Narrow Perivitelline Space Research Articles

Human metaphase II oocytes with narrow perivitelline space have poor fertilization, developmental, and pregnancy potentials.

To analyze the fertilization, developmental, and pregnancy potentials in oocytes with narrow perivitelline space. Perivitelline space (PVS) of oocytes was evaluated at the time of ICSI, and those without sufficient PVS were judged as oocytes with narrow PVS (NPVS oocytes), and those with sufficient PVS formation were judged as oocytes with non-narrow PVS (non-NPVS oocytes). The analysis included 634 NPVS oocytes from 278 cycles and 12,121 non-NPVS oocytes from 1698 cycles. The fertilization and developmental potentials of NPVS and non-NPVS oocytes were compared by calculating odds ratios using a mixed-effects logistic regression model. We also compared the embryo transfer outcomes of those used for single vitrified-warmed blastocyst transfer after developing into the blastocyst stage. NPVS oocytes had higher odds ratios for degeneration (adjusted odds ratio [aOR], 1.555; 95% confidence interval [CI], 1.096-2.206; p = 0.0133) and 0PN (aOR, 1.387; 95% CI, 1.083-1.775; p = 0.0095), resulting in a lower 2PN rate (aOR, 0.761; 95% CI, 0.623-0.929; p = 0.0072). Even embryos with confirmed 2PN had lower odds ratios for cleavage (aOR, 0.501; 95% CI, 0.294-0.853; p = 0.0109) and blastocyst development (Gardner criteria; CC-AA) rates (aOR, 0.612; 95% CI, 0.476-0.788; p = 0.0001). Blastocysts developed from NPVS oocytes had significantly lower odds ratios for clinical pregnancy (aOR, 0.435; 95% CI, 0.222-0.854; p = 0.0156) than those developed from non-NPVS oocytes. Oocytes with NPVS have low fertilization and developmental potential, as well as low likelihood of pregnancy.

Light and electron microscopic morphology of the fertilized egg and fertilized egg envelope of Poropanchax normani, Poeciliidae, Teleostei

We examined the morphology of the fertilized egg and the fine structure of fertilized egg envelopes of Poropanchax normani belonging to the family Poeciliidae, also known as Norman’s lampeye using light and electron microscopes. The fertilized eggs with narrow perivitelline space were found to be spherical and demersal, additionally containing small oil droplets in the vitelline membrane. Further, a bundle of adhesive filaments was observed to be present on one side of the fertilized egg. These filaments possessed remarkably high elasticity and were approximately 1-3 mm in length. The size of the fertilized egg was determined to be about 1.49 ± 0.07 mm (n = 30). The outer surface appeared smooth, and adhesive filaments originating at different location of the surface of the envelope were found to be distributed around the egg envelope and were joined together to form a single long bundle in scanning electron microscopic observation. A peak-like structure formed of several straight wrinkles was observed around the micropyle. However, the complete structure of the micropyle could not be studied due to the depth at which it was located. Additionally, the total thickness of the egg envelope was ascertained to be approximately12.5–14.5 μm. The egg envelope consisted of two distinct layers, an outer electron dense layer and an inner lamellar layer, further consisting of 10 sublayers of varying thicknesses. Collectively, it was observed that the morphological characteristics of the fertilized egg, fine structures surrounding the micropyle, outer surface, adhesive structure consisting adhesive filaments, and sections of fertilized egg envelope displayed species specificity.

P-153 Oocytes with narrow perivitelline space have poor fertilization and developmental potentials after ICSI

Abstract Study question Do oocytes with narrow perivitelline space have poor clinical outcomes after ICSI? Summary answer After ICSI, oocytes with narrow perivitelline space have an increased degeneration rate and decreased rates of 2PN and embryo development. What is known already Several studies reported that oocytes with dysmorphologies, such as zona pellucida (ZP) abnormalities and cytoplasmic inclusions (vacuoles, smooth endoplasmic reticulum cluster, refractile bodies), have poor developmental potential in IVF/ICSI. In research on perivitelline space (PVS), many studies have focused on the PVS with fragmentation as well as large PVS. On the other hand, oocytes with narrow perivitelline space (narrow PVS oocytes) are considered to have an immature cytoplasm, but there are few reports on the relationship between narrow PVS oocytes and clinical outcomes. In this study, we retrospectively analyzed the effect of narrow PVS oocytes on ICSI outcomes. Study design, size, duration This retrospective single-center study analyzed 11149 MII oocytes that underwent ICSI between January 2018 and October 2021. We observed the PVS of MII oocytes during ICSI, and oocytes with sufficient PVS between the ZP and cytoplasm were determined to be non-narrow PVS oocytes. Oocytes without sufficient PVS from any angle (PVS was observed only around the first polar body) were defined as narrow PVS oocytes. Participants/materials, setting, methods After ICSI, oocytes were cultured in ONESTEP medium (NakaMedical, Tokyo, Japan). Embryos that developed into blastocysts were used for single vitrified-warmed blastocyst transfer (SVBT). We compared the rates of degeneration, 2PN, cleavage, blastocyst formation, good-grade (Gardner’s criteria ≥BB) blastocyst, top-grade blastocyst (Gardner’s criteria=AA), and clinical pregnancy (presence of a gestational sac) between oocytes with narrow and non-narrow PVS. Logistic regression analysis with consideration of patient age, BMI, and basal AMH was performed for each outcome. Main results and the role of chance Of the 11149 MII oocytes, 570 and 10579 were determined to be narrow and non-narrow PVS oocytes, respectively. Narrow PVS oocytes showed significantly higher degeneration rates (aOR: 1.52, 95% CI: 1.12–2.06, p<0.01) and lower 2PN rates (aOR: 0.77, 95% CI: 0.64–0.93, p<0.01) after ICSI compared to non-narrow PVS oocytes. Furthermore, rates of cleavage (aOR: 0.52, 95% CI: 0.31–0.87, p<0.05), blastocyst formation (aOR: 0.56, 95% CI: 0.45–0.70, p<0.01), good-grade blastocyst formation (aOR: 0.59, 95% CI: 0.46–0.76, p<0.01), and top-grade blastocyst formation (aOR:0.625, 95% CI:0.45–0.86, p<0.01), were significantly lower in the narrow PVS oocytes. Of the blastocysts developed, 32 and 1439 blastocysts from narrow PVS oocytes and non-narrow PVS oocytes, respectively, were used for SVBT. The clinical pregnancy rate was not significantly different between blastocysts developed from narrow (aOR: 0.52, 95% CI, 0.22–1.22, p = 0.131) and non-narrow PVS oocytes. However, in blastocysts developed from narrow PVS oocytes, clinical pregnancy was confirmed only in top-grade blastocysts (58.8% [10/17]), and blastocysts of other grades did not result in pregnancy (0% [0/15]). Limitations, reasons for caution The limitation of this study is that it was a retrospective analysis conducted at a single IVF center. It is necessary to confirm the reproducibility at other facilities because the evaluation of PVS differs among embryologists and IVF centers. Therefore, a prospective multicenter study is needed. Wider implications of the findings We found that the narrow PVS oocytes showed poor outcomes after ICSI. While a good pregnancy rate could be expected if a top-grade blastocyst from such oocytes was obtained and transferred, the embryonic development rate of narrow PVS oocytes is low. Trial registration number Not applicable

P-199 Effect of the area of oocyte perivitelline space on the fertilization and embryo development following intracytoplasmic sperm injection

Abstract Study question Does the area of oocyte perivitelline space have an effect on fertilization and embryo development following intracytoplasmic sperm injection? Summary answer The area of oocyte perivitelline space has not an effect on the fertilization but the embryo development following intracytoplasmic sperm injection. What is known already Oocyte perivitelline space has a lot of variation at intracytoplasmic sperm injection (ICSI). Some researchers reported that the characteristics of perivitelline space (large or small) affect embryo development, pregnancy, and implantation. However, these studies did not accurately calculate the area of perivitelline space. Therefore, little information is available on the effect of the area of oocyte perivitelline space on fertilization and embryo development following ICSI. The purpose of this study was to calculate and classify the area of oocyte perivitelline space and investigate the effect of the area of perivitelline space on fertilization and embryo development following ICSI. Study design, size, duration 1. We retrospectively investigated 634 mature oocytes that were conducted ICSI between January 2021 and December 2021. The area of each oocyte perivitelline space was defined from between the area of circle calculated from the inner layer of zona pellucida and cytoplasm and divided into 3 groups (-9%, 10-19%, 20%-). 2. We retrospectively calculated the diameter of an inner layer of zona pellucida and cytoplasm and compared it with the 3 groups (-9%, 10-19%, 20%-). Participants/materials, setting, methods 1. The fertilization, survival, good quality day-3 embryo, blastocyst, good quality blastocyst rates following ICSI were compared with the 3 groups (-9%, 10-19%, 20%-). 2. The average diameter of an inner layer of zona pellucida and cytoplasm of each oocyte for the 3 groups (-9%, 10-19%, 20%-) were compared. The data were analyzed by Fisher’s exact test, residual analysis, one-way ANOVA test, with Bonferroni correction as appropriate to determine the statistical differences among groups. Main results and the role of chance 1. The survival rates of perivitelline space -9%, 10-19%, 20%- groups were 100% (109/109), 96% (363/378), 94% (138/147), the fertilization rates were 89% (97/109), 88% (331/378), 86% (127/147), the good quality day-3 embryo rates were 56% (54/97), 70% (232/331), 70% (89/127) respectively. No significant difference was observed between these comparison items. The blastocyst rates of perivitelline space -9%, 10-19%, 20%- groups were 51% (47/92), 69% (222/321), 82% (93/114), the good quality blastocyst rates were 22% (20/92), 40% (129/321), 52% (59/114) respectively. The blastocyst and good quality blastocyst rates of perivitelline space -9% group showed significantly lower results. On the other hand, the blastocyst and good quality blastocyst rates of perivitelline space 20%- group showed significantly higher results. 2. The average diameter of an inner layer of zona pellucida of perivitelline space -9%, 10-19%, 20%- groups were 125 ± 4 µm, 129 ± 5 µm, 136 ± 6 µm, the average diameter of the cytoplasm of perivitelline space were 121 ± 4 µm, 119 ± 4 µm, 118 ± 4 µm respectively. Significant differences were observed in all pairs of groups of the average diameter of an inner layer of zona pellucida and cytoplasm. Limitations, reasons for caution The area of oocyte perivitelline space was calculated at only one plane. Wider implications of the findings Oocytes with narrow perivitelline space might have a wide region of adhesive between the cytoplasm surface and an inner layer of the zona pellucida which resulted in a smaller diameter of the zona pellucida and lower blastocyst rate by forming cytoplasmic fragments (Yumoto K et al. JARG. 2020 ;37(6):1349-1354.). Trial registration number Not Applicable

EFFECT OF MORPHOLOGICAL ABNORMALITIES OF PROTRUDING AND STRATIFIED ZONA PELLUCIDA ON FERTILIZATION RATE

In general, oocyte maturation is judged by the presence or absence of the first polar body. However, there are cases in which it is difficult to determine the released polar body due to morphological abnormalities of the zona pellucida. We have reported cases of pregnancy and delivery from oocytes with abnormal zona pellucida, which are characterized by protruding or stratified zona pellucida and a narrow perivitelline space. However, since the potential effect of zona pellucida abnormalities on fertilization rate has not been clarified, we compared the fertilization rate of oocytes with and without zona pellucida abnormalities. In addition, since there were oocytes with different shapes of the zona pellucida, such as protruding or stratified, we compared the difference in fertilization rates for each morphological group. In this study, we examined 218 MII stage oocytes among 12 patient cases with abnormal zona pellucida retrieved at our clinic from January 2013 to December 2019, and compared the normal fertilization rate of ICSI and IVF with that of 186,534 MII stage oocyte from 10,444 cases without abnormal zona pellucida retrieved during the same period. The oocytes that showed zona pellucida abnormalities were classified into two groups: those with protruding zona pellucida and those with stratified zona pellucida, and the normal fertilization rates were compared. Test for significance differences was performed using the chi-square test. The normal fertilization rates of oocytes with abnormal zona pellucida were 61.4% for ICSI(124/202) and 6.3% for IVF(1/16), which were significantly lower than those of oocytes without abnormal zona pellucida, which were 80% for ICSI(126685/158309) and 65% for IVF(18356/28225). (P<0.05) When comparing oocytes with abnormal zona pellucida, the normal fertilization rates were 60.7% (51/84) for protruding oocytes and 63.6% (70/110) for stratified oocytes, with no significant difference between the two groups. The fertilization rate in cases with abnormal zona pellucida is significantly lower than in cases without abnormal zona pellucida. This may be due to the fact that the polar body is difficult to see due to the narrow perivitelline space, and therefore the oocytes that cannot be accurately judged for maturity may be fertilized. In addition, there was no difference in fertilization rate between the two groups of protruding and stratified oocytes, suggesting that the difference in shapes does not affect the fertilization results.

DOES THE MORPHOLOGICAL EVALUATION OF NUCLEAR AND CYTOPLASMIC MATURATION OF MII OOCYTES CORRELATE WITH ICSI OUTCOMES?

Some oocytes with the first polar body and appear to be mature have immature nuclear or cytoplasm. If no spindle is observed by polarized light microscopy, nuclear maturation may be immature. On the other hand, oocytes with narrow perivitelline space (PVS) are considered immature cytoplasm. Therefore, the spindle may reflect nuclear maturation, and the PVS may reflect cytoplasmic maturation. In this study, we analyzed how they relate to ICSI outcomes.

Stages of embryonic development in the live-bearing fish, Gambusia holbrooki.

Divergent morphology and placentation of Poeciliids make them suitable model for investigating how evolutionary selection has altered and conserved the developmental mechanisms. However, there is limited description of their embryonic staging, despite representing a key evolutionary node that shares developmental strategy with placental vertebrates. Here, we describe the embryonic developmental stages of Gambusia holbrooki from zygote to parturition using freshly harvested embryos. We defined 40 embryonic stages using a numbered (stages 0-39; zygote to parturition, respectively) and named (grouped into seven periods, ie, zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and parturition) staging system. Two sets of quantitative (ie, egg diameter, embryonic total length, otic vesicle closure index, heart rates, the number of caudal fin rays and elements) and qualitative (ie, three-dimensional analysis of images and key morphological criteria) data were acquired and used in combination to describe each stage. All 40 stages are separated by well-defined morphological traits, revealing developmental novelties that are influenced by narrow perivitelline space, placentation, internal gestation, and sex differentiation. The principal diagnostic features described are quick, reliable, and easy to apply. This system will benefit researchers investigating molecular ontogeny, particularly sexual differentiation mechanisms in G. holbrooki.

Ultrastructure of the fertilized egg envelope from Melanotaenia praecox, Melanotaeniidae, Teleostei

We examined the morphology of fertilized egg and ultrastructures of fertilized egg envelopes of dwarf rainbowfish (Melanotaenia praecox) belong to Melanotaeniidae using light and electron microscopes. The fertilized eggs were spherical with adhesive filament, transparent, demersal, and had a narrow perivitelline space and small oil droplets. The size of fertilized egg was 1.02 ± 0.18 mm (n = 30), and there were two kinds of adhesive filament on the fertilized eggs. The long and thick (diameter 12.22 ± 0.52 μm, n = 20) adhesive filaments were only at the area of animal pole, and short and thin (diameter 1.99 ± 0.23 μm, n = 20) adhesive filaments were around the long filaments. A micropyle was conical shaped with adhesive filament and located near the animal pole of egg. The outer surface of fertilized egg was rough side. Also, the total thickness of the fertilized egg envelope was about 7.46 ± 0.41 μm (n = 20), the fertilized egg envelope consisted of two layers, an inner lamellae layer and an outer layer with high electron-density. And the inner layer was 8 layers. Collectively, these morphological characteristics and adhesive property of fertilized egg with adhesive filaments, and ultrastructures of micropyle, outer surface, and section of fertilized egg envelope are showed species specificity.

Molecular and Morphological Identification and Seasonal Distribution of Eggs of Four Decapterus Fish Species in the Northern South China Sea: A Key to Conservation of Spawning Ground

Although species of Decapterus form important pelagic fisheries in the northern South China Sea, information on their spawning grounds is limited because identification of fish eggs based on their morphology is difficult. We identify eggs of four Decapterus species (D.macrosoma, D.maruadsi, D.macarellus, and D.tabl) with DNA barcodes from the fishery resources surveys in Spring and Autumn 2018 in Xisha islands, and Spring and later summer-Autumn 2019 along the continental shelf of the northern South China Sea, and describe egg morphology. Of 1405 fish eggs with obtained COI sequences, 81 were successfully attributed to four Decapterus species; eggs of each are spherical, have a smooth chorion and narrow perivitelline space, and can be partly differentiated by diameter, melanophore drops on the oil globule, and the notum of the embryo. Seasonal distributions of eggs reveal spawning grounds, with that of D.maruadsi located mainly off the Pearl River estuary in spring; eggs of D. maruadsi rarely co-occur with those of D.macrosoma. Spawning grounds of D.macrosoma are probably further south, where water temperature and salinity are higher. Spawning periods of these four Decapterus species overlap slightly. Spawning habitat D.maruadsi has been lost, and the spawning season of D.macrosoma has extended. Identification of Decapterus eggs using DNA barcodes can assist with the identification of eggs using traditional morphological approaches. Spatial and temporal information on the distributions of Decapterus eggs can be used for improved conservation of spawning grounds and fisheries management in the northern South China Sea.

Ultrastructure of the fertilized egg envelopes in Ancistrus cirrhosus, Loricariidae, Teleostei

We examined the morphology of fertilized egg and ultrastructures of fertilized egg envelopes of Ancistrus cirrhosus belong to Loricariidae using light and electron microscopes. The fertilized eggs formed a mass on the spawning place and were yellowish, spherical, non-transparent, demersal, adhesive, and a narrow perivitelline space. But, the adhesiveness of fertilized eggs was disappeared after spawning excluding contact parts. The micropyle with funnel shape was surrounded by 15–19 furrow lines of egg envelope in a spoke-like pattern. The outer surface of egg envelope has smooth side and inner surface of egg envelope was rough with grooves. Also, the total thickness of the fertilized egg envelope was about 32.58 ± 0.85 μm (n = 20), and the fertilized egg envelope consisted of three layers, an outer adhesive electron-dense layer, a middle layer with low electron density and an inner electron-dense layer with grooves in counter structure from other most teleost. Collectively, these morphological characteristics and adhesive property of fertilized egg, and ultrastructures of micropyle, outer surface, and section of fertilized egg envelope are showed species specificity.

Embryonic and early larval development of two marine angelfish, Centropyge bicolor and Centropyge bispinosa.

Marine angelfish (family: Pomacanthidae) are among the most sought-after fish species in the saltwater aquarium trade. However, there is a lack of information in the literature on their early ontogeny. The objective of this study was to describe the embryonic and early larval development of two dwarf angelfish, the bicolour angelfish, Centropyge bicolor and the coral beauty angelfish, Centropyge bispinosa. The eggs of these two species were obtained from spontaneous spawning of the broodstock fish in captivity and incubated at 26.0 ± 0.2°C throughout the study. Fertilized eggs (n = 15) of both species are transparent, pelagic and spherical; the mean diameters of the eggs were measured at 703.6 ± 7.8 μm for C. bicolor and 627.6 ± 7.8 μm for C. bispinosa. The eggs of both species possessed a narrow perivitelline space, smooth and thin chorion, a homogenous and non-segmented yolk as well as a single oil globule. Overall, the observed embryonic development pattern of C. bicolor and C. bispinosa was very similar, and the main difference was the embryonic pigmentation pattern, which only became evident close to hatching. Larvae of both species started hatching at 13 h 30 min after fertilization, and the larval characteristics of both species also showed high levels of similarities. However, the mouth opening time for C. bicolor was 72 h after hatching (AH) and 96 AH for C. bispinosa. In general, the observed early ontogeny of C. bicolor and C. bispinosa also resembled that of other Centropyge species documented in the literature.

Embryonic and Larval Development and Some Reproductive-Biology Features of Dendrochirus zebra (Scorpaenidae)

Embryonic and larval development of zebra turkeyfish Dendrochirus zebra is described to the transition of the larvae to exogenous feeding. Mature sex products are obtained after double hormonal injections of the fish. A detailed, illustrated morphological description of developmental stages is presented. The changes of body pigmentation and number of muscular segments after hatching of the embryo from the egg envelope are recorded. Fertilized eggs are characterized by slightly irregular spherical shape; narrow perivitelline space; smooth, transparent, and unstructured egg envelope; and homogenous, transparent, and colorless yolk ~0.79 (0.74−0.81) mm in diameter. A single lipid droplet 0.15 (0.146−0.153) mm in diameter is located in the yolk. The droplet is colorless or with a yellowish pink tinge. The duration of the cellular cycle during the period of synchronous cleavage is approximately 27 min, and the duration of the incubation period is 25.5 h. At hatching, the prelarvae body length is 1.6–1.7 mm. The transition of the larvae to the exogenous feeding occurs at 2.33–2.42 mm body length by the fifth day after hatching.

REPRODUÇÃO DO ARIACÓ, Lutjanus synagris (LINNAEUS, 1758), SOB CULTIVO, EM RESPOSTA A INDUÇÃO HORMONAL

O presente trabalho teve como objetivo realizar a indução da desova do ariacó, Lutjanus synagris, em cativeiro utilizando a técnica de tratamento hormonal com gonadotrofina coriônica humana (hCG). Foi utilizado um total de 72 exemplares de L. synagris, sendo 24 fêmeas e 48 machos, com pesos médios respectivos de 0,381±0,154 kg e 0,353±0,105 kg, em três tratamentos com oito repetições. Em cada tratamento (T), as fêmeas receberam doses hormonais distintas de: T1=1.000, T2=1.250 e T3=1.500 UI de hCG/kg de peso corpóreo, sendo que os machos receberam dose única de 500 UI. As desovas ocorreram entre oito e doze horas após cada indução hormonal, e os ovos fertilizados apresentaram como características: forma esférica, transparência, espaço perivitelínico estreito, córion claro, vitelo homogêneo e não segmentado, sem pigmentação, flutuantes com gota de óleo visível. Os valores médios do diâmetro dos ovócitos, ovos e gotas de óleo foram de T1: 425±16, 659±10 e 139±2 µm; T2: 427±15, 661±14 e 140±5 µm; T3 422±10, 667±20 e 141±5 µm, respectivamente. O maior e menor números de ovócitos liberados foram observados nos tratamentos T2, com 556.661±209.171 ovos e T1, com 405.797±230.812 ovos. A maior taxa de fertilização for observada em T1, com 75±10%, seguida de T2 e T3 com 66±7% e 65±17%, respectivamente.

Pregnancy with oocytes characterized by narrow perivitelline space and heterogeneous zona pellucida: is intracytoplasmic sperm injection necessary?

This retrospective study analyzed fertilization protocols and pregnancy outcomes for oocytes with with narrow perivitelline space and heterogeneous zona pellucid (NPVS/HZP). In 63 in-vitro fertilization cycles filled with NPVS/HZP oocytes (abnormal oocytes group) and 521 cycles with normal oocytes (normal oocytes group), major clinical and laboratory parameters were recorded and compared in different fertilization cycles (conventional IVF cycles, rescue ICSI cycles, and traditional ICSI cycles). NPVS/HZP oocytes meant lower MIIoocytes rates in both IVF and ICSI cycles compared with normal oocytes (p < 0.05). The 2PN rates for abnormal oocytes were significantly lower than those for normal oocytes in both conventional IVF cycles (58.8% VS 71.3%, P < 0.05) and rescue ICSI cycles (58.0% VS 78.0%, P = 0.0000). The high-quality embryo rates in normal oocytes groups were significantly higher than those in abnormal oocytes groups in different fertilization cycles (52.2% VS 35.0%, P < 0.01; 42.9% VS 23.9%, P < 0.001; 50.6% VS 31.0%, P = 0.0000, respectively). No clinical pregnancy was obtained from abnormal oocytes in 11 conventional IVF cycles. The clinical pregnancy rates in rescue ICSI and traditional ICSI cycles were comparatively lower in abnormal oocytes groups, but there was no significant difference as compared with normal oocytes groups (35.0% VS 48.1% and 26.7% VS 50.7%, P > 0.05, respectively). Retrieval of oocytes characterized by NPVS/PZP from cycle to cycle was one of the reasons for obscure infertility. ICSI may be the right way to avoid fertilization failure and get pregnancy in women with NPVS/HZP oocytes.

Computer-Assisted Oocyte Morphometry Before ICSI: Correlation of Oocyte Measurements With Fertilization and Embryo Development

The present study aimed to correlate morphometric parameters of the oocytes with the occurrence of fertilization following intracytoplasmic sperm injection (ICSI). In a prospective, controlled cohort design, women (n = 32) who were candidates for ICSI had oocytes (n = 258) collected and submitted to morphometric evaluation using the Cronus3 software program. The morphometric parameters obtained were oocyte diameter, perivitelline space width, zona pellucida thickness, and first polar body diameter. The median oocyte diameter was similar in cases in which fertilization occurred compared with those in which fertilization failed (75.2 and 75.9 μm, respectively; P = .218). The 2 groups also had similar measurements of perivitelline space, zona pellucida, and first polar body. However, the best quality zygotes identified by a morphological score resulted from oocytes with larger diameter (75.6 vs 74.0 μm; P < .01) and narrow perivitelline space (5.3 vs 7.1 μm; P < .01). Embryo development, as assessed by cleavage at second day of culture, was not significantly associated with oocyte morphometric parameters. These findings suggest that morphometric parameters of the oocytes do not correlate with the occurrence of fertilization following ICSI but may assist in selecting oocytes more likely to originate high-quality zygotes.

267 ULTRASTRUCTURAL CHARACTERISTICS OF NON-MATURED AND IN VITRO MATURED OOCYTES COLLECTED FROM FOLLICULAR, LUTEAL, AND INACTIVE OVARIES OF DOMESTIC CAT (FELIS CATUS) DURING BREEDING AND NON-BREEDING SEASON

The aim of this experiment was to describe the ultrastructural characteristics of non-matured (NM) and in vitro matured (IVM) cumulus–oophorus complexes (COC) recovered from adult queens during the breeding season (BS; July, August, and September) and the non-breeding season (NBS; January, February, and March) in southeast Brazil. Transmission electronic microscopy was performed in NM COC immediately after harvest, and IVM COC were matured for 36 h before transmission electronic microscopy. All IVM COC during both seasons were at metaphase II stage. Specimens were divided into COC from inactive ovaries, follicular ovaries, and luteal ovaries recovered during BS and NBS. During NBS, NM follicular and inactive COC presented a narrow perivitelline space covered with microvilli, which were less evident in NM luteal COC. Cumulus-cell projections penetrated the zona pellucida forming gap junctions with the oolemma in all NM COC. In the cytoplasm of NM inactive COC, lipid droplets and vesicles were evenly distributed in the ooplasm except for in the cortical zone, where clusters of mitochondria were observed. Non-matured luteal COC were also characterised by peripheral mitochondrial clusters, but greater clusters could also be seen centrally in the cytoplasm. In contrast, NM follicular COC were characterised by evenly distributed mitochondria within the ooplasma. In NM inactive and follicular COC, cortical granules were seen only in the central region of the cytoplasm, but the electron density of these organelles appeared to be low and Golgi complexes were often seen in association with these granules. The density of cortical granules in NM follicular COC was higher, but they were also present in central regions of the ooplasm. In all NM COC, a well-developed Golgi complex was observed. In all IVM COC, mitochondria clusters were no longer observed, and these organelles presented an even distribution toward the ooplasm. High-density cortical granules were present in the peripheral region of all IVM COC, although a small number could still be observed in central region of the ooplasm of IVM follicular COC. The perivitelline space was more prominent in IVM COC. During BS, COC characteristics were similar to NM COC during NBS; the presence of mitochondrial clusters associated with lipid droplets, well-developed cortical granules, and the presence of nuage could be observed, except that all cortical granules located in the central region of the cytoplasm of NM follicular COC during BS. A great amount of smooth endoplasmic reticulum could be verified in NM follicular and luteal COC. During BS, all IVM COC presented uniform distribution of mitochondria and peripheral distribution of cortical granules in the ooplasma. These results indicate that IVM was efficient in inducing the morphological changes necessary for cytoplasmic maturation of cat COC, independently of the ovarian status. The authors acknowledge FAPESP.

Reproductive Characteristics and Egg Development in Flounder (Pleuronectes flesus luscus) in the Southern Black Sea

Spawning time, total fecundity, egg size, fertilization and hatching rates, and egg development of the flounder, Pleuronectes flesus luscus, were investigated in six wild female broodstock (mean wt 394.4±226.8 g). Spawning lasted 33 days from December 29 to January 30. Mean total fecundity was 171.4±109 x 103 eggs per female. Newly ovulated eggs were spherical and buoyant, with a diameter of 1.075-1.213 mm (avg 1.156±0.025 mm), a colorless transparent chorion, a slightly yellowish unsegmented yolk, and a narrow perivitelline space, without an oil globule. Fertilization and hatching rates were 17.2±15.7% and 51.5±27.6%, respectively. Hatching occurred after 117 h of incubation at 9.8-11oC. There were variations in egg size between batches, with the size tending to decrease during the spawning season (p&lt;0.05).

Egg Development of Paralichthys orbignyanus (Valenciennes, 1839)

Eggs of the Southern Brazilian flounder Paralichthys orbignyanus were obtained through spawning induction and reared in laboratory. The eggs released were free-floating or pelagic. Other important characteristics were: spherical shape, smooth chorion, narrow perivitelline space, and a single oil globule. Egg and oil globule diameter were about 0.792 ± 0.008 mm and 0.114 ± 0.003 mm, respectively. The blastodisc was observed approximately 1 h after fertilization and followed by blastodisc cleavage. Blastula stage started after about 5 h and gastrula stage after 9 h. Approximately 20 h after fertilization, blastopore closure was observed. Neurula or early embryo occurred after 24 h. Cardiac beats and movements of the free embryonic tail were recorded after 40 h of incubation. Hatching occurred after an incubation period of 40-50 h at temperatures ranging from 18 to 20 ºC. Newly hatched larvae were about 2.04 ± 0.024 mm long and quite undeveloped, with a large yolk sac with a posterior oil globule and sparse pigmentation. These results were greatly similar to those obtained by previous studies regarding congener species. This is one of the first steps towards the artificial propagation of this species under controlled conditions.

Ultrastructure of follicles after vitrification of mouse ovarian tissue

Ultrastructure of follicles after vitrification of mouse ovarian tissue

Ultrastructure of bovine blastocysts following cryopreservation: Effect of method of blastocyst production

The objective of this study was to describe the ultrastructure of blastocysts derived by in vivo and in vitro methods and to investigate how the morphology is affected by exposure to cryoprotectant (10% glycerol) or cryopreservation by conventional slow freezing. In vivo derived blastocysts were characterized by a narrow perivitelline space (PvS), a continuous cover of numerous stacked microvilli (MV) on the plasma membrane, a well-defined system of cell-to-cell coupling and a large population of round or elongated mitochondria with numerous transverse cristae. Exposure of these blastocysts to cryoprotectant was manifested by shrinkage of the blastocysts and swelling of the mitochondria. Cryopreservation resulted in further shrinkage, damage to the MV, and accumulation of cellular debris. In comparison, the in vitro matured (IVM)/in vitro fertilized (IVF) in vivo cultured blastocysts displayed a wider PvS; they appeared to possess less MV and all blastocysts displayed some cellular debris in their PvS. There was also a decrease in the number of junctional contacts between the trophoblastic cells. The reaction of these blastocysts to exposure to cryoprotectant was similar to that of the in vivo derived blastocysts. However, they appeared to be more susceptible to cryopreservation. The totally in vitro produced (IVP) blastocysts displayed a wider PvS, no stacking of the MV, increased numbers of lipid droplets and a further reduction in the junctional contacts between trophoblastic cells. The IVP blastocysts sustained breakage of the zona pellucida on exposure to cryoprotectant and were extremely sensitive to cryopreservation, losing all cell structure and organization. The findings of the present study indicate that in vivo derived blastocysts possess certain structural characteristics that confer a greater tolerance on them to exposure to cryoprotectant and cryopreservation.