recovery and transfer, cooled-shipped embryos, embryo freezing, oocyte freezing, oocyte collection and transfer, intracytoplasmic sperm injection (ICSI), and sexed semen. Embryo transfer procedures have been constant for many years and the costs have not changed. The major change has been the ability to store embryos at 5 C for 12–24 hours and transport them to recipient stations. Embryo freezing has become more common using the technique of vitrification of embryos >300 μm or deflating embryos >300 μm before freezing. Oocyte vitrification has resulted in poor pregnancy rates although the technique works well in women. The ability to collect oocytes from mares and fertilize them by sperm injection has revolutionized the veterinarian’s approach to infertility in the mare and/or stallion. A transvaginal approach can be used to collect oocytes from preovulatory follicles and unstimulated follicles 5–25 mm in size. Although traditional in vitro fertilization does not work well in the horse, ICSI can be used to produce blastocysts which, upon nonsurgical transfer into recipients, provide a pregnancy rate similar to fresh embryos collected from donor mares. Sorting sperm by flow cytometry into X- and Y-bearing spermatozoa has been shown to provide about a 50% pregnancy rate with freshly sorted sperm but only 12% with sorted, frozen/thawed stallion sperm. It is likely that more advanced reproductive techniques will be developed in the future. Their acceptance will depend on how well they work, perceived need, cost, and, to some extent, the breed associations.     

Effect of Ca Ionophore On Blastocyst Production Following Intracytoplasmic Sperm Injection in Caprine Oocytes

The aim of the present investigation was to study the effect of calcium ionophore activation on blastocyst production following intracytoplasmic sperm injection (ICSI) in in vitro‐matured Caprine oocytes. A total of 470 in vitro‐matured oocytes were selected and randomly divided in to three groups. Cumulus oocyte complexes (COCs) recovered by slicing the Caprine ovaries were matured in TCM199 supplemented with 10% foetal bovine serum (FBS) + 10% follicular fluid + FSH (5 μg/ml) + LH (10 μg/ml) + estradiol (1 μg/ml) + EGF (10 ng/ml) + BSA (3 mg/ml) for 27 h in humidified atmosphere at 38.5°C with 5% CO₂ in CO₂ incubator. After 27 h of culture, selected COCs (n = 470) were separated from cumulus cells by treating with 0.1% hyaluronidase enzyme and passing repeatedly through a fine pipette and randomly divided into three groups. In group 1, (n = 168) matured oocytes were injected with injection micropipette without sperm as control. In group 2, (n = 152) capacitated spermatozoa were injected into cytoplasm of in vitro‐matured oocytes through injection micropipette. In group 3, (n = 150) capacitated spermatozoa were injected into cytoplasm of in vitro‐matured oocytes through injection micropipette and then activated with 5 μm Ca ionophore for 5 min. The oocytes of all groups were then culture in RVCL media for embryo development. The cleavage rate was observed after 48–72 h of injection. The cleavage rate and blastocyst production in group 1, 2 and 3 were 0.00 and 0.00, 18.42 and 3.57 and 61.33% and 16.30%, respectively. The result indicated that mechanical activation failed to induce cleavage in in vitro‐matured Caprine oocytes, whereas chemical activation of intracytoplasmic sperm‐injected in vitro‐matured Caprine oocytes showed significantly higher cleavage rate and blastocyst production as compare to non‐activated oocytes.     

Seasonality and photoperiod influence in vitro production of buffalo embryos

Buffalo is considered short-day breeder in tropical and subtropical part of the world and seasonality and photoperiodism impart major influence on its fertility. However, its impact on in vitro embryo production (IVEP) remains elusive. Therefore, this study investigated the effect of seasonal variations and photoperiodism on morphological and molecular parameters of IVEP in buffalo. For this purpose, we conducted two different experiments on the oocytes obtained by aspirating follicles from abattoir derived ovaries. In Exp. I, retrospective analysis was performed for oocyte recovery, blastocyst and hatching rate, during four consecutive seasonal periods (i.e. January–March, April–June, July–September and October–December). In Exp. II, oocytes from peak breeding and non-breeding seasons were subjected to 24 hr in vitro maturation and evaluated for polar body extrusion to assess maturation rate. Results showed that embryo development was markedly low during second quarter (April–June) and maximum during fourth quarter (October–December) of the year; referred as non-breeding and breeding seasons, respectively. Comparative data analysis demonstrated that poor oocyte quality is major reason for lesser efficiency of embryo production during non-breeding season than peak breeding season as suggested by poor oocyte recovery (2.31 ± 0.10 vs. 3.65 ± 0.27) and maturation rate (33.32 ± 2.1 vs. 63.15 ± 7.31). Subsequently, comparative gene expression analysis of blastocysts during peak breeding season significantly upregulated pluripotency gene (OCT-4) and downregulated heat shock protein 90, as compared to non-breeding season. Therefore, it could be divulged from the present study that seasonal variations and photoperiodism have profound effect on oocyte quality and subsequent embryo development. It is recommended to find suitable additives for in vitro maturation that could mitigate seasonal effects.     

Advances in Holding and Cryopreservation of Equine Oocytes and Embryos

Methods for holding of oocytes and embryos during shipment as well as for their cryopreservation can greatly aid equine reproductive management. Oocytes can be held at room temperature overnight or at cooler temperatures for two nights without affecting maturation or embryo development after intracytoplasmic sperm injection. In contrast, methods for cryopreservation of equine oocytes that support high rates of embryo development have not yet been established. Equine embryos may be held overnight at temperatures from 5°C to 19°C without reduction in viability, but longer holding periods, or higher holding temperatures, may be detrimental. Small equine embryos (<300 μm), either in vivo derived or in vitro produced, can be slow frozen or vitrified successfully. In the last decade, methods have been developed to allow in vivo–derived expanded blastocysts, up to Day 8, to be vitrified successfully after blastocoele collapse. These methods of shipment and preservation allow mare owners in remote locations to have access to sophisticated assisted reproductive technologies.     

Optimization of in vitro embryo production and zygote vitrification for the indigenous Vietnamese Ban pig: The effects of different in vitro oocyte maturation systems

The Vietnamese Ban pig is a precious genetic resource that needs to be preserved. In vitro embryo production from in vitro matured (IVM) oocytes is an important tool for the utilization of cryopreserved porcine sperm. The aim of this study was to compare two media for the IVM of Ban pig oocytes. Immature oocytes were subjected to IVM either in a non‐defined (TCM‐199 + pig follicular fluid) or in a defined base medium (POM + epidermal growth factor). At the end of IVM, the oocytes were in vitro fertilized (IVF) with frozen Ban sperm. Ten hours after IVF, the oocytes were either subjected to orcein staining to check fertilization and maturation status or cultured in vitro for 7 days. There was no difference between the two IVM media in terms of percentages of oocyte maturation and blastocyst production. However, the percentage of male pronuclear formation after IVF and the total cell numbers in blastocysts were higher with the defined system. Zygotes obtained by the two IVM systems survived vitrification at similar rates. In conclusion, the two IVM systems were both effective for the production of Ban pig embryos; however, better embryo quality was achieved with the defined one.     

Effect of Mineral Block Supplementation on In Vivo Digestibility and In Vitro Gas Production With Equine Fecal Bacteria

The effects of a mineral block for horses on in vivo digestibility and in vitro fermentability with equine fecal inoculum were evaluated. Fifty healthy horses from three groups (lactating mares n = 19, working horses n = 18, and maintenance horses n = 13) were randomly assigned to two treatment groups (with or without the mineral block; Ca 10.0%, P 12.0%, Zn 12.1 mg/kg, Cu 2,050 mg/kg, Mn 4,050 mg/kg, Se 30 mg/kg, and I 105 mg/kg). Dry matter digestibility was estimated with an internal marker. Samples of diet were incubated with equine fecal bacteria with varying amounts of mineral block (0, 1.1, 3.6, and 6.2 mg/g dry matter [DM]) to record gas production and to estimate in vitro DM digestibility. The results showed that mineral supplementation with the blocks increased in vivo DM digestibility (P < .01) in all groups, but there was an interaction (P < .01) with a greater response in the maintenance horses (55.5% vs. 78.0%) compared to lactating mares (62.8% vs. 79.6%) and working (70.3% vs. 75.1%). Block consumption was lowest in the lactating mares (12.8 g/d), intermediate in the working horses (44.6 g/d), and highest in the maintenance horses (74.2 g/d). The mineral supplementation did not affect the kinetics of gas production but tended (P = .10) to improve the in vitro DM digestibility (37.01% vs. 38.34%). Mineral block supplementation increased dry matter digestibility in horses. The unsupplemented control diet was deficient in several minerals, and block intake was not proportional to the mineral requirements.     

In vitro Developmental Competence of Adult Sheep Oocytes Treated with Roscovitine

The efficiency of in vitro sheep embryo production is still low compared to that observed in vivo and in other species. In this context, meiotic inhibition strategies emerged as a promising alternative to improve this biotechnology. So, this study aimed to evaluate, for the first time, the effects of roscovitine on in vitro maturation of sheep oocytes and their subsequent embryo development. For this, cumulus–oocyte complexes (COCs) were cultured for 6 h in the presence (Rosco) or absence (Control) of 75 μm roscovitine and, subsequently, in vitro matured (IVM) for 18 h with gonadotropins. At 0 (Immature), 6 and 24 h of culture, the nuclear status of oocytes was evaluated by Hoechst staining. Embryo cleavage and blastocyst formation were recorded 30 h after in vitro fertilization and on day 7 of culture, respectively. Blastocyst quality was evaluated by differential staining. At 6 h, the GV rate in the Rosco treatment (93.8%) was similar to that observed in the Immature oocytes (94.9%) and significantly higher compared to Control (41.3%). After IVM for 18 h, a high and similar proportion of oocytes from Rosco (93.6%) and Control (88.4%) reached the MII stage. In both treatments, approximately 70% of oocytes cleaved and 50% of them developed up to blastocyst. The mean percentage of blastocyst cells, embryoblast, trophoblast and pyknosis did also not differ between Control and Rosco. In conclusion, roscovitine, at the studied experimental conditions, was efficient to reversibly inhibit the meiosis of adult sheep oocytes without detrimental effect on development and quality of the in vitro produced embryos.     

Effect of vitrification procedures on the subsequent development of in vitro matured swamp buffalo oocytes following in vitro fertilization

Nowadays, the efficiency of buffalo oocytes cryopreservation is still low. The purpose of this study was to evaluate effects of two combinations of cryoprotectant agents (CPAs) and two vitrification devices for vitrification of swamp buffalo oocytes on their survival after vitrification warming, and subsequent developmental ability after in vitro fertilization. In vitro matured (IVM) oocytes were vitrified by either Cryotop (CT) or solid surface vitrification (SSV) interacting with vitrification solution A (VA) or B (VB). In the VA or VB solution exposed test, the oocytes showed similar survival rates, but decreased blastocyst rates after in vitro fertilization compared with that of untreated oocytes. After vitrification, the CT method combined with VA solution yielded a higher survival rate (91.3 ± 5.84%) of vitrified oocytes than that combined with VB solution (69.8 ± 4.19%–75.8 ± 4.55%); however, all the vitrification treatments showed lower blastocyst rates (1.1 ± 0.07%–5.2 ± 0.24%) compared with that of untreated oocytes (18.0 ± 1.09%). Our results indicated that combined vitrification treatments in this study did not improve the decreased ability of vitrified oocytes developing to the blastocyst stage.     

影响猪ICSI转基因技术效率的主要因素研究

以猪体外成熟卵子和冷冻解冻的死精子为材料,以pEGFP-N1为模式基因,探讨注射台温度、激活后6-DMAP的处理和精子与PEGFP-N1孵育液添加BSA(牛血清白蛋白)对精子胞质内注射(ICSI)转基因效率的影响。结果表明:注射台温度为30℃时的阳性率为40.07%,而38.5℃时为20.97%,差异极显著(P<0.01)。添加BSA的囊胚转基因率为55.56%,对照组为33.33%,差异极显著(P<0.01)。6-DMAP处理组与对照组的转基因率分别为52.53%和26.25%,差异极显著(P<0.01);而且6-DMAP处理组的囊胚率(9.96%)显著高于(P<0.05)对照组(2.30%)。研究表明注射台温度对转基因效率有明显影响,温度高转基因率低;精子与PEGFP-N1孵育液添加BSA对转基因胚胎发育有一定促进和保护作用,有利于提高囊胚转基因率;激活后用6-DMAP处理能提高转基因率和囊胚率。     

玻璃化冷冻牛卵母细胞单精子注射后体外发育能力的研究

实验研究了不同成熟培养时间的牛卵母细胞玻璃化冷冻及胞质内单精子注射(ICSI)后的受精效果。结果表明:成熟后的新鲜牛卵母细胞按照ICSI注射方法穿刺而不注射精子组与未经穿刺的对照组相比,孤雌激活后的卵裂率、囊胚发育率及囊胚细胞数无显著差异(P>0.05);成熟培养16h(MⅠ)和23h(MⅡ)卵母细胞冷冻解冻后形态正常率均显著低于新鲜对照组(76.66%、87.33%vs100.0%)(P<0.05),冷冻解冻后二者分别成熟培养至24h,ICSI后胚胎的囊胚发育率(5.29%、14.41%)显著低于新鲜对照组(24.40%)(P<0.05);成熟培养23h与成熟培养16h的卵母细胞冷冻解冻后形态正常率及ICSI后囊胚发育率(14.41%vs5.29%)均有显著性差异(P<0.05)。实验证明,ICSI操作不会影响卵母细胞发育潜力;玻璃化冷冻影响卵母细胞解冻后形态正常率以及ICSI后胚胎的发育能力;成熟培养23h比16h的卵母细胞冷冻保存后经ICSI的胚胎发育潜力高。     

Live foals produced from sperm-injected oocytes derived from pregnant mares

Recently, in vitro fertilization (IVF) in the horse has met with less than anticipated results. Various problems associated with equine IVF include: (1) the inability to collect large numbers of good quality oocytes, (2) the alteration of the zona pellucida associated with in vitro maturation of equine oocytes, and (3) the improper preparation of equine sperm cells for IVF of these oocytes. Therefore, this study was conducted to achieve fertilization via sperm injection of equine oocytes and to produce live offspring from this IVF procedure. Oocytes were collected by transvaginal ultrasound-guided oocyte retrieval procedures from early pregnant mares of mixed breeds (day 14 to day 70 of pregnancy) and were matured in vitro and subjected to intracytoplasmic sperm injection (ICSI). Injected oocytes were then cultured for 48 hours in either TCM-199 or P-1 medium (glucose and phosphate-free medium) supplemented with 15% fetal bovine serum. Cleavage rates for embryos cultured in the two culture media were different (47% vs. 63% in TCM-199 and P-1, respectively). Also, four Grade 1 embryos were surgically transferred into the oviducts of four recipient mares (one embryo/mare) at 48 hours post-ICSI, with three pregnancies (75%) developing as ultrasonically demonstrated by the presence of an embryonic vesicle in the uterine body by day 16 post-ICSI. On June 23rd one live filly was born after 328 days of gestation and subsequently, a second healthy filly was born after 319 days of gestation. To our knowledge, this is the first report of live foals resulting from in vitro fertilization (via ICSI) of in vitro matured oocytes recovered from pregnant mares using an efficient, repeatable transvaginal ultrasound-guided procedure.     

Beta‐mercaptoethanol supplementation of in vitro maturation medium does not influence nuclear and cytoplasmic maturation of equine oocytes

In vitro embryo production in the horse is still not as efficient as in other species. Oxidative stress negatively affects oocyte and embryo culture. To attenuate/minimize the oxidative stress, antioxidants such as low‐molecular thiol compounds can be added to culture media. Beta‐mercaptoethanol (BME) has been shown to improve maturation and embryo development in different species. The aim of this study was to investigate whether the addition to maturation medium of BME at common (0.1 mM) and high (0.7 mM) concentration could improve oocyte maturation also in the horse. Equine oocytes recovered from slaughterhouse ovaries were used. Meiotic configuration after in vitro maturation (IVM) and early embryo production after intracytoplasmic sperm injection (ICSI) were considered as criteria for assessing nuclear and cytoplasmic maturation, respectively. A total of 1,076 oocytes were analysed over two experiments: 848 (control n = 293, BME 0.1 n = 270, BME 0.7 n = 285) were stained with Hoechst 33342 and examined for nuclear stage after 26 hr of IVM, and 228 MII oocytes were fertilized by ICSI (control n = 83, BME 0.1 n = 65, BME 0.7 n = 80). Cleavage rates were determined after 60 hr of culture. Unlike results obtained in other species, the addition of BME did not influence maturation rates (51.9% control vs 55.6% BME 0.1 mM and 55.1% BME 0.7 mM), nor cleavage rates after ICSI (38.6% vs 38.5% and 41.3%, respectively). In conclusion, the addition of BME at 0.1 and 0.7 mM to the maturation medium, in our culture conditions, has no effect on nuclear and cytoplasmic maturation of equine oocytes.     

In vitro production of porcine zygotes using intracytoplasmic injection of vitrified sperm

The objectives of this study were to evaluate if vitrified porcine spermatozoa are able to maintain their capacity to produce zygotes in vitro using intracytoplasmic sperm injection (ICSI) and to evaluate the zygote development in two in vitro atmospheric conditions: 5% CO₂ and tri‐gas. A group of porcine oocytes maturated in vitro were injected with vitrified‐warmed sperm (treatment group) and another group, with sperm diluted and conserved at 17°C (control group). To evidence parthenogenetic activation, some oocytes were submitted to a Sham test. The injected oocytes were cultured in G1 medium at 38°C, 100% humidity and 5% CO₂ or tri‐gas. No significant differences (p > .05) were observed in embryo development between the oocytes injected with vitrified‐warmed sperm (31.8%; 36/113), and those injected with semen diluted and conserved at 17°C (35.5%; 32/90), when cultured in 5% CO₂ or under tri‐gas atmosphere (42.9%; 39/91 vs. 34.2%; 26/76, respectively). No significant differences (p > .05) were observed in the percentage of pronuclei (PN) obtained between 5% CO₂ and tri‐gas, within each treatment either. Of the 52 oocytes submitted to the Sham test, only two presented a female PN (activation) indicating that the PN observed in the treatment group were a product of fertilization and not parthenogenetic activation. To conclude, porcine sperm vitrified using spheres, at a concentration of 5 × 10⁶ spermatozoa/ml in TALP medium with 1% bovine serum albumin (BSA), conserve condensed and intact chromatin capable of producing early embryo development up to the pronuclear stage.     

Vitrification of bovine matured oocytes and blastocysts in a paper container

In the present study, we aimed to determine the applicability of a paper container for the vitrification of in vitro matured (IVM) bovine oocytes. In experiment 1, IVM oocytes were exposed to vitrification solution (20% dimethylsulfoxide (DMSO), 20% ethylene glycol (EG), and 5 mol/L sucrose), using a two‐step method, for 30 s; loaded onto either a paper container or Cryotop; and stored in liquid nitrogen. No significant difference (P < 0.05) in the survival and blastocyst formation rates after in vitro vitrification was observed between the paper container and Cryotop. In experiment 2, IVM oocytes were exposed to either a two‐ or three‐step vitrification solution. The three‐step vitrification solution was not significantly different from the two‐step solution in terms of oocyte survival, cleavage and blastocyst rates. In experiment 3, in vitro produced blastocysts were graded according to the manual of the International Embryo Transfer Society (grades 1 and 2) and vitrified using the two‐ and three‐step methods. For grade 2 blastocysts, the three‐step method showed significantly higher (P < 0.05) survival and hatched blastocyst rates than the two‐step method, whereas for grade 1 blastocysts, no significant difference was observed. In conclusion, the paper device and three‐step technique are suitable for oocytes and embryo vitrification.     

Laminin‐111 Inhibits Bovine Fertilization but Improves Embryonic Development in vitro,and Receptor Integrin‐β1 is Involved in Sperm–Oocyte Binding

This study detected the distribution of laminin during embryonic formation by immunofluorescence. To determine the possible function of laminin on developmental ability of in vitro fertilized embryos, the presumptive zygotes were divided and transferred to CR1aa medium supplemented with different concentrations (0 μg/ml, 5 μg/ml, 10 μg/ml and 20 μg/ml) of laminin. To explore the association with sperm–oocyte fusion, oocytes and/or sperm were pre‐incubated with laminin or anti‐β1 antibody before insemination. Laminin was absent in mature oocytes and could be detected first at the 8‐cell stage and then displayed an increasing tendency. Adding 10 μg/ml laminin to the culture medium improved embryonic development including cleavage rate, blastocyst rate, total cell numbers in the blastocyst and cell numbers in the inner cell mass. Laminin inhibited sperm–oocyte fusion when incubated with oocytes and/or sperm before in vitro fertilization, and only integrin‐β1 of sperm was involved in sperm–oocyte binding. Inhibition may be caused by blocking β1, but why laminin inhibits fertilization is still unknown. The results suggest that laminin plays an important role during embryonic formation and has a negative function in sperm–oocyte fusion, but improves embryonic development. However, only integrin‐β1 is involved in sperm–oocyte binding.     

Cryopreservation of immature oocytes of the indigeneous Vietnamese Ban Pig

We report the cryopreservation of oocytes from Ban miniature pigs which are endemic in Vietnam. Immature cumulus‐oocyte complexes were collected from antral follicles of 7–8 mo old female cyclic Ban pigs and vitrified in micro‐drops. Oocyte morphology, lipid content, post‐warming survival, nuclear maturation, and embryo development were compared to those of oocytes from commercially slaughtered Landrace × Large white hybrid pigs. The size of oocytes in the two breeds was similar. However, significantly lower amounts of intracellular lipid were detected in Ban oocytes. There was no difference (p > 0.05) between Ban and Landrace × Large white oocytes in percentages of post‐warming survival (93.1 ± 3.4% vs. 70.7 ± 16.7%, respectively) and nuclear maturation after in vitro maturation (80.4 ± 5.1% vs. 90.0 ± 1.3% respectively). Similarly, cleavage (30.8 ± 7.8% vs. 10.3 ± 6.1%, respectively) and blastocyst development rates (9.4 ± 5.0% vs. 0.79 ± 0.79, respectively) were not different (p > 0.05) between vitrified Ban and Landrace × Large white oocytes after in vitro fertilization and embryo culture. In conclusion, high survival and maturation rates were achieved after vitrification of immature Ban oocytes and their cryo‐tolerance was similar to that of Landrace × Large white oocytes, despite the difference in lipid content. We succeeded to generate reasonable rates of blastocysts from vitrified Ban oocytes by in vitro fertilization.     

Sucrose assists selection of high‐quality oocytes in pigs

We investigated whether high‐quality in vitro matured (IVM) oocytes can be distinguished from poor ones based on the morphological changes after treatment with hyperosmotic medium containing 0.2 mol/L sucrose in pigs. We hypothesize that IVM oocytes maintaining round shape have higher quality than mis‐shapened oocytes following dehydration. Oocyte quality was verified by determining embryonic developmental competence using in vitro fertilization, nuclear transfer and parthenogenetic activation. In all cases, the round oocytes had greater (p < .05) developmental competence than that of mis‐shapened oocytes in terms of blastocyst rate and total cell number in blastocysts obtained after 6 days of in vitro culture. We also confirm that round aged oocytes are higher in quality than mis‐shapened aged oocytes. In an attempt to find out why high‐quality oocytes maintain a round shape whereas poorer oocytes become mis‐shapened following sucrose treatment, we examined the arrangement of actin microfilaments and microtubules. Abnormal organization of these cytoskeletal components was higher (p < .05) in mis‐shapened oocytes compared to round oocytes after 52 hr of IVM. In conclusion, sucrose treatment helps selection of high‐quality oocytes, including aged oocytes, in pigs. Abnormal cytoskeleton arrangements partly explain for low developmental competence of mis‐shapened oocytes.