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In vitro embryo production using oocytes of prepubertal females is named JIVET (juvenile in vitro embryo technologies). The aim of the JIVET is to increase genetic gain by shortening the generation interval in breeding programs. In this chapter we describe the methodology currently used in our laboratory for producing in vitro embryos from prepubertal sheep (90-120 days old) and goats (30-45 days old).Briefly, we obtain cumulus-oocyte complexes (COCs) from slaughtered female ovaries. These COCs are in vitro matured in TCM199 with 10% (v/v) fetal bovine serum (FBS) at 38.5 °C in a humidified air atmosphere with 5% CO 2 for 24 h. The next day we collect ejaculates from males of proven fertility and select the most motile spermatozoa by density gradient. Following coincubation with mature COCs, presumptive zygotes and embryos are cultured in SOF for 8 days at 38.5 °C, 90% N 2 , 5% CO 2 , 5% O 2 , and 100% humidity to obtain blastocysts.

Melatonin is a universal antioxidant that improves in vitro embryo production in several species. The aims of this study were to determine the melatonin concentration in the ovarian follicular fluid (FF) of juvenile goats and the effect of melatonin during in vitro maturation (IVM) on embryo development. The FF melatonin concentration was 0.57--1.07×10-9 M, increasing with follicular diameter. Oocytes were matured, fertilised and cultured under conventional conditions. Blastocyst development, embryo quality and levels of reactive oxygen species (ROS) and reduced glutathione were assessed. In Experiment 1 different melatonin concentrations (10-3, 10-7, 10-9, 10-11 M) were added to the IVM medium, which contained cysteamine as antioxidant, and no differences were observed. In Experiment 2, melatonin (10-7 M) was tested in the presence or absence of cysteamine (experimental groups: melatonin, cysteamine, melatonin+cysteamine, non-antioxidant). The melatonin group presented a higher blastocyst rate than the non-antioxidant group (28.9 vs 11.7%; P<0.01) and a higher total cell number than the cysteamine group (225.1 vs 129.0; P<0.05). Oocytes from the melatonin and cysteamine groups had lower ROS levels than those from the non-antioxidant group. This study shows that melatonin is an interesting tool for improving oocyte competence in juvenile goats as it increases embryo production and quality.

In this study we assessed the concentration of linoleic acid (LA) and linolenic acid (ALA) in follicular fluid of prepubertal goats according to follicle size (<3mm or ≥3mm) by gas chromatography and tested the addition of different LA and ALA (LA:ALA) concentration ratios (50:50, 100:50 and 200:50µM) to the IVM medium on embryo development, mitochondrial activity, ATP concentration and relative gene expression (RPL19, ribosomal protein L19; SLC2A1, facilitated glucose transporter 1; ATF4, activating transcription factor 4; GPX1, glutathione peroxidase 1; HSPA5, heat-shock protein family A 70 kDa; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DNMT1, DNA methyltransferase 1; GCLC, glutamate-cysteine ligase catalytic subunit; SOD1, superoxide dismutase 1). Oocytes were in vitro matured, fertilised or parthenogenetically activated and zygotes were cultured following conventional protocols. LA concentration ranged from 247 to 319µM and ALA concentration from 8.39 to 41.19µM without any effect of follicle size. Blastocyst production from the different groups was: control FCS (22.33%) and BSA (19.63%), treatments 50:50 (22.58%), 100:50 (21.01%) and 200:50 (9.60%). Oocytes from the 200:50 group presented higher polyspermy and mitochondrial activity compared with controls and the rest of the treatment groups. No differences were observed in ATP concentration or relative expression of the genes measured between treatment groups. In conclusion, the low number of blastocysts obtained in the 200:50 group was caused by a high number of polyspermic zygotes, which could suggest that high LA concentration impairs oocyte membranes.

Objective: To compare blastocyst production, after IVF and ICSI, from sheep oocytes of various quality. Sham-injected oocytes and parthenogenetic activated oocyte groups were considered as control., Design: Prospective experimental study., Setting: University., Animal(s): Three- to 6-month-old sheep., Intervention(s): Oocyte quality was assessed with the use of brilliant cresyl blue (BCB) stain. Adenosine triphosphate content was measured. Intracytoplasmic sperm injection and IVF were performed and blastocyst development and cell numbers were analyzed., Main Outcome Measure(s): Adenosine triphosphate content, embryo development and blastomere numbers., Result(s): After IVF, BCB-stained (BCB+) oocytes developed up to the blastocyst stage at higher percentages and with more cells per embryo (24.1% vs 4.0% and 69.7 vs 43.9, respectively) than unstained (BCB-) oocytes. Using intracytoplasmic sperm injection, no differences were found in blastocyst production (14.3% vs 11.8%) and number of cells per embryo (71.1 vs 54.3) between BCB+ and BCB- oocytes. Adenosine triphosphate content was higher before in vitro maturation than after in both types of oocytes. Brilliant cresyl blue-stained oocytes had more adenosine triphosphate content than BCB- oocytes., Conclusion(s): Brilliant cresyl blue-stained oocytes show more adenosine triphosphate content than BCB- oocytes. Results from IVF were affected by the oocyte quality while ICSI did not produce differences in embryo development or blastomere numbers., (Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

This study examines the effectiveness of the cryotop vitrification method for the cryopreservation of goat blastocysts. To determine the effects of embryo development stage and donor age on in vitro survival rates, good-quality blastocysts from adult and prepubertal goats were sorted into non-expanded, expanded, hatching and completely hatched. In vitro produced (IVP) blastocysts were derived from prepubertal goat oocytes by slicing of ovaries from slaughtered animals while adult goat oocytes were collected by the laparoscopic ovum pick up (LOPU) method. Blastocysts were vitrified/warmed using the cryotop technique. Survival rates were determined in terms of blastocoele re-expansion at 3 and 20 h post-warming. For prepubertal goats, survival rates at 3h post-warming were significantly higher when expanded blastocysts (78.3%) were vitrified/warmed compared to hatched blastocysts (57.4%), whereas non-expanded (62.5%) or hatching blastocysts (71.4%) showed similar rates. For adult goats, survival rates were significantly higher after warming in expanded (36.4%), hatching (75%) or hatched (50%) blastocysts when compared to non-expanded (0%) blastocysts. When survival rates were assessed at 20 h post-warming, no differences were observed when we compared non-expanded (45.8%), expanded (56.5%), hatching (64.3%) and hatched (50.5%) blastocysts from prepubertal goats; and for blastocysts from adult goats, survival rates were only significantly lower for the non-expanded stage (0%) compared to the other stages. For adult versus prepubertal blastocysts at the same developmental stage, our data indicate significantly higher survival rates at 3 h post-warming for non-expanded and expanded blastocysts from prepubertal goats over their counterparts from adult goats. At 20 h post warming, survival rates were only higher for non-expanded blastocysts from prepubertal goats versus adult goats. Collectively, our data reveal that blastocysts produced in vitro from prepubertal goats return similar survival rates regardless of their development stage, whereas blastocysts derived from adult goats are best for vitrification at the expanded, hatching or hatched stage., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This study examines the effects of adding insulin-transferrin-selenium (ITS) and/or L-ascorbic acid (ASC) to a conventional medium for maturing prepubertal calf oocytes on chromosome organization, cortical granule (CG) distribution, and embryo development to the blastocyst stage. Cumulus-oocyte complexes (COCs) were matured in medium TCM 199 containing PVA and EGF (control), and supplemented with ITS and/or ASC for 12 or 24 h at 38.5 °C in a 5% CO(2) atmosphere. Calf oocytes matured with ITS + ASC or ASC for 12 h showed significantly higher percentages of peripherally distributed CG (83.3% and 86.2% respectively) than control oocytes (71.4%) or those matured with ITS alone (71.4%). No effects on chromosome organization were detected. Conversely, 24 h of supplementation did not affect CG distribution patterns, while the addition of ASC gave rise to significantly higher percentages of oocytes showing a normal alignment of their chromosomes (72.9%) compared to controls (58.7%). At 48 hpi, similar cleavage rates were observed among treatments regardless of the treatment time. However, the presence of ITS + ASC for 12 h rendered significantly higher blastocyst rates than those recorded in the remaining groups. Supplementation for 24 h with ITS or ITS + ASC had no significant effects on the percentage of blastocysts obtained, while the presence of ASC significantly reduced the proportions of embryos developing to the blastocyst stage. Our data suggest that ITS plus L-ascorbic acid supplementation during the first 12 h of in vitro maturation improves cytoplasm maturation and the developmental competence of embryos produced from prepubertal calf oocytes., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Two experiments were designed to determine the ability of in vitro-cultured blastocysts at different stages of development to survive the vitrification procedure using cryotop devices. Day 7 and Day 8 embryos were classified as non-expanded, expanded or hatching and/or hatched blastocysts. In the first experiment, we examined the survival rate of vitrified-warmed blastocysts after 3 h incubation in synthetic oviducal fluid (SOF) medium. In the second experiment, vitrified-warmed blastocysts were evaluated using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) technique to detect nuclei with damaged DNA. In both experiments, results for cow and calf blastocysts were compared. No differences in survival rates were observed after vitrification of Day 8 expanded (52.4%) and hatched (50%) cow blastocysts or Day 8 expanded (54.5%) and hatched (59.4%) calf blastocysts. When embryos were vitrified on Day 7, survival rates of 78.4% and 66.7% were observed after warming expanded and hatched cow blastocysts, respectively, compared with rates of 80% and 76.9%, respectively, for calf blastocysts. Lowest survival rates were recorded for non-expanded blastocysts (26%-54%) compared with the other developmental stages, particularly those vitrified at Day 8 (

Our previous studies have shown that the addition of 100 mircroM cysteamine to the in vitro maturation (IVM) medium increased the embryo development of prepubertal goat oocytes. The aim of the present study was to evaluate the effect of adding different concentrations of cysteamine to the IVM medium and to the in vitro embryo culture medium (IVC) on the embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue (BCB) test. Oocytes were exposed to BCB and classified as: oocytes with a blue cytoplasm or grown oocytes (BCB+) or oocytes without blue cytoplasm or growing oocytes (BCB-). In Experiment 1, oocytes were matured in a conventional IVM medium supplemented with 100 microM, 200 microM or 400 microM cysteamine. In Experiment 2, oocytes were matured with 400 microM cysteamine and following in vitro fertilization (IVF) were cultured in SOF medium supplemented with 50 microM and 100 microM cysteamine. In Experiment 1, BCB+ oocytes matured with 100 microM and 200 microM cysteamine showed higher normal fertilization and embryo development rates than BCB- oocytes. Oocytes matured with 400 microM cysteamine did not present these differences between BCB+ and BCB- oocytes. In Experiment 2, the addition of 50 microM and 100 microM cysteamine to culture medium did not affect the proportion of total embryos obtained from BCB+ oocytes (35.89% and 38.29%, respectively) but was significantly different in BCB- oocytes (34.23% and 29.04%, respectively, P < 0.05). In conclusion, the addition of 400 microM cysteamine to the IVM improved normal fertilization and embryo development of BCB- oocytes at the same rates as those obtained from BCB+ oocytes. The proportions of morulae plus blastocyst development were not affected by the treatments.

Our previous studies have shown that larger and more competent oocytes can be selected using the brilliant cresyl blue (BCB) test. The objective of this study was to assess, in BCB-selected oocytes, the effect on the embryo development of prepubertal goat oocytes of the addition to in vitro culture (IVC) medium of either glutathione (GSH) alone or GSH in combination with glucose. Oocytes were exposed to 26 mM BCB and were classified as: oocytes with a blue cytoplasm or grown oocytes (BCB+) and oocyteswithout blue cytoplasm or growing oocytes (BCB-). Oocytes were matured in TCM-199 with 100 microM cysteamine. Presumptive zygotes were cultured in synthetic oviductal fluid (SOF) in the presence or absence of 1 mM glutathione (experiment 1) for 7 days (8 days post-insemination, p.i.). In experiment 2 we tested the addition to culture of 2.78 mM glucose at day 5 p.i. BCB+ oocytes showed higher percentages of nuclear maturation than the BCB- and control groups (82.6%, 55.7% and 74.7%, respectively). The percentage of polyspermic oocytes was higher in BCB- than BCB+ oocytes. Supplementation of SOF medium with 1 mM GSH did not affect embryo development but the percentage of total embryos developed after culture was higher in BCB+ oocytes than in BCB- oocytes independently of the GSH supplementation. Glucose, alone or with GSH, added at 5 days p.i. did not affect embryo development. In conclusion, prepubertal goat oocytes were unable to develop beyond the 8-cell stage embryo under the culture conditions in this study.

Experiments were carried out to develop and improve in vitro culture systems for IVM-IVF prepubertal goat oocytes. Cumulus oocyte complexes (COC) were obtained by slicing ovaries from slaughtered prepubertal goats. Oocytes were matured in TCM-199 supplemented with 20% estrous goat serum (EGS) + 10 micrograms/mL FSH + 10 micrograms/mL LH + 1 microgram/mL estradiol 17 beta for 27 h at 38.5 degrees C in 5% CO2 in air. Matured oocytes were placed in drops of TALP- fert medium supplemented with hypotaurine (1 microgram/mL) and inseminated with freshly ejaculated spermatozoa following capacitation as described by Younis et al. (69) but with 100 micrograms/mL heparin. At 24 h post insemination the ova were transferred to various in vitro culture media, and early embryo development was evaluated until Day 8 post insemination. Specifically, in the studies described here, we have compared the effects of (Experiment 1) co-culture systems using oviductal ephitelial cells (OEC) and cumulus cells (CC), both caprine and bovine; (Experiment 2) the presence of serum and/or OEC; (Experiment 3) 4 culture media (TCM199, Ham's F10, CZB abd SOF) for co-culture with OEC; and (Experiment 4) conditioned medium with OEC. In Experiment 1, the percentage of morulae plus blastocysts was higher in culture with OEC, both caprine and bovine (15.1 and 14.8%, respectively) than with CC (4.1 and 6.7%, respectively). In Experiment 2, the OEC with EGS did not improve the percentage of morulae and blastocysts obtained with OEC alone (14.3 and 23.1% respectively). In Experiment 3, this percentage was higher using OEC with TCM-199 compared to CZB medium (21.3 and 12.3%, respectively) and in Experiment 4, the results were 3.7, 11.2 and 21.3% for TCM-199 without cells, Conditioned Medium and co-culture with OEC, respectively.

The aim of this study was to evaluate the utility of the brilliant cresyl blue (BCB) test as an indirect measure of oocyte growth to select competent prepubertal sheep oocytes for in vitroembryo production. The BCB stain allows the determination of glucose–6-phosphate dehydrogenase (G6PD) activity, an enzyme with decreased activity in oocytes that have finished their growth phase. Oocytes were obtained after slicing the surface of lamb ovaries (2–5 months old) obtained from a local abattoir. Oocytes with more than 3 compact cumulus layers and homogeny cytoplasm were selected and stained with different concentrations of BCB diluted in PBS (13-, 26-, 39-, and 52-μM BCB) during 60min at 38.5°C in a humidified air atmosphere. Oocytes were classified into groups depending on their cytoplasm coloration: oocytes with blue cytoplasm or grown oocytes (BCB+) and oocytes without blue coloration or growing oocytes (BCB–). Oocytes were matured in an enriched TCM-199 medium for 24h at 38.5°C and 5% CO2in a humidified atmosphere. Oocyte diameter was also measured. Matured oocytes were partially denuded and transferred to fertilization medium (SOF) supplemented with 10% of oestrous sheep serum. Fresh semen was kept at room temperature (25°C) for 1h. Highly motile spermatozoa were selected by using Ovipure density gradient (Nidacon EVB S.L.), and oocytes were fertilized with 1×106spmL-1. After 20h postinsemination, presumptive zygotes were cultured for 8 days in SOF with 10% of fetal bovine serum at 38.5°C, 5% CO2, and 90% N2. Data was analysed by performing Fisher’s exact test for blastocyst production and ANOVA with Tukey’s post-test for oocyte diameter. Table 1 shows the percentage of BCB-stained oocytes and their embryo development. In this study oocytes exposed during 60min to 13-μM BCB showed a higher percentage of embryos reaching blastocyst stage than did those in the control group (≤0.01). In other species such as goats (Rodriguez-Gonzalez et al.2002 Theriogenology 57(5), 1397–1409) and cattle (Alm et al.2005 Theriogenology 63(8), 2194–2205), the best protocol for the oocyte selection was the use of 26-μM BCB during 90min. Oocyte diameter showed significant differences between BCB– with BCB+ and control group (110μm, 134μm, and 121μm, respectively, ≤0.001). In conclusion, using 13μM of BCB during 60min is a suitable technique for increasing embryo blastocyst rates using lamb oocytes. Table 1.Effect of BCB1concentration on embryo development of lamb oocytesThe grant sponsor was the Spanish Ministry of Science and Innovation, Code: AGL2007-60227-CO2-01. [ABSTRACT FROM AUTHOR]

The aim of this study was to test a FISH approach using ovine-painted probes specific for the chromosomes X and Y, on goat interphase and metaphase nuclei of blastocyst cells. Oocytes of prepubertal goats were recovered at a slaughterhouse and selected by morphological criteria. Oocytes were matured in TCM-199 supplemented with hormones, serum and cysteine at 38.5°C for 27 h. IVM-oocytes were fertilized in vitroand the presumptive zygotes were cultured for 10 days in SOF with 10% FCS at 38.5°C, 5% CO2and 5% O2. The blastocyst nuclei were spread using a modified Tarkowski method (1966). Briefly, individual embryos were immersed into hypotonic solution for 5 min, followed by fixative solution of methanol/acetic acid (Carnoy''s solution) until the embryos acquired a transparent appearance. Next, the embryos were transferred to a Superfrost plus Slide (Menzel Gläser, Braunschweig, Germany) in a small droplet mixture of distilled water and Carnoy The zona pellucida and the blastomere cytoplasm dissolved gradually and Carnoy solution was added dropwise to the slide before the nuclei dried out. The morphology and total number of nuclei in each embryo were analyzed under phase contrast microscope and stored at -18°C. Embryos with appropriate fixation were then subjected to hybridization with ovine-painted probes specific to chromosomes X (Fluorocrom green-FITC) and Y (Fluorocrom orange-TAMRA) (Chrombios-Molecular Cytogenetics GmbH, Raubling, Germany) according to the manufacturers protocol and adjusted for the caprine species. Briefly, slides were then incubated at 60°C for 1 h. The chromosomal DNA was then denatured by immersing slides in 70% formamide/30% 2 × SSC at 70°C for 1.5 min, and immediately dehydrated in an ethanol series (70%, 90%, and 100%), 4 min duration per solution and air dried. In parallel, X- and Y-probes were added to the hybridization solution (50% deionized formamide, 10% dextran sulfate, 2 × SSC) and denatured at 75°C for 10 min. Aliquots (0.5 to 1.5 μL) of this solution were placed on each slide and sealed with a coverslip and glue prior to incubation at 37°C (Hybrite; Vysis Inc, Dowers Grove, IL, USA). After 22 to 24 h the coverslip was removed and the slides were washed three times. The first and third washes were performed with 2 × SSC at room temperature while the second wash was in a 0.4 × SSC/0.1% Tween at 73°C for 3 min. Nuclear DNA was counterstained with diamino-phenyl-indole solution (DAPI) and examined with a fluorescence microscope (Olympus BX61, Olympus America Inc., Melville, NY, USA) equipped with appropriate filters. From a total of 69 blastocysts, 11 355 blastomeres were analyzed and 7,825 were correctly hybridized (68.9%). The results of the embryo sexing were: 24 embryos XX, 11 XY, 22 polyploid embryos (of which 13 presented more than 80% of cells XX), 3 haploid embryos (X0), 2 tetraploid embryos, and 5 no result. In summary, goat blastocysts were successfully sexed using FISH with painted ovine X- and Y-specific probes. Grant sponsor Spanish Ministery of Science and Innovation.Code: AGL2007-60227-CO2-01. [ABSTRACT FROM AUTHOR]

The prepubertal goat ovary presents a large number of small oocytes with a compromised competence to develop up to blastocyst stage. In pigs (Wu et al.2006), using growth medium (GM) composed by low hormone concentrations, ascorbic acid, and insulin transferrin selenium (ITS) during the first 24h of in vitromaturation (IVM) improved embryo development of small oocytes. The aim of this study was to test the GM in small prepubertal goat oocytes in order to increase blastocyst yield. The cumulus–oocyte complexes (COC) were recovered from prepubertal (1–2 months old) goat ovaries by slicing. The COC with a compact cumulus and homogeneous cytoplasm were selected and classified into 2 categories based on oocyte diameter: <125μm and ≥125μm. The ≥125μm oocytes were matured in groups of 25 to 30 COC/100μL drops of conventional IVM medium covered with mineral oil for 24h (Treatment A). This medium was TCM-199 supplemented with 10% donor bovine serum, 10μgmL-1FSH, 10μgmL-1LH, 1μgmL-117β-oestradiol, and 100μM cysteamine. The <125μm oocytes were distributed into 3 experimental groups: Treatment B, COC matured in the conventional IVM medium; Treatment C, COC cultured in GM (TCM-199, 10% donor bovine serum, 0.04μgmL-1FSH, 0.04μgmL-1LH, 0.004μgmL-117β-oestradiol, 100μM cysteamine, 100μgmL-1ascorbic acid, and 5μLmL-1ITS) for 12h before placement for other 12h in the conventional IVM medium, all drops of growth or maturation medium were covered with mineral oil; Treatment D, COC cultured during the first 12h in GM and other 12h into the conventional medium supplemented with 100μgmL-1ascorbic acid and 5μLmL-1ITS. After IVM, oocytes were fertilized for 24h with a sperm concentration of 4×106spzmL-1. Presumptive zygotes were cultured in SOF for 9 days. The cleavage rate was evaluated at 48h post-insemination and blastocyst percentages at the final in vitroembryo culture (treatments A, B, C: 5 replicates; treatment D: 4 replicates). The results are shown in the Table 1. Cleavage and embryo development did not show different results when we compared small oocytes matured in GM to those matured in conventional IVM medium. However, the biggest oocytes (≥125μm) showed the highest percentage of blastocyst development. The current study shows that the culture of small prepubertal goat oocytes in GM does not improve blastocyst yield. Table 1.Effect of growth medium on embryo development of small oocytes (<125μm) from prepubertal goats [ABSTRACT FROM AUTHOR]

Two experiments were designed to determine the ability of 7 and 8 day in vitro-produced blastocysts to survive to the vitrification procedure. Embryos were classified as early blastocysts, expanded, or hatching/hatched blastocysts. Vitrification was done using cryotop devices as described Du et al.(2007). After warming, blastocysts were incubated for 3 h in SOF medium. In the first experiment, we examined the developmental competence of early blastocysts, expanded blastocysts, and hatching/hatched blastocysts after vitrification using the Cryotop method. In the second experiment, warmed blastocysts that had been vitrified on cryotops were fixed in 4% formaldehyde and incubated with TUNEL staining for detecting DNA damaged nuclei. The percentage of TUNEL positive and negative blastomeres was assessed by confocal microscopy. In all experiments cow and calf blastocysts were compared. When the results according to the developmental stage were analyzed, no differences in the survival rates after vitrification of expanded and hatched blastocysts were observed at Day 8 from cow and calf blastocysts. After warming, survival rates of 52.4 and 50% were noted in the groups of expanded and hatched blastocysts respectively from Day 8 cow blastocysts. Similar results were observed in the groups of expanded (54.5%) and hatched (59.4%) blastocysts from Day 8 calf blastocysts. When embryos were vitrified at Day 7, survival rates of 78.4 and 66.7% were observed after warming expanded and hatched blastocysts from cows. In calves, a significant increase in the survival up to 83.3 and 80% was observed after warming expanded and hatched blastocysts. The lowest survival rates were observed in early blastocysts (from 26 to 51%), particularly in those vitrified at Day 8 (≤40%). Following vitrification, cell death was monitored in blastocysts 3 h after warming by TUNEL labelling of cells with damaged DNA. The TUNEL staining procedure was undertaken on Day 7 calf (n= 23) and cow (n= 25) blastocysts, as well as on Day 8 calf (n= 22) and cow (n= 30) blastocysts. When taking into account the stage of blastocyst development, there was a trend toward higher DNA integrity index after warming of expanded and hatched blastocysts compared with early blastocysts in calf and cow groups. So, cell damage was minimal in those blastocysts vitrified at expanded and hatched stage and rates were comparable with those from control fresh blastocysts. These findings suggest that the Cryotop technique seems to be particularly useful for blastocysts presenting a high degree of expansion (expanded and hatched blastocysts), mainly those blastocysts vitrified and warmed at Day 7. [ABSTRACT FROM AUTHOR]

Abstract: Developmental competence of oocytes from prepubertal females is lower than those from adult females. Oocyte development competence is positively related to follicular diameter. Most of the follicles of prepubertal goat ovaries are smaller than 3 mm. The aim of this study was to compare oocytes of two follicle sizes (< 3 mm and ≥ 3 mm) from prepubertal goats with oocytes from adult goats in relation to their in vitro production and quality of blastocysts. Oocytes from prepubertal goats were obtained from slaughterhouse ovaries and selected according to the follicle diameter whereas oocytes from adult goats were recovered in vivo by LOPU technique without prior selection of follicle size. COCs were IVM for 27 h, IVF at the conventional conditions with fresh semen and presumptive zygotes were cultured in SOF medium for 8 days. Blastocysts obtained were vitrified and after warming their blastocoele re-expansion and the ploidy by FISH technique were assessed. We found significant differences between blastocysts yield of oocytes recovered from follicles smaller than 3 mm of prepubertal goats compared to those from adult goats (5.45% vs 20. 83%, respectively) however, these differences disappear if oocytes were recovered form large follicles (18.07%). A total of 28 blastocysts were analysed and 96.43% showed mixoploidy. Age did not affect the number of embryos with abnormal ploidy or blastocyst re-expansion after warming. Furthermore, the percentage of diploid blastomeres per embryo was similar in the 3 groups studied, adult, prepubertal from follicles ≥ 3 mm and < 3 mm (68.6%, 80.8% and 73.6%, respectively). In conclusion, IVP of blastocysts coming from follicles larger than 3 mm of goats 45 days old were not different to the blastocysts produced from adult goats, both in terms of quantity and quality. [Copyright &y& Elsevier]

Abstract: Oocytes secrete soluble paracrine factors called Oocyte Secreted Factors (OSFs) which regulate the cumulus cell phenotype. Follicle populations in ovaries from prepubertal females have smaller diameters than their adult counterparts. Oocytes from small follicles are less competent than those from large follicles. The aim of this study was to investigate, in prepubertal goats, the effect of OSFs secreted by denuded oocytes (DOs) from small (<3 mm) or large (≥3 mm) follicles during IVM on embryo development and the blastocyst quality of cumulus-oocyte complexes (COCs) from small follicles and to determine if GDF9 participates in this process. Treatment groups were: (A) COCs non selected by their follicle size (control group); (B) cumulus oocytes complexes from small follicles (SFCOCs), (C) cumulus oocytes complexes from small follicles co-cultured with denuded oocytes from small follicles (SFCOCs + SFDOs), and (D) cumulus oocytes complexes from small follicles co-cultured with denuded oocytes from large follicles (SFCOCs + LFDOs). The effect of the addition of kinase inhibitor SB-431542, which antagonizes GDF9, was tested in A, C, and D treatment groups. Co-cultured SFCOCs with SFDOs or LFDOs significantly augmented the blastocyst rate in comparison to SFCOCs alone (15.77%, 17.39% vs. 10.31%, respectively). Blastocysts from SFCOCs + LFDOs group showed higher rates of tetraploid nuclei than blastocysts from SFCOCs and the control group (14.43% vs. 5.45% and 5.24%, respectively; P < 0.05). However, we did not observe differences in the hatching rate, mean cell number or embryo cryotolerance (P > 0.05) between the four treatment groups. The addition of SB-431542 during IVM did not have any effect on blastocyst rate (P > 0.05). In conclusion, in prepubertal goats, COCs with a low embryo developmental competence as a consequence of follicle size can be improved by coculturing them with denuded oocytes from both small and large follicles. GDF9 does not seem play a role in this improvement. [Copyright &y& Elsevier]

Abstract: The aim of this study was to assess the following parameters in prepubertal goat oocytes of different follicle diameter (≥3 mm, <3 mm, control): oocyte diameter, early (Annexin-V) and late (TUNEL) apoptosis, embryo development and chromosomal ploidy of these blastocysts using Fluorescence In Situ Hybridization (FISH). Before in vitro maturation, oocytes were measured and stained with Annexin-V or TUNEL. The rest of the oocytes were matured, fertilized, and cultured in vitro for 8 days. Oocytes from follicles of ≥3 mm showed greater mean oocyte diameter (128.27 ± 7.20 μm vs. 125.35 ± 7.59 μm), higher percentages of TUNEL positive (42.86 vs. 24.23%), higher cleavage (47.85 ± 3.98 vs. 23.07 ± 2.44 %) and blastocyst rates (19.77 ± 3.04 vs. 4.11 ± 1.10 %) than oocytes from follicles of <3 mm.. Blastocyst mean cell numbers did not show differences between follicular groups (123.83 ± 49.62 vs. 104.29 ± 36.09 for follicles of ≥3 mm and <3 mm, respectively). A total of 54 blastocysts with 7084 nuclei were hybridized with specific probes to chromosomes X and Y. Ninety-eight percent (98%) of the embryos presented at least one cell carrying an abnormal number of chromosomes, but 78% of them presented less than 25% of chromosomal abnormal cells. No differences in the percentage of blastocysts with abnormal ploidy were found in embryos produced from oocytes of different follicle diameter. [Copyright &y& Elsevier]

Abstract: Oocyte quality is the main factor that determines blastocyst yield; any factor that could affect it, such as apoptosis, could impair subsequent embryonic development. Our aim was to investigate the incidence of apoptosis in prepubertal goat oocytes and cumulus cells, assessed by Annexin-V staining and TUNEL assay, and their effect on embryo development. Oocyte-cumulus complexes (COCs) from slaughtered females were collected and classified depending on COC morphology as: Healthy (H) and Early Atretic (EA). Each one of these groups was classified depending on oocyte diameter: A: 110–125μm, B: 125–135μm and C: >135μm. The COCs were IVM for 27h, IVF with fresh semen and IVC for 8 days after insemination. Apoptosis analyses were performed before and after maturation. Annexin-positive oocytes decreased with diameter in the EA class (immature oocytes: A: 42.6%; B: 30.3%; C: 21%; IVM-oocytes: A: 17.5%; B: 4.8%; C: 0%), while TUNEL assay showed a decrease of apoptosis in the largest oocytes before and after IVM only in Healthy oocytes (immature oocytes: A: 51.5%; B: 43.3%; C: 12.1%; IVM-oocytes: A: 31.7%; B: 12%; C: 0%). Blastocyst rate increased with increasing oocyte diameter, and it was higher in H than in EA oocytes (Healthy; A: 0%; B: 5.3%; C: 14.4%; Early atretic: A: 0.3%; B: 4.1%; C: 5.1%). Oocyte diameter and COC morphology had no effect on the percentage of apoptosis in blastocyst cells. In conclusion, oocyte developmental competence in prepubertal goats is influenced by oocyte diameter and COC morphology. [Copyright &y& Elsevier]

Abstract: ICSI and embryo biopsy are routine methods used for assisted reproduction. However, their impact on embryo quality is still poor studied. Moreover, oocyte size is also a crucial factor for blastocyst production. In this study effect of oocyte size, ICSI and embryo biopsy was assessed in terms of incidence of apoptosis and blastocyst development. IVM-oocytes from prepubertal goats were fertilized by ICSI or IVF. Embryos obtained were divided depending on oocyte size, biopsied at day-4 post-insemination/injection and cultured for additional 4–5 days. Apoptotic cell number was assessed by TUNEL staining in day-4 embryos and blastocysts obtained. In each diameter group, ICSI did not affect embryo development, blastocyst cell number and embryo apoptotic grade in comparison to IVF. Embryo biopsy did not affect blastocyst rate and apoptotic cell number, but decreased blastocyst cell number (P =0.0018). Moreover, there was a negative relationship between blastocyst cell number and apoptotic grade (P <0.05). In conclusion, ICSI and embryo biopsy do not have negative effect on embryo quality and development. However, oocyte size has a positive relationship on blastocyst yield and quality. [Copyright &y& Elsevier]