Search

Intra-follicular oocyte transfer (IFOT) is a promising and innovative technique for in vivo embryo production previously described for equines and bovines. The aim of this study was to assess the feasibility of IFOT in the ovine species. Two preliminary in vivo and in vitro trials were performed to test the optimal procedures and timing for IFOT. In the in vivo trial, follicular growth was monitored with transrectal ultrasonography in ten adult ewes to preliminarily determine the ovulation and ideal timing for IFOT. The in vitro trial assessed i) the optimal inner diameter of the injection needle and ii) the recovery rate and integrity of injected cumulus-oocyte complexes (COCs) after follicle aspiration. For IFOT and embryo collection, five ewes were synchronized by CIDR insertion. Forty hours after CIDR removal, in ewes under sedation and general anesthesia, the ovaries were exposed by laparotomy, and the preovulatory follicle was injected with COCs previously collected from ovaries obtained from an abattoir. At 4 h after surgery, fully recovered ewes were housed in a paddock with a ram of proven fertility. Crayon marking on ram's chest was used to detect mating. Ovulation was assessed 40 h after the transfer of oocytes by transrectal ultrasonography. On day 6 after IFOT, embryo collection was performed by uterine flushing. In the in vitro testing, injection of >5 mm follicles with a 28 G needle loaded with 30 COCs in a 5 μL volume resulted in higher recovery rates and better preservation of COCs integrity. In the in vivo trial, ultrasound scanning revealed that ovulation occurred between 60 and 72 h after CIDR removal in all animals. In one ewe subjected to IFOT, 22/24 oocytes were effectively injected into the preovulatory follicle, but no embryos were collected after flushing. In the remaining four animals, 85/102 oocytes were injected, and six cleaved embryos, 12 morulae and 1 blastocyst were collected, including native embryos. This preliminary investigation indicated that IFOT in ovine species resulted in ovulation, fimbrial capture, tubal transport of heterologous oocytes and in vivo embryo production. Further studies are needed to optimize the embryo recovery rate and develop less invasive techniques for oocyte injection and uterine flushing, such as through a laparoscopic or transcervical approach.

A complete assessment of morphological and functional characteristics of ram semen during refrigeration is necessary to optimize the process of semen manipulation and storage. The aim of this study was to describe changes in main predictive parameters of ram semen diluted in a commercial soy lecithin-based extender (OVIXcell), during long term liquid storage at 4 °C. Ejaculates of 5 Sarda rams were collected, pooled and diluted in OVIXcell. Samples were cooled at 4 °C and stored at this temperature until 96 h. At 0-24-48-72-96 h semen samples were analysed for the following parameters: motility [computer assisted sperm analysis (CASA)]; integrity of cytoplasm membrane and acrosome (PI/PSA staining); DNA fragmentation index [DFI(%); sperm chromatin structure assay (SCSA)]; levels of reactive oxygen species (ROS; H2DCFDA staining). Effect of time of storage on main parameters and correlations among them were assessed respectively by ANOVA and Pearson's correlation coefficient. Primary and secondary motility parameters were significantly affected by time of storage (P 0.05). Long term storage did not affect the levels of DFI(%) (P > 0.05) while ROS production significantly increased from 48 to 96 h (P

L-ergothioneine levels were measured in amniotic fluid of pregnant sheep after natural mating and transfer of vitrified/thawed in-vitro produced embryos. Amniotic fluids were collected between 60 and 65 and 80-85 days of gestation and analysed by an ultra-performance liquid chromatographic (UPLC)method with fluorescence detection. L-Ergothioneine concentrations ranged between 0.23 and 9.36 μmol/L and were significantly higher in pregnancy obtained by the transfer of vitrified/thawed in-vitro produced embryos. Conversely, no significant changes in amniotic fluid L-ergothioneine concentrations were observed according to the stages of pregnancy considered in this study. These findings suggest that L-ergothioneine concentrations, are not affected as much by the gestational age, but rather by the method used to induce the pregnancy. On the whole, the measurement of L-ergothioneine in amniotic fluid could serve as a useful biomarker of oxidative stress and/or inflammatory state in pregnancy.

In vitro embryo production (IVP) and cryopreservation are associated with a high incidence of pregnancy complications and fetal abnormalities that may be linked with alterations of placental development. The amniotic fluid is partly derived from the transport of water and solutes across the placenta and provides the fetus with amino acids (AAs), which are the building blocks for biomolecules involved in physiological growth and development. To better understand the anomalies associated with IVP pregnancies, the present study was conducted to test the hypothesis that amniotic concentrations of AAs differ in pregnancies derived from vitrified/thawed (V/T) IVP embryos compared with gestations obtained with natural mating (NM) in sheep. Amniotic fluid was sampled in ewes that were pregnant after transfer of V/T IVP embryos and that had conceived with NM between Days 60 and 65 (V/T, n = 6; NM, n = 11) and between Days 80 and 85 (V/T, n = 5; NM, n = 14) of gestation via ultrasound-guided amniocentesis. Concentrations of 16 AAs in the amniotic fluid were measured using high-performance liquid chromatography. From Days 60 to 65 of gestation, concentrations of cystine, phenylalanine, and isoleucine were lower in V/T compared with NM ewes. From Days 80 to 85 of pregnancy, the mean concentrations of cystine and lysine were lower in the V/T versus NM groups. The total AA concentration per ewe was similar between the groups from Days 60 to 65 and 80 to 85 of gestation and decreased by 55% from Days 60 to 65 and 80 to 85 of gestation in all ewes. The most abundant AA from Days 60 to 65 of gestation was alanine in both groups, whereas from Days 80 to 85, the most abundant AAs were alanine in NM and glycine in V/T ewes; cystine was the less abundant detectable AA in all ewes at both stages of gestation. Results report that V/T IVP embryos have decreased concentrations of individual AAs in the amniotic fluid during the second trimester of gestation possibly because of an impaired placental vasculogenesis or because of a reduced placental transport. These novel findings are relevant to unravel the mechanisms responsible for the issues of pregnancies achieved with the transfer of IVP and cryopreserved embryos.

Cryopreservation-induced modifications of zona pellucida (ZP) have been explored to a lesser extent compared to other oocyte compartments. Different methods have been applied to identify ZP changes, but most of them are invasive and measure only few properties of ZP. Raman microspectroscopy (RMS) is a powerful technique for studying the molecular composition of cells but to date few studies have been performed on the oocytes using this method. The aim of the present study is to investigate the structural modifications of ZP of vitrified/warmed in vitro matured ovine oocytes by means of RMS. Cumulus-oocyte complexes were recovered from the ovaries of slaughtered adult sheep, matured in vitro and vitrified following the Minimum Essential Volume method using cryotops. ZPs of vitrified/warmed oocytes (VITRI), were exposed to vitrification solutions but not cryopreserved (CPA-exp) and untreated oocytes (CTR) were analyzed by RMS. We focused our analysis on the ZP protein and carbohydrate components by analyzing the 1230-1300 cm(-1) amide III region and the 1020-1140 cm(-1) spectral range in RMS spectra, respectively. The spectral profiles in the ranges of proteins and carbohydrates were comparable between CTR and CPA-exp ZPs, whereas VITRI ZPs showed a significantly altered protein secondary structure characterized by an increase in β-sheet content and a decrease in the α-helix content. A significant modification of the carbohydrate components was also observed. This study demonstrates that vitrification of ovine oocytes induces biochemical changes of ZP related to the secondary structure of proteins and carbohydrate residues. Cryoprotectants do not strongly alter the molecular composition of ZP which is affected mainly by cooling. Raman technology offers a powerful and non-invasive tool to assess molecular modifications induced by cryopreservation in oocytes.

The present study was conducted to determine the effect of okadic acid (OA), a potent inhibitor of seronine/treonine 1 and 2A phosphatase, on meiotic resumption and progression in canine oocytes with different diameters. Cumulus-oocyte complexes were collected from ovaries of bitches at different oestrous phases. In Experiment 1, to determine the optimal concentration of OA (0.5 or 2 μM), the oocytes were pre-incubated for 1, 3, and 20 h in TCM 199 supplemented with 20% SCE and thereafter cultured in the same medium without OA. In Experiment 2, the selected oocytes were divided into three groups according to their diameter:110 μm, 110-120 μm,120 μm, and pre-incubated in OA 0.5 μM for 1 h. Oocytes were cultured in vitro as previously described. After 72 h of IVM, in Experiment 1, significantly more oocytes reached MII stage with 0.5 μM for 1 h (30.8% P0.001%) for oocytes cultured in other OA condition and in control group. In Experiment 2, OA induced a significantly higher incidence of MII oocytes in the 110-120 μm and120 μm groups (P0.001) compared to control group, but a significantly higher proportion of the oocytes120 μm pre-incubated with OA progressed to MII (51.3% P0.001). In contrast, smaller oocytes (110) did not develop to MII stage with or without OA. In conclusion, treatment of canine oocytes with 0.5 μM for 1 h, improves meiotic maturation. The culture of fully grown (120 μm) oocytes with OA at the onset of in vitro maturation can result in a higher frequency of meiotic maturation.

Parthenogenetic activation of the mammalian oocyte constitutes an essential step to a number of oocyte- or embryo-related technologies. Mammalian parthenotes are useful tools for studying the roles of paternal and maternal genomes in early mammalian development and are considered potential candidates for an ethical source of embryonic stem cells. We investigated the in vitro developmental competence of pre-implantation ovine embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) together with the expression of a panel of fourteen genes at different times of development. IVF and PA embryos showed similar developmental competence. No differences in gene expression were observed between PA and IVF two cell-stage embryos, while PA morulae showed a significantly higher expression of IGF2 . At the blastocyst stage, parthenotes exhibited up-regulation of TP-1 , CDC2 , and IGF2 transcripts and significantly lower levels of AQP3 , ATP1A1 , H2A.Z , hsp90beta , and OCT4 , while NANOG , BAX , CCNB1 , CDH1 , GAPDH , and IGF2R displayed similar expression patterns in the two groups. Our study indicates that oocyte parthenogenetic activation does not impair in vitro pre-implantation development to the blastocyst stage, but affects the gene expression status of the embryo after the activation of its own genome.

The purpose of this study was to assess the viability (rates of re-expanding and hatching in vitro), of in vitro derived ovine blastocysts using vitrification and warming/rehydration media containing fetal calf serum (20% FCS) or polyvinyl alcohol (0.1% PVA), and the incorporation of labelled methionine in protein synthesised during the first 4h after cryopreservation. In experiment 1, after 60 h culture in TCM-199 supplemented with 10% FCS, the hatching rates of blastocysts that had been vitrified, warmed, and rehydrated in media containing only PVA (p/p) were significantly (P

This study was conducted to determine the in vitro effects of three different cadmium concentrations (0, 2, and 20 microM CdCl(2)) on oocyte maturation, fertilisation, and acrosome integrity and sperm viability in sheep. Cumulus-oocyte complexes were recovered from ovaries of slaughtered sheep and sperm were collected by artificial vagina from adult rams. The oocyte maturation rate was significantly affected (P0.001) by Cd at both concentrations, with a metaphase II (MII) rate of 96.8, 63.8, and 32.0% for 0, 2, and 20 microM cadmium, respectively. In the second experiment, the presence of Cd significantly decreased (P0.01) the rate of oocytes resting in MII after 24-h postmaturation culture, compared with the control group (93.8 versus 29.0 and 19.8%, respectively, for 0, 2, and 20 microM Cd). Oocytes cultured with Cd 2 microM showed a higher activation rate (59.5%, P0.001) with one or two pronucleus than with 0 and 20 microM Cd (6.2 and 22.9%, respectively). During fertilisation the presence of fertilised oocytes was decreased in both culture systems with Cd compared with the control (76.1, 25.9, and 4.7% for 0, 2, and 20 microM Cd, respectively; P0.001) while polyspermy was increased in the 2 microM Cd group (23.5 for 2 microM versus 6.7 and 0%, respectively, for 0 and 20 microM groups). In both experiments Cd significantly increased (P0.001) the rates of oocyte degeneration. In the third experiment, Cd 20 microM significantly decreased (P0.01) the viability rate (35.6%) of spermatozoa compared with 2 microM (57.6%) and 0 microM (54.4%) while Cd 2 microM increased (P0.01) acrosome-reacted spermatozoa (45.2%) compared with 20 microM (32.5%) and control (31.9%). The results suggest that in vitro cadmium at the lowest dose tested affects the physiological function of both ovine gametes but at higher dose tested can compromise cell viability.

The ability to mature and fertilize oocytes of endangered species may allow us to sustain genetic and global biodiversity. The first objective of this study was to compare the effect of two different culture media and two different incubation times on in vitro maturation (IVM) of domestic cat oocytes. The second objective was to determine the developmental competence of in vitro matured cat oocytes after intracytoplasmic sperm injection (ICSI) with cat spermatozoa. Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 medium or in synthetic oviductal fluid (SOF), both of which were supplemented with cysteamine, BSA, FSH, LH. Nuclear maturation was assessed after 24 h and 40 h of incubation. Results of IVM showed that the percentage of oocytes reaching MII after 24 h and 40 h of incubation were significantly higher (P0.001) after culture with SOF (88/110, 80% and 159/192, 82.8%) than TCM 199 (86/129, 66.7% and 58/90, 64.4%). Oocytes (n = 231) matured in vitro in SOF for 24 h were fertilized by ICSI with frozen-thawed epididymal cat spermatozoa. After ICSI, one group of oocytes (n = 129) was activated with ethanol, and a second group (n = 102) was not activated. The developmental competence of all ICSI oocytes was examined after 7 days of in vitro culture. After 28 h of culture, the cleavage frequency of ICSI-activated oocytes was significantly higher (P0.001) than that of IC

Oocyte cryopreservation, despite its impact on conservation of genetic resources, is not yet an established technology. Several problems need to be solved before this technology can be applied regularly. Chilling membrane susceptibility and formation of ice due to the large volume of the cell are the major problems observed. However, during the last years, several studies were done to obtain viable oocytes after cryopreservation. The addition of molecules known to stabilize membranes and the creation of freezing systems with rapid cooling through the transition phase have yielded a good percentage of viable immature and mature oocytes. More recently, storage of female gametes was achieved by cryopreservation of cortical ovarian tissue. The possibility of restoring fertility by transplantation of frozen ovarian tissue or its long-term culture invitro represents an important future means of preserving the fertility of patients and of storing the gametes of rare animals.

In this study multiple ovulation and embryo transfer (MOET) technology was tested as a method for increasing the number of offspring obtained from superovulated mouflons and then using Sardinian ewes as recipients. Two experiments were carried out over consecutive years. In Experiment 1, female mouflons received a standard superovulatory treatment during both breeding and anoestrous seasons. Sarda sheep, used as controls, received the same treatment. Mean superovulatory response (corpora lutea and large follicles) was higher in the domestic sheep than in the mouflons (4.8 vs. 10.1 and 4.2 vs. 8.8 in breeding and anoestrous seasons, respectively; P < 0.05). A high percentage of mouflons showed early luteal regression which negatively affected recovery rate (35% and 30% in mouflons vs. 69% and 71% in sheep) and the yield of embryos suitable for transfer (37% and 25% in mouflons vs 74% and 69% in sheep; P < 0.05). In Experiment 2, ten mouflons were treated by the same superovulatory protocol and divided into two groups. In the first (Group 5), embryos were recovered earlier by oviductal flushing and cultured in vitro with oviductal cells in CZB medium until the morula/blastocyst stage; in the second (Group 6), the usual embryo recovery time was followed. Recovery rate was higher in the former (89% vs. 31%; P < 0.01) than in the latter. After 4 days of culture, 53% of embryos reached compact morula or early blastocyst stage (16/30). Lambing rate was 57% for mouflon embryos transferred immediately and 56% for those cultured in vitro for 4 days; the lambing rate in the sheep control group was 71%. The length of gestation was longer in ewes carrying mouflons than in those carrying lambs (155 vs. 148 days).

We assessed the viability of vitrified ovine blastocysts in vitro and in recipient ewes at different degrees of synchronization. Expanded ovine blastocysts obtained from superovulated Sarda ewes were exposed to a vitrification solution at 20 °C according to the following procedures: the blastocysts were added into 200 μl glycerol (1.4 mol) for 5 min, then into 200 μl glycerol (1.4 mol) and ethylene glyco (l 3.6 mol) for 5 min. These embryos were transferred into 25 μl glycerol (3.4 mol) and ethylene glycol (4.6 mol) and loaded into the center of 0.25-ml straws separated from air bubbles by 2 columns of 90 μl sucrose solution (0.5 mol). The straws were plunged immediately into liquid nitrogen (LN 2 ). The basic vitrification and thawing solution was phosphate buffered saline (PBS) supplemented with 20% fetal calf serum (FCS). The embryos were thawed by agitating the straws in a water bath at 20 °C, and expelling them into Petri dishes and mixing them, and placing the embryos into 200 μl sucrose solution (0.25) mol for 5 min. The percentage of hatched blasticysts, after exposed and vitrified-thawed embryos, was 96.8% (3031) and 82.8% (5364), respectively, after 4 d of coculture in TCM 199 medium with 10% FCS in 5% CO 2 humidified atmosphere in air at 39 °C. Vitrified-thawed blastocysts, which re-formed the blastocoelic cavity after 24 h of co-culture as in the in vitro experiment, were surgically transferred into the uterine horn of recipients (2 embryos per ewe) at 6, 7 and 8 d after estrus. The pregnancy and lambing rates in recipient ewes at 6, 7 and 8 d after estrus were 72.7%, 90%, 54.5% and 72.7%, 80%, 45.4%, respectively. Both pregnancy and lambing rates differed significantly (P

1. Hemoglobin (Hb) switching in the perinatal life of wild mouflon (Ovis musimon) was characterized by the replacement of Hb F by 60% levels of Hb C, and subsequently of Hb C by Hb B. 2. The recently discovered Hb M variant was not replaced by Hb C; thus, Hb BM heterozygote newborns synthesized 30% Hb C at the expense of Hb B. 3. Hybrid B mouflon x B sheep synthesized only 5% Hb C at birth but were able to produce 30% Hb C in adult life following induced anemia. 4. Adult BB and BM mouflons, after the same extent of induced anemia, synthesized HB C levels similar to those produced at birth. The results indicate a mouflon beta-globin gene cluster arrangement similar to those of sheep and goat, the beta C gene having an intermediate expression. Results also suggest a selective disadvantage in hybrid animals.