Tubulin posttranslational modifications in in vitro matured prepubertal and adult ovine oocytes.

Microtubules (MTs), polymers of alpha/beta-tubulin heterodimers, are involved in crucial functions in eukaryotic cells. MTs physiology can be influenced by a variety of post-translational modifications (PTMs), including tyrosination, detyrosination, delta 2 modification, acetylation, polyglutamylation, polyglycylation. In mammalian oocytes, MTs are essential for meiosis, regulating the formation of meiotic spindle and chromosomes movements. Considering that the patterns of tubulin PTMs (tyrosination, detyrosination, acetylation, polyglutamylation and delta 2 modification) have not been investigated in ovine oocytes, this study has been designed to investigate their presence and quantification in in vitro matured (IVM) adult and prepubertal ovine oocytes. Oocytes from adult and lamb Sarda ewes, regularly slaughtered at the local abattoir, were in vitro matured, fixed, and processed by indirect immunofluorescence and confocal microscopy analyses at metaphase II stage. Our results revealed a well detectable signal for total, tyrosinated and acetylated α-tubulin in meiotic spindle of both sheep and lamb oocytes. On the other hand, no immunopositivity were appreciable for detyrosinated, polyglutamylated, and delta 2 tubulin in meiotic spindle of both sheep and lamb oocytes. As regard the tyrosinated and the acetylated α-tubulin PTMs, through the quantification of the fluorescence intensity, we did not find significant differences in their expression in meiotic spindle of sheep, while in lamb the acetylated tubulin levels were predominant in comparison with tyrosinated. Our results in addition to investigating for the first time the different tubulin PTMs in the spindle organization of ovine oocytes, showed a different microtubule pattern between adult and prepubertal oocytes. The microtubule cytoskeleton survey may thus suggest further cues to better understand skill-related problems in in the acquisition of oocyte competence.

Changes in renal hemodynamics of undernourished fetuses appear earlier than IUGR evidences.

The present study used a sheep model of intrauterine growth restriction, combining maternal undernutrition and twinning, to determine possible markers of early damage to the fetal kidney. The occurrence of early deviations in fetal hemodynamics which may be indicative of changes in blood perfusion was assessed by Doppler ultrasonography. A total of 24 sheep divided in two groups were fed with the same standard grain-based diet but fulfilling either their daily maintenance requirements for pregnancy (control group; n=12, six singleton and six twin pregnancies) or only the 50% of such quantity (food-restricted group; n=12; four singleton and eight twin pregnancies). All the fetuses were assessed by both B-mode and Doppler ultrasonography at Day 115 of pregnancy. Fetal blood supply was affected by maternal undernutrition, although there were still no evidences of brain-sparing excepting in fetuses at greatest challenge (twins in underfed pregnancies). However, there were early changes in the blood supply to the kidneys of underfed fetuses and underfed twins evidenced decreases in kidney size.

Glucogenic treatment creates an optimal metabolic milieu for the conception period in ewes.

This study determined the influence of a short-term glucogenic nutritional treatment on circulating concentrations of glucose, insulin, insulin-like growth factor 1 (IGF-1), nonesterified fatty acids (NEFA), and urea, and on their correspondent levels in follicular fluid (FF) collected 12 h after the end of the treatment. After estrous synchronization with intravaginal progestagen-impregnated sponges, 20 Sarda ewes were randomly allocated into two experimental groups (GLU and WAT) and, from day 7 to day 10 (day 0 = day of sponge removal), the GLU group was gavaged with a glycogenic mixture, whereas the WAT group was gavaged with water (control group). Follicular development was stimulated by FSH administration from day 8 to 10. At day 11, ovaries were collected and follicular fluid processed. Plasma changes were assessed from day 6 to 11. In GLU group, circulating concentration of glucose (P < 0.0001), insulin (P < 0.0001), and IGF-1 (P < 0.01) rose significantly, whereas NEFA and urea concentrations decreased (P < 0.0001), as compared with controls. In particular, in FF the higher glucose concentrations found in GLU ewes compared with controls (P < 0.0001) were not accompanied by any increase in insulin and IGF-1 concentrations. NEFA (P < 0.0001) and urea (P < 0.0001) were lower in FF of GLU than WAT group, although NEFA clearance in the ovary proved to be less efficient than at the systemic level. No significant difference between groups was found in FF concentrations of pregnancy-associated plasma protein A (a protease regulating the levels of free IGF-1 in follicles), glutathione, and in its total antioxidant capacity. These results suggest that glycogenic mixture administration creates a suitable follicular microenvironment for the conception period in dairy ewes.

Glucogenic supply increases oocyte developmental competence in sheep.

The present study aimed to determine the influence of a glucogenic supply on oocyte developmental competence. Oestrous cycles were synchronised in 22 Sarda ewes by the insertion (Day 0) of one intravaginal progestagen-impregnated sponge that was removed after 6 days. After removal, the ewes were randomly allocated into two experimental groups (treated and control ewes) and, from Day 7 to Day 11, treated ewes received oral administration of a glucogenic mixture, whereas control animals received water. Follicular development was stimulated by FSH administration from Days 8 to 10. Glucose metabolism was assessed from Days 7 to 11, whilst follicle and corpus luteum growth dynamics and functionality were evaluated between Days 6 and 11. At Day 11 ovaries were collected and processed for in vitro embryo production. Glucogenic treatment increased both the plasma levels of glucose, progesterone, oestradiol and the number of 2-3-mm follicles (P < 0.05). Higher fertilisation and blastocyst rates (P < 0.05) were obtained after IVM of oocytes recovered from treated ewes compared with control ones. In conclusion, glucogenic treatment modifies follicle and corpus luteum functionality and improves oocyte quality, as evaluated by in vitro developmental kinetics and blastocyst output.

Effect of "ice blockers" in solutions for vitrification of in vitro matured ovine oocytes.

Polymers have been used as a substitute for serum in vitrification solutions for embryos and oocytes. This study was designed to replace serum with defined commercial macromolecules in vitrification solution for in vitro matured ovine oocytes. Oocytes were cryopreserved in two vitrification solutions (16.5 percent ethylene glycol + 16.5 percent dimethyl sulphoxide) supplemented with 1 percent of SuperCool X-1000 and 1 percent SuperCool Z-1000 (Ice Blockers) or 20 percent foetal calf serum (FCS). After warming, oocytes viability and developmental potential after processing for in vitro embryo production were assessed. The number of viable oocytes (87.4 percent and 85.9 percent), cleaveage rates (21.4 percent and 19.6 percent) and blastocyst development rates (4.8 percent and 4.5 percent) were similar for Ice Blockers and FCS, respectively. On the basis of these findings, it may be concluded that combined use of Ice Blockers (SuperCool X-1000 and SuperCool Z-1000) as supplementation in vitrification solution offers similar results to serum for vitrification of in vitro matured ovine oocytes.

Differences in semen freezability and intracellular ATP content between the rooster (Gallus gallus domesticus) and the Barbary partridge (Alectoris barbara).

This study aimed to compare viability, ATP content, and DNA integrity of rooster (Gallus gallus domesticus) and Barbary partridge (Alectoris barbara) fresh and frozen spermatozoa in order to identify factors possibly related to differences in semen freezability. Ejaculates were obtained from March to May by the abdominal massage method from 3 adult roosters and 12 adult Barbary partridges. Semen was frozen with different cryoprotectants using Lake's diluents as a base medium: 1) glycerol 11%; 2) glycerol 11% and trehalose 70 mmol/L; 3) dimethylacetamide (DMA) 6%; 4) DMA 6% and trehalose 70 mmol/L. Both fresh and frozen semen showed a lower viability and higher intracellular ATP concentrations in the Barbary partridge compared with the rooster (P < 0.05). In the Barbary partridge, semen viability after thawing did not differ among the 4 media used, but glycerol showed positive effects in avoiding a significant loss of ATP after thawing, compared with DMA containing media (P < 0.05). On the other hand, in the rooster a higher viability was recorded when semen was frozen in glycerol containing media compared to DMA (P < 0.0001), while ATP values significantly decreased after thawing (P < 0.05) without showing any differences among the semen frozen in the 4 different media. DNA integrity, as evaluated by the comet assay, was assessed only in frozen semen. In the Barbary partridge, mean scored parameter did not differ significantly among semen frozen in the 4 different media. In the rooster DNA fragmentation was higher in DMA ctr medium compared with the other media and with values found in Barbary partridge semen frozen in the same medium (P < 0.001). In both species, the addition of trehalose did not show any positive effects on viability, ATP levels and DNA integrity after thawing. In conclusion, species-related differences in semen features exist between the rooster and the Barbary partridge and the wide variation observed in ATP levels may account for differences in semen freezability between the two species.

Recovery of COCs from ovaries with high follicle numbers enhances in vitro embryo yield in sheep.

The aim of the present study was to investigate the correlation between the number of ovarian follicles and in vitro embryo development and quality in sheep. Sarda ewe ovaries were classified according to the number of follicles: or=8 (High). IVM, IVF and IVC were performed under standard conditions. Cleavage rate and blastocyst development were assessed 48 h after fertilization and on Days 6, 7 and 8 of culture, respectively. Expanded blastocysts were vitrified; blastocoel re-expansion and hatching rates were assessed at 8, 16 and 72 h post-thawing and hatched blastocysts were analyzed with the TUNEL assay. In a subset of thawed blastocysts the incorporation of amino acids was evaluated. The proportion of ovaries varied significantly among the three groups (ANOVA F=12.20, P=0), and more ovaries (59%) were assigned to the Low group than to the Intermediate (28%; ANOVA F=8.19, P=0.009) and High group (13%; ANOVA F=18.63, P=0), (High vs. Intermediate F=6.31, P=0.020). The three groups statistically differed in the proportion of total blastocysts (chi(2)(2)=22.616, P=0.00), of blastocysts produced on Days 6 (chi(2)(2)=6.829, P=0.033) and 7 (chi(2)(2)=6.810, P=0.033), while no difference was found in the proportion of blastocysts obtained on Day 8 (chi(2)(2)=3.874, P=0.144) of culture after fertilization. A higher proportion of total blastocysts was obtained from the High (44%) compared with the other two groups (Low: 28%, chi(2)(2)=22.629, P=0; Intermediate: 33%, chi(2)(2)=7.266, P=0.007), while the Low and Intermediate groups did not statistically differ either in the total blastocyst output (chi(2)(2)=3.384, P=0.066), nor in the number of blastocysts produced on Days 6 (Low: 7%, Intermediate: 9%; chi(2)(2)=0.874, P=0.35), 7 (Low: 14%, Intermediate: 16%, chi(2)(2)=1.256, P=0.26) and 8 (Low: 6%, Intermediate: 7% chi(2)(2)=0.554, P=0.45) of culture. The High group produced a significantly higher percentage of embryos on Days 6 (High: 13%, Low: 7%; chi(2)(2)=6.840, P=0.009) and 7 (High: 21%, Low: 14%; chi(2)(2)=6.806, P=0.009) of culture post-insemination than the Low group. The three categories did not differ in the blastocoel re-expansion (chi(2)(2)=0.095, P=0.95) and hatching rates (chi(2)(2)=0.754, P=0.68) after 72 h post-warming, in the total number of cells per blastocyst (ANOVA F=1.12, P=0.337) and in the (F=0.46, P=0.639) incorporation of amino acids. The number of TUNEL-positive cells per embryo was higher (ANOVA F=4.32, P=0.022) in the Low group compared to the other groups. In conclusion, high ovarian follicle number enhances in vitro embryo output in sheep, but has no effect on blastocyst quality.

A new selection criterion to assess good quality ovine blastocysts after vitrification and to predict their transfer into recipients.

The feasibility to accurately select viable embryos would be valuable for improving pregnancy rates and avoiding futile transfer attempts. The aim of our study was to assess if in vitro-produced embryo quality could be determined by the timing of blastocoelic cavity re-expansion after vitrification, warming, and in vitro culture using sheep as a model. Blastocysts were produced in vitro, vitrified/warmed, and cultured in TCM-199 plus 10% FCS for 72 hr. Embryos were divided into two groups: re-expanded within 8 hr (A) and from 8 to 16 hr (B) of IVC after warming. Fast re-expanded blastocysts showed higher in vitro hatching rates and total cell number calculated on the hatched blastocysts compared with slow re-expanded ones (P < 0.01). Peroxide status evaluation (P < 0.01) and TUNEL test (P < 0.05) revealed a higher number of positive cells in group B compared with group A. The quantitative analysis of protein synthesis revealed a higher synthesis in fast compared with slow re-expanded embryos (P < 0.05). Quantitative RT-PCR showed that 90-kDa Heat Shock Protein beta was more expressed in group A than in group B (P < 0.05), while the quantity of P34(cdc2), Cyclin b, Aquaporin 3, Na/K ATPase, and Actin did not differ between the two groups. Pregnancy rates after transfer to synchronized recipients were higher in fast compared to slow re-expanded blastocysts (P < 0.05). Our results evidenced that timing of blastocoelic cavity re-expansion after vitrification/warming and in vitro culture can be considered as a reliable index of in vitro produced embryo quality and developmental potential.

Effect of vitrification solutions and cooling upon in vitro matured prepubertal ovine oocytes.

The vitrification procedure effects on molecular and cytoskeletal components and on developmental ability of in vitro matured prepubertal ovine oocytes were evaluated. MII oocytes were divided into three groups: (1) vitrified in cryoloops (VTR); (2) exposed to vitrification solutions and rehydrated without being plunged into liquid nitrogen (EXP); (3) without further treatment as a control (CTR). Two hours after treatment, membrane integrity, assessed by propidium iodide/Hoechst staining, was lower in VTR and EXP than in CTR (70.6%, 88.5% and 95.2%, respectively). Cleavage rate after fertilization was statistically different among all groups (21.4%, 45.4% and 82.8% for VTR, EXP and CTR groups respectively; P<0.01). Blastocyst rate in VTR (0.0%) and EXP (2.8%) groups was lower (P<0.01) than in CTR (22.8%). Maturation promoting factor activity was lower (P<0.01) in VTR and EXP groups compared with CTR at both 0 h (82.2%, 83.6% and 100%, respectively) and 2 h (60% and 53.9% and 100%, respectively) after warming. Immediately after warming VTR and EXP oocytes showed a lower rate of normal spindle and chromosome configuration compared to CTR (59.1%, 48.0% and 83.3%, respectively; P<0.01). After 2 h of culture in standard conditions the percentage of oocytes with normal spindle and chromosome organization decreased in both VTR and EXP groups compared to CTR (36.4%, 42.8% versus 87.5%, respectively). In conclusion the exposition to the tested cryoprotectant solution and the vitrification in cryoloops modified cytoskeletal components and alter biochemical pathways that compromise the developmental capacity of prepubertal in vitro matured ovine oocytes.

A low oxygen atmosphere during IVF accelerates the kinetic of formation of in vitro produced ovine blastocysts.

Among the factors that affect in vitro embryo development, oxygen atmosphere is considered to be of great influence. In this study, we evaluated the influence of two different oxygen atmospheres during in vitro fertilization (IVF) of ovine oocytes on their developmental capacity and quality assessed by cryotolerance. Cumulus oocyte complexes derived from ovaries of slaughtered sheep were matured in vitro and subsequently fertilized under low (5%) or high (20%) oxygen atmospheres, and cultured in SOF + aa + 0.4% BSA in 5% CO2 and 5% O2 up to blastocyst stage. The cleavage rates obtained in the fertilization system at 20% O2 were significantly higher than those obtained in the 5% O2 fertilization system (61.2% vs 50.8%; p < 0.01). The distribution of cleaved oocytes at 22, 26 and 40 h of culture intervals was not different in the low or high O2 atmosphere (31.4%, 26.4% and 42.1% vs 28.0%, 29.3% and 42.7% respectively). Blastocysts output on the 6th day post-fertilization (dpf) was significantly higher when oocytes were fertilized under 5% O2 concentration (63.04% in 5% O2 vs 47.36% in 20% O2), while on the 7th dpf the higher number of blastocysts was obtained in the 20% O2 system (35.10%.in 20% O2 vs 26.09% in 5% O2). After vitrification no differences were observed between low or high oxygen atmosphere in the viability rates of blastocysts obtained on day 6 (93.6% vs 96.5%), on day 7 (46.3% vs 41.7%) and on day 8 (11.1% vs 6.6%). After differential staining, no significant differences were observed in the total cell number and inner cell mass and trophoblastic cells ratio of blastocysts produced on 6 dpf (189.6 +/- 51.3 and 0.260 +/- 0.07 vs 223.3 +/- 45.6 and 0.277 +/- 0.09), on 7 dpf (168.3 +/- 25.1 and 0.316 +/- 0.06 vs 172.1 +/- 33,6 and 0.320 +/- 0.06) and on 8 dpf (121.2 +/- 23,8 and 0.302 +/- 0.03 vs 117.0 +/- 35.1 and 0.313 +/- 0.04) under low or high oxygen atmosphere respectively). In conclusion, our data suggest that low oxygen atmosphere during IVF affects positively the production of high quality ovine blastocysts.

Effects of trehalose co-incubation on in vitro matured prepubertal ovine oocyte vitrification.

Our aim was to evaluate if loading prepubertal ovine oocyte with trehalose would impact on their further developmental potential in vitro and if it would improve their survival to vitrification procedures. COCs matured in vitro with (TRH) or without (CTR) 100mM trehalose were tested for developmental potential after in vitro fertilization and culture. Trehalose uptake was measured by the antrone spectrophotometric assay. No differences were recorded between the two experimental groups in fertilization rates (91.1 CTR vs 92.5% TRH), cleavage rates calculated on fertilized oocytes (96.1 CTR vs 95.4% TRH), first cleavage kinetic (56.1 CTR vs 51% TRH), and blastocyst rates (14.3 CTR vs 13.0% TRH). Anthrone assay revealed that in TRH group trehalose concentration/oocyte was 2.6microM. MII oocytes were then vitrified using cryoloops in TCM 199 containing 20% FCS, sucrose 0.5M, 16.5% Me(2)SO, 16.5% EG and plunged in LN(2). After warming, oocytes from TRH and CTR groups were tested for membrane integrity using the propidium iodide (PI)/Hoechst differential staining, and for developmental ability after in vitro fertilization. Trehalose in maturation medium affected membrane resistance (P<0.01) to vitrification/warming but not fertilization and cleavage rates. The differential staining showed a lower number of PI positive cells in TRH group compared to CTR one (14.3 vs 24.7%, respectively). Fertilization rates and cleavage rates did not differ between the two groups (55.3 and 41% for TRH and 47.7 and 41.7% for CTR, respectively). In conclusion trehalose in maturation medium stabilizes cell membranes during vitrification/warming of prepubertal ovine oocytes but does not affect fertilization and cleavage rates after warming.

Cryopreservation of European Mouflon (Ovis Gmelini Musimon) semen during the non-breeding season is enhanced by the use of trehalose.

The influence of trehalose on European mouflon spermatozoa cryopreservation during the non-breeding season was tested. Semen was frozen in two different extenders: (a) recommended Tris-based ram extender (CTR); (b) CTR extender supplemented with trehalose 0.147 mm (TRH). Sperm viability and acrosome integrity were assessed using propidium iodide and fluorescein isothiocynate labelled Pisum Sativum agglutinin. Trehalose significantly enhanced sperm viability after thawing compared with CTR extender (62.7% vs 51.8%; p < 0.05), whereas no differences were observed on acrosome integrity (42.9% vs 42.1%). Trehalose influence was also evidenced in the in vitro fertility test performed with sheep oocytes matured in vitro. Both fertilization rates (60.9% TRH vs 43.6% CTR; p < 0.05) and cleavage rates (58% TRH vs 39.8% CTR; p < 0.001) were higher for trehalose frozen semen compared with control extender frozen semen. A higher percentage of zygotes resulting from fertilization with trehalose cryopreserved semen presented the first cleavage earlier if compared with the group fertilized with control semen (48.7% vs 31.5%, respectively; p < 0.01). This result was confirmed by embryo kinetic development. Fertilization with trehalose cryopreserved semen leaded to an higher percentage of blastocysts (40.2% vs 27.8% CTR; p < 0.05), and enhanced in particular the number of blastocysts that developed on the day 6th of culture (28.6% vs 17% CTR; p < 0.05). Our data demonstrated that, during mouflon non-breeding season, trehalose extender enhances spermatozoa viability and its in vitro fertilizing capacity, allowing the production of an higher number of blastocysts.

Vitrification devices affect structural and molecular status of in vitro matured ovine oocytes.

We evaluated the effect of three different cryodevices on membrane integrity, tubulin polymerization, maturation promoting factor (MPF) activity and developmental competence of in vitro matured (IVM) ovine oocytes. IVM oocytes were exposed during 3 min to 7.5% DMSO and 7.5% ethylene glycol (EG) in TCM199 and 25 sec to 0.5 M sucrose, 16.5% DMSO and 16.5% EG, loaded in open pulled straws (OPS), cryoloops (CL) or cryotops (CT) and immersed into liquid nitrogen. Untreated (CTR) or exposed to vitrification solutions but not cryopreserved (EXP) oocytes were used as controls. After warming, double fluorescent staining evidenced a lower membrane integrity in vitrified groups compared to the controls (P < 0.01). After in vitro fertilization and culture OPS and CL groups evidenced a lower cleavage rate than CT and controls (P < 0.01) while blastocysts were obtained only in CL and EXP, at a lower rate than CTR (P < 0.01). All vitrified groups showed alterations in spindle conformation, which were partially recovered in OPS and CT groups. MPF activity was lower in treated compared to CTR and CT showed the lowest value (P < 0.01). After 2 hr culture MPF activity was restored in all groups except CT. Parthenogenetic activation was higher in treated compared to CTR and CT evidenced the highest value. Our results indicate that cryodevice influences not only the ability to survive cryopreservation but is also associated with molecular alterations which affect developmental competence.

Relations between relative mRNA abundance and developmental competence of ovine oocytes.

The present study was conducted to investigate the relation between in vitro developmental competence and the expression of a panel of developmentally important genes in germinal vesicle (GV) stage oocytes. One-month-old prepubertal and adult sheep oocytes were used as models of low and high quality gametes, respectively. Cumulus-oocyte complexes (COCs) derived from lambs and ewes were in vitro matured and fertilized, and their cleavage rate at 22, 26, and 32 hr post fertilization and the blastocyst yield were observed to assess their developmental potential. In parallel, the relative abundance (RA) of 11 genes was analyzed by semi-quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) assay in the two groups of oocytes. We observed similar maturation and fertilization rates in the two groups, but a significant lower rate of cleaved prepubertal oocytes (P < 0.05), a general delay in the timing of their first division (P < 0.01), and a lower blastocysts production (P < 0.05). The analysis of gene expression evidenced no difference in the RA of four transcripts [superoxide dismutase (SOD), ubiquitin, beta-actin, cyclin B] in the two classes of oocytes, but a statistically lower RA of seven messenger RNAs (mRNA) [Na(+)K(+)ATPase, p34(cdc2), Glucose-transporter I (Glut-1), Activin, Zona Occludens Protein 2 (PanZO2), Poli(A)Polymerase (PAP), E-Cadherin (E-Cad)] in the prepubertal oocytes compared to the adult ones. The present data show for the first time in the ovine species that the lower developmental competence is associated with deficiencies in the mRNAs storage during the oocyte growth.

Effects of progestagens on follicular growth and oocyte developmental competence in FSH-treated ewes.

Previous research has reported evidence for negative effects of progestagens on follicular growth and oocyte competence. In the present study, negative effects of progestagens on follicular growth and oocyte developmental competence were assessed. During the breeding season, 20 Sarda ewes were treated with two doses of cloprostenol, 10 days apart, to assure the presence of a corpus luteum (CL). On day 5 after the second cloprostenol dose, 10 ewes were treated with a progestagen sponge while 10 females remained untreated. Starting on day 7 after the second cloprostenol dose, all the ewes were treated with 6 equal doses of 24 I.U. of FSH (Ovagen, ICP, NZ), every 12h. The number of follicles > or =2mm in diameter increased (P<0.0005) in all the ewes from 24 h before to 60 h after the first FSH dose (from 12.8+/-1.1 to 23.4+/-1.3 in treated and from 12+/-0.6 to 22+/-1.2 in untreated ewes, n.s.). There were no significant differences in follicle dynamics between groups, but concentrations of estradiol in control ewes were higher than in the progestagen group (P<0.05). Twelve hours after the last FSH dose, oocytes were collected by ovum pick-up. Recovery rates were lower for progestagen-treated ewes (71.1 versus 83%; P<0.001). After IVP procedure, cleavage rate was also lower in the progestagen group (39.1 versus 82.6%; P<0.001). Furthermore, blastocysts output revealed that oocyte developmental competence was lower in progestagen group (17.3 versus 30.4%; P=0.245), although differences were not significant. These results suggest deleterious effects from progestagen on oocyte developmental competence and set the basis for new protocols for in vitro embryo production.

Association between numbers of ovarian follicles in the first follicle wave and superovulatory response in ewes.

The aim of this study was to examine the variability in the number of ovarian follicles in sheep and to determine if the average number of follicles per day influences the response to superovulation and resulting embryo quality. Ewes (n=83) were synchronized and the number of follicles (> or =2 mm diameter) in the ovaries were counted daily between Days 0 and 4 of the oestrous cycle using transrectal ultrasonography. Fourteen to 21 days later, 47 ewes were randomly chosen from the group and were treated with an intravaginal progestagen pessary for 12 days and superovulated with 1500 IU eCG administered as a single injection 10 days after sponge insertion. Ewes were mated and reproductive tracts were recovered after slaughter on Day 6 of pregnancy. The number of corpora lutea was counted, uterine horns were flushed and the morphology and developmental stage of the recovered oocytes/embryos was assessed. The mean daily number (+/-S.D.) (> or =2 mm diameter) of follicles per ewe was 8.5+/-2.8 (ranging between 3 and 16). After superovulation animals with few follicles (Low group: <8 follicles/day; n=21) had fewer (P<0.005) corpora lutea, total structures (unfertilized oocytes and embryos), good quality and total embryos compared to animals with many follicles (High group: > or =8 follicles/day; n=23). No difference was found in the proportion of good quality embryos (relative to the total number; Low 0.68+/-0.11 versus High 0.79+/-0.08; P=0.21) between the two groups, or the recovery rate, the number of unfertilized oocytes or the number of poor quality embryos per animal. We conclude that ewes with a higher number of follicles (> or =8) during the first follicular wave had a better superovulatory response (in terms of corpora lutea and high quality embryos) 2-3 weeks later; however, there was no relationship between the number of follicles and the proportion of good quality embryos per animal.

Delay on the in vitro kinetic development of prepubertal ovine embryos.

In the present study we characterize the developmental potential of prepubertal and adult ovine oocytes, analyzing the developmental speed to two-cell and blastocyst stages and its relationship with hatching from the zona pellucida, development after vitrification and the number and allocation of inner mass and trophoblastic cells. Prepubertal and adult ovine oocytes were matured and fertilized in vitro and first cleavage rates at 22, 26 and 32 h were recorded. Cleaved oocytes were cultured and blastocyst production was assessed at 6-9 days post-fertilization (dpf). Blastocysts from the two sources obtained on different days were divided into two groups: the first was vitrified, warmed and cultured in vitro to evaluate re-expansion of the blastocoelic cavity; blastocysts of the second were cultured separately to allow for hatching and count of trophoblastic and inner mass cells of hatched blastocysts by differential staining. We observed a significantly lower rate (P < 0.01) of cleaved prepubertal oocytes at 22 and 26 h after fertilization while it was higher (P<0.01) at 32 h than in the adult ones. Adult blastocyst production was significantly lower (P < 0.01) in prepubertal than in adult groups and began on the seventh dpf, later (P < 0.01) than in the adult group, where they appeared on the sixth dpf. Prepubertal blastocysts hatched at a lower rate than the adult ones (P < 0.01) and in both experimental groups faster blastocysts showed a higher (P < 0.01) hatching rate. Similarly, prepubertal derived blastocysts showed lower viability after vitrification (P < 0.01) compared to the adult counterparts, and in particular slower embryos had reduced viability after vitrification compared to the fastest (P < 0.01). Cell number was not different between blastocysts of both groups obtained at 6 and 7 dpf, which were higher (P < 0.01) than those obtained at 8 and 9 dpf. The ICM/trophoblast cell ratio was similar in 6- and 7-day obtained blastocyst and increased (P < 0.01) in those obtained 1 or 2 days later. These findings show that differences in kinetic development between prepubertal and adult derived embryos reflect differences in developmental capacity of the oocytes from which they derive and could be indicative of embryo quality.

GnRH antagonist enhance follicular growth in FSH-treated sheep but affect developmental competence of oocytes collected by ovum pick-up.

This study evaluates the in vitro developmental competence of oocytes collected by ovum pick up (OPU) from sheep treated with GnRH antagonists (GnRHa) and high doses of FSH. Eighteen Sarda ewes were treated with progestagen sponges (day 0). On day 7, 10 ewes received 3 mg of GnRHa s.c., while 8 served as control receiving saline. On day 10, all animals were treated with 96 IU of ovine FSH in four equal doses given i.m. every 12 h. We monitored follicular development by ultrasonography, twice daily from day 7 to 11, and found that GnRHa induced a significant increase in the number of total follicles in 72 h (11.7+/-0.9 to 21+/-2.4, r(2)=0.598, P<0.0001), while this number remained stable in control sheep. We found that FSH induced a significant rise in the number of follicles in both groups; but always higher (P<0.05) in GnRHa treated sheep, confirming that GnRHa enhances ovarian response to exogenous FSH stimulation. Twelve hours after the last FSH dose, oocytes were collected by OPU. Recovery percentage, morphological quality, ability to resume meiosis, fertilization and cleavage were similar in oocytes from treated and untreated sheep. However, the final blastocysts output was lower in GnRHa group (10.1% versus 27.4% in control group; P<0.05). In addition, re-expansion rates after vitrification, thawing and in vitro culture were lower in GnRHa treated ewes, although differences did not reach statistical significance (55.5% versus 74.1% in GnRHa treated and in control sheep, respectively).

Induction of the presence of corpus luteum during superovulatory treatments enhances in vivo and in vitro blastocysts output in sheep.

This report offers the results of two experiments developed to test possible benefitial effects of the presence of corpus luteum (CL) on in vivo and in vitro sheep embryo production; using two different breeds treated with two different protocols by two different teams at two different centres. In the first trial, estrus was synchronized in 11 ewes with two doses of cloprostenol, 10 days apart. On day 1 after estimated ovulation, sheep were treated with progestagen sponges and superovulated with eight decreasing doses (26.4 units NIH-FSH-S1 x 3, 22.0 units x 2, and 17.6 units x 3) of ovine FSH injected twice daily. Ovulation rate and number of embryos obtained in vivo were compared to those from 12 control ewes without cloprostenol treatment. Presence of a CL improves the number of transferable embryos (7.4+/-0.6 versus 4.1+/-0.6 in control ewes, P < 0.05). The second trial investigated the effects of the presence of CL on embryos produced in vitro from six ewes bearing CL and six ewes without CL at start a superovulatory treatment consisting of 96 units of ovine FSH administered in four equal doses given every 12 h. There were not detected effects of the CL on the number and size of follicles or in the number, morphology and ability to resume meiosis of their oocytes. However, oocytes from ewes with CL showed higher rates of fertilization (73.5 versus 45.5%, P < 0.005), higher development to blastocyst (35.8 versus 19.3%, P < 0.01) and higher hatching rates after vitrification (80.0 versus 25.0%, P < 0.05).

M-phase promoting factor (MPF) and mitogen activated protein kinases (MAPK) activities of domestic cat oocytes matured in vitro and in vivo.

This work was undertaken in order to examine M-phase promoting factor (MPF) and mitogen-activated protein kinases (MAPK) activities during meiotic progression of cat oocytes cultured in two different media for two different incubation times and preovulatory cat oocytes that reached MII in vivo. Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 or SOF for 24 h and 40 h. In vivo matured oocytes were recovered by follicular aspiration from ovaries of domestic cats ovariectomized 24 h to 26 h after hormonal treatment. Results showed that the kinetic of MPF and MAPK activity was similar during meiotic progression of cat oocytes matured in TCM 199 and SOF. After 24 h of incubation, MII oocytes had significantly (p < 0.001) higher MPF and MAPK levels than MII oocytes cultured for 40 h in both culture media. MPF and MAPK activity was significantly (p < 0.01) lower in the oocytes matured in vitro than in those matured in vivo. This study provides evidence that the two different maturation media did not determine differences in MPF and MAPK fluctuations and levels during meiotic progression of cat oocytes and that the time of maturation influenced the level of the two kinases. Moreover, it shows that MPF and MPK activity is higher in in vivo matured oocytes than in in vitro matured oocytes, suggesting a possible incomplete cytoplasmic maturation after culture.