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Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos® and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization.

Mosquitoes (Diptera: Culicidae) of the Anopheles (Cellia) Myzomyia Series are important malaria vectors in Africa, India and Southeast Asia. Among 10 named species of Myzomyia known from the Oriental Region, seven form the An. minimus group. Even for expert taxonomists, the adults of these species remain difficult to identify morphologically. For technical staff of malaria control programmes, confusion may extend to misidentification of species that are not formally within the minimus group. For identification of specimens from Indochina (Cambodia, Laos, Vietnam), we describe a multiplex polymerase chain reaction (PCR) assay, based on rDNA internal transcribed spacer 2 (ITS2) sequences, that employs a cocktail of primers to identify An. minimus Theobald sibling species A and C (sensu; Green et al., 1990) and three other species in the An. minimus group (An. aconitus Donitz, An. pampanai Buttiker & Beales, An. varuna Iyengar), as well as An. jeyporiensis James, also belonging to the Myzomyia Series. As the test is DNA-based, it can be applied to all life stages of these mosquitoes for ecological investigations and vector incrimination studies. This PCR assay is simpler, quicker, cheaper and more readily interpreted than previous assays.

Reproductive activity in swamp buffalo is characterized by a clearly demonstrated anestrus season. The aim of the present study was to evaluate season effect on the oocyte collection, in vitro maturation, and somatic cell nuclear transfer. The ovaries collected from a slaughterhouse were divided into 3 groups according to the collection period: (1) G1: from January to April; G2: from May to August, which is characterized by higher climate temperature and low reproductive activity; and G3: from September to December. Cumulus–oocyte complexes (COCs) were aspirated from follicles 2-6 mm in diameter using an 18-gauge needle, washed in HEPES-buffered TCM-199 (Sigma-Aldrich, St Louis, MO, USA), and classified following 3 different quality levels: A (with 4–6 layers of cumulus cells), B (with 2–3 layers of cumulus cells), and C (few or without cumulus cells). The oocytes of A and B categories were used for IVM in maturation media currently used in cattle (TCM-199 medium + 10% fetal bovine serum) with an increase of FSH concentration (30 �g mL-1) and estradiol-17β (3 �g mL-1). Maturation was carried out at 39�C in a water-saturated incubator, under 5% CO2 for 22 h. The oocytes were observed for the cumulus expanding and the presence of polar body (PB). The oocytes with PB were used for further enucleation and cell nuclear transfer using buffalo quiescent fibroblast cells and the technique described previously (Nguyen et al. 2000 Theriogenology 53, 235). The percentages of intact and fused oocytes as well as reconstructed embryos developed to blastocyst stage were compared for the oocytes from G1 and G2. The results indicated that the average number of good quality COCs collected per ovary for the G1, G2, and G3 period were 6.00 � 4.08 (n = 426), 2.93 � 2.55 (n = 346), and 4.78 � 1.05 (n = 445), respectively. The percentages of A and B oocytes were 62.4% (1.58 � 0.51 vs. 2.17 � 1.54), 63.2% (0.90 � 0.32 vs. 0.95 � 0.50), and 54.7% (1.12 � 0.25 vs. 1.49 � 0.53), respectively; the maturation rate was 55.08%, 56.28%, and 52.16%, respectively. There were no significant differences between G1 and G2 in the percentage of intact and fused oocytes (93.7% and 59% for G1; 100% and 60% for G2, respectively), but the rate of embryos developed to blastocyst stage was higher for oocytes from G1 (18.5% vs. 10.2%). In conclusion, in swamp buffalo, the hot season affected significantly the number of oocytes collected per animal and the subsequent results of somatic cell nuclear transfer. The optimal period for working with buffalo oocyte is from January to April. This work was aupported by a grant from the Vietnam-Italy 3AB3 Project.

The Ban minipig is a local breed characterized by small ovaries with a scant number of follicles available for in vitro maturation (IVM). The combination of eCG and hCG has been used successfully to control estrus in pig breeding programs. In this paper we present the first results of IVF in this breed in comparison with 2 types of oocyte preparation: (1) from animals not receiving gonatotropin treatment (group 1, n = 9); and (2) from animals receiving an injection of mixed pregnant mare serum gonadotropin and hCG, 300 IU/animal, for 3 days before oocyte collection (group 2, n = 4). All animals were 1 to 3 years old and with body weights that varied from 8 to 12 kg. At the time of collection, the ovaries were observed for follicle development; the cumulus–oocyte complexes (COCs) were aspirated using a 18-gauge needle. COCs of categories A (with more than 4 layers of cumulus cells) and B (with 2 to 4 layers of cumulus cells) were collected and matured in vitro as described previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) at 39�C under 5% CO2 in air. Matured oocytes with expanding cumulus cells were inseminated using male Ban minipig epididymal semen frozen by the methods reported by Kikuchi et al. (1998 Theriogenology 50, 615–623). The frozen–thawed spermatozoa were pre-incubated for 1 h in modified medium-199 adjusted to pH 7.8 in the incubator at 37�C. The capacitated spermatozoa were diluted and added to drops of fertilization medium (Fig-FM; Suzuki et al. 2002 Int. J. Androl. 123, 135–142) containing oocytes; the final concentration of sperm was 106/mL. After 3 h of co-incubation, attached spermatozoa and cumulus cells were removed from oocytes and the oocytes were the cultured in vitro as described previously (Kikuchi et al. 2002). The results obtained from 4 replicates showed that the number of follicles with a diameter larger than 2 mm and the rates of oocytes categorized as A and B were significantly lower (P < 0.05; ANOVA test) in the nontreated animals (0.0 and 67.5%, respectively) than in the treated group (25.5 and 87.1%, respectively). The rates of oocytes with a clearly expanding cumulus obtained after IVM were 78.6 (n = 136) and 88.1% (n = 101) for groups 1 and 2, respectively. The rates of cleaved embryos and embryos developed to the compact morula stage were 47.2 and 9.1% (n = 39), respectively, for group 1; and 89.1 and 18.8% (n = 101), respectively, for group 2. In conclusion, gonadotropin treatment before the collection of oocytes is recommended for application of IVM–IVF to local Ban minipigs. This work was supported by a grant from the VAST-Japan Society for the Promotion of Science Project.

Production of cloned nonhuman primate embryos has been reported using mature oocytes obtained from donors treated in vivo with a high dose of recombinant human FSH (r-hFSH, 35 IU per day for 10 days). The disadvantages of this approach are the high cost of hormones and the need to use the oocytes shortly after collection. Our study aimed to investigate the possibility of using initial in vivo treatment with a reduced FSH dose followed by in vitro culture for long periods of up to 3 days to produce mature monkey oocytes as host ooplasts for somatic cell nuclear transfer (SCNT). Adult female long-tailed Macaque (Macaca fascicularis) monkeys were treated with r-hFSH (Serono, Aubonne, Switzerland, 35 IU per day, i.m.) either for 10 days with an injection of hCG (1000 IU, i/m) 34 h before oocyte collection (G.I) or with only r-hFSH for 7 days (G.II). Cumulus oocyte complexes (COCs) were collected by follicular aspiration and then cultured in TCM-199 medium (GIBCO) supplemented with estradiol-17β, FSH, LH, and 10% FCS at 39°C in an incubator with 5% CO2 in air. The maturation rate based on the level of cumulus expansion and the presence of the first polar body was recorded at the moment of collection and during 24 h, 48 h, and 72 h of in vitro maturation (IVM). For SCNT, the mature Metaphase II oocytes were separated from cumulus cells and selected for enucleation in the presence of cytochalasin B (Sigma, St. Louis, MO, USA). Skin fibroblasts obtained from adult monkeys were cultured in DMEM+ 10% FCS and induced to quiescence in DMEM 0% FCS 2 days before use. A single cell was transferred under the zona of each enucleated oocyte. Couplets were fused with two direct current (DC) pulses of 220 V/mm for 25 μs in Zimmerman medium. Fused oocytes were cultured in medium containing cyclohexamide for 6 h before placing them into monkey culture medium (Cook, Brisbane, Australia). The average number of oocytes collected per animal were 21.2 (n = 18) and 18.6 (n = 12) for the G.I and G.II treatments, respectively. For G.I, the rate of COCs with fully expanded cumulus was 42% at collection and was maximal (80%) at Day 1 of IVM. For G.II, fully expanded cumulus was not observed at the time of collection and during the first 2 days of IVM, but 75% of COCs had full cumulus expansion by Day 3 of IVM. The rates of intact and fused oocytes were 50.3% for G.I and 55.4% for G.II. From the fused oocytes, 67.8% and 64.4% developed to the 4- to 8-cell stages at Days 2–3 after nuclear transfer for G.I and G.II, respectively. From these data, it can be concluded that this approach can be applied to optimize production of mature oocytes for non-human primate SCNT and ART (assisted reproductive technologies) programs. This work was supported by AIRE-Development.

Increasing the diary population and milk production is a goal of many tropical developing countries. We report in this paper an attempt to develop a system of intercontinental shipping for transfer of fresh crossbred Bos taurus×Bos indicus IVF embryos into local Laisind (Bos indicus) recipients as a way to produce tropical dairy calves with highly improved milk productivity. The production of embryos was done at BOMED, Inc (Madison, WI, USA). Oocytes collected from ovaries of Holstein (Bos taurus) at slaughter and semen from milking Gir (Bos indicus) were used for IVP. Cleaved embryos were selected for air shipping in portable incubators at Day 4 (Group 1), Day 3 (Group 2) or Day 2 (Group 3) after IVF. The duration of shipping varied from 60 to 65h. Embryo transfer was done in Vietnam. Laisind cows (Yellow cattle×Red Sindhi) with body weight more then 280kg and normal reproductive activity were selected for treatment of estrous synchronization with double 11-day interval injection of PG2α (Intervet, Boxmeer, The Netherlands) and single injection of eCG (SABC Vietnam) two days before the second injection of PG2α. Timing of injections was calculated according to the IVF schedule. Embryos collected from portable incubators were transferred to a CO2 incubator for further culture at 39°C. Two experiments were carried out: (1) transfer of embryos without sexing;; (2) transfer of embryos after biopsy and sex determination by PCR. In experiment 2, compact morulae or morula-blastocysts were selected for sex determination. Four to five blastomeres were aspirated from each embryo using a cutting pipette and an aspiration pipette of 30-μm diameter. PCR was done as previously described (Uoc et al., 1999 J. Biology). After biopsy, embryos were kept in culture for one day to observe the development in vitro. Embryos developed to morula-blastocyst or hatching blastocyst at Day 7 or Day 8 after IVF were transferred nonsurgically to recipients with estrus detected in the period from 0 to 12h before or after the starting IVF. Pregnancy was confirmed by rectal palpation 3 months after embryo transfer. The average rate of embryos developed into morula-blastocyst was more then 50% (Table 1) and there were no significant differences among different shipping groups. For experiment 2, more then 87 % of embryos biopsied developed in vitro to expanding and hatching embryos. The average rate of female embryos was 56.3%. The pregnancy rate at 3 months was more then 44% (n=188). The first group of calves was born without unusual birthing problems. In conclusion, the system of embryo transfer using intercontinental shipping of fresh IVF embryos and local Bos indicus recipients can be applied for production of dairy calves. Supported by grant from the AIRE-Development agency. Table 1 Development in vitro of IVP embryos