Salvatore Naitana

Polyvinyl alcohol as a defined substitute for serum in vitrification and warming solutions to cryopreserve ovine embryos at different stages of development

The purpose of this study was to assess the viability of ovine embryos after replacing fetal calf serum (FCS) with polyvinyl alcohol (PVA) in vitrification and warming solutions. Ovine embryos were obtained from superovulated Sardinian breed ewes at 4, 5, 6, and 7 days after insemination. All vitrification and warming solutions were prepared using buffered saline solution with 20% FCS (group a) or 0.1% PVA (group b). Embryos were vitrified in 20 microliters of glycerol 3.4 M + ethylene glycol 4.6 M and loaded into the centre of 0.25 ml straws between two columns of sucrose solution (0.5 M), and plunged immediately into liquid nitrogen. After being warmed in a water bath at 35 degrees C for 10 s, the vitrified embryos were moved to 0.25 M sucrose solution for 3 min. Embryos were cultured in TCM-199 after washing with 10% FCS and sheep oviductal epithelial cells up to hatching or re-expansion of the blastocoelic cavity. No significant difference in the viability rates was observed between embryos vitrified/warmed in PVA or FCS solutions. In both groups, the rate of in vitro viability was (P < 0.01) lower at the precompacted and compacted morula stages than at the expanded, hatching or hatched blastocyst stage. In both groups, early blastocysts were less viable than expanded (P < 0.01), hatching or hatched blastocyst (P < 0.05). There was no significant difference in survival rates at days 14 (79 and 76%) and 45 (63 and 59%) after transfer into sychronised recipients between vitrified expanded blastocysts of groups a and b, respectively. These results suggest that it is possible replace serum with PVA in vitrification and warming solutions without reducing in vivo and in vitro viability.

Melatonin protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner

Abstract:  Cryopreservation harms spermatozoa at different levels and thus impairs their fertilizing ability. The role of melatonin in protecting spermatozoa from different kind injuries has been widely reported. Thus, this study tested whether the addition of melatonin to ram semen freezing extender could exert a protective effect and ameliorate postthawing sperm function. Melatonin was added to recommended ram extender to yield five different final concentrations: 0.001, 0.01, 0.1, 1, and 10 mm. A control group without melatonin supplementation was included. Spermatozoa viability, motility parameters, and intracellular ATP concentrations were evaluated both before and after cryopreservation, while DNA integrity and in vitro fertilizing ability were evaluated only after thawing. Obtained results showed that the concentration of 1 mm melatonin led to higher viability rates, higher percentages of total motile and progressive motile spermatozoa, higher percentages of spermatozoa with average rapid and medium velocity, higher intracellular ATP concentrations, and higher DNA integrity among semen frozen in control and melatonin‐supplemented extenders (P < 0.05). In addition, results obtained after the IVF test showed that at 1 mm concentration, melatonin led to a faster first embryonic division and to higher total cleavage rates compared to the other experimental groups (P < 0.05). No difference in embryo output was observed among the six experimental groups. In conclusion, the addition of melatonin to ram semen freezing extender protected spermatozoa during cryopreservation in a dose‐dependent manner. These results are likely to be mediated by its well‐known antioxidant properties, even if a direct action of the indolamine cannot be ruled out.

Intracytoplasmic sperm injection of in vitro matured oocytes of domestic cats with frozen-thawed epididymal spermatozoa.

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 (P<0.001) 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 (P<0.001) than that of IC

Cell Coupling and Maturation-Promoting Factor Activity in In Vitro-Matured Prepubertal and Adult Sheep Oocytes

Abstract We examined some differences between prepubertal and adult ovine oocytes; in particular we analyzed the functional status of the cumulus-oocyte complex, protein synthesis during in vitro maturation, and because no information is available on prepubertal and adult sheep, maturation-promoting factor (MPF) fluctuations throughout meiotic progression both in prepubertal and adult sheep oocytes. After 24 h of maturation, percentages of MII oocytes were similar between prepubertal and adult animals. Electron microscopy examinations showed that prepubertal oocytes had fewer transzonal projections than adult oocytes. Methionine uptake was significantly lower in prepubertal cumulus-enclosed oocytes examined through meiotic progression. On the contrary, denuded prepubertal oocytes showed a higher methionine incorporation in the first 4 h of incubation compared with adult oocytes. We also found some differences in MPF activity between prepubertal and adult oocytes at MII stage. In fact, prepubertal MII oocytes had a significantly lower level of MPF activity than adult oocytes did and, after fusion with germinal vesicle oocytes, they were unable to induce nuclear breakdown and chromosome condensation 1–2 h post-fusion, whereas adult MII oocytes could induce these processes. Our findings show that the lesser competence of prepubertal oocytes could be due to morphological anomalies and alterations in physiological activity and that oocytes do not reach full developmental competence until puberty.

Exogenous melatonin positively influences follicular dynamics, oocyte developmental competence and blastocyst output in a goat model

Abstract:  The role of melatonin in modulating mammalian reproduction is of particular interest; however, its effects on ovarian follicles and their oocytes still remain to be characterized. This study determined the influence of melatonin treatment on follicular growth patterns and on in vitro oocyte developmental competence. In a first experiment, the effects of melatonin supplementation on follicular dynamics were evaluated using daily transrectal ultrasonographies for 21 days, in 7 multiparous Sarda goats receiving a subcutaneous implant of 18 mg of melatonin and in 5 control untreated does. Melatonin caused more follicular waves (5.2 ± 0.2 versus 4 ± 0.3; P < 0.05) as the waves were shortened at around 2 days when compared with the non‐melatonin treated control goats (P < 0.001). Oocyte developmental competence was evaluated in a second experiment by applying procedures for in vitro embryo production. There were no significant differences in the total number of oocytes obtained from 6 control (n = 192) and 7 melatonin‐treated (n = 265) goats given follicle stimulating hormone to induce follicular development. Differences in oocyte developmental competence between the two groups became evident after in vitro fertilization and culture; melatonin increased the rate of cleaved oocytes in comparison with control animals (82.5 versus 63.4%; P < 0.001), advanced timing of embryo development and enhanced blastocyst output (31.5 versus 16.3%; P < 0.01). However, blastocyst quality, as evaluated by cryotolerance and gene expression analysis, was not found to be different between the groups. In conclusion, in vivo melatonin treatment is beneficial for increasing ovarian follicle turnover and improving oocyte developmental competence and kinetics of the blastocyst.

OOCYTE CRYOPRESERVATION AND OVARIAN TISSUE BANKING

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 throughout 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 in vitro represents an important future means of preserving the fertility of patients and of storing the gametes of rare animals.

Influence of cadmium exposure on in vitro ovine gamete dysfunction

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 (P < 0.001) 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 (P < 0.01) 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%, P < 0.001) 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; P < 0.001) 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 (P < 0.001) the rates of oocyte degeneration. In the third experiment, Cd 20 microM significantly decreased (P < 0.01) the viability rate (35.6%) of spermatozoa compared with 2 microM (57.6%) and 0 microM (54.4%) while Cd 2 microM increased (P < 0.01) 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.

Recipient synchronization affects viability of vitrified ovine blastocysts

Abstract 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

Expression pattern of zygote arrest 1 (ZAR1), maternal antigen that embryo requires (MATER), growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes in ovine oocytes and in vitro-produced preimplantation embryos

The expression patterns of four maternal effect genes (MEG), namely zygote arrest 1 (ZAR1), maternal antigen that embryo requires (MATER), growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), were determined in ovine oocytes and in vitro-produced preimplantation embryos. The existence of ZAR1 and MATER in ovine species has not been reported previously. Reverse transcription-polymerase chain reaction was performed on germinal vesicle and IVM MII oocytes, as well as in in vitro fertilised and cultured two-, four-, eight- and 12/16-cell embryos, morulae and blastocysts. Quantification of gene expression by real-time polymerase chain reaction showed the highest abundance of all transcripts analysed in the immature oocyte. During the following stages of preimplantation development, the mRNAs examined exhibited different patterns of expression, but often significant decreases were observed during maturation and maternal-embryonic transition. The transcription of the four genes did not resume with activation of the genome.

Defined media for vitrification, warming, and rehydration: effects on post-thaw protein synthesis and viability of in vitro derived ovine embryos

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<0.05) lower than those vitrified in medium containing PVA with warming and rehydration in medium containing FCS (p/s). Blastocysts that were vitrified in medium containing FCS and warmed and rehydrated in medium with PVA (s/p) had hatching rates that were significantly lower (P<0.01) than those vitrified, warmed, and rehydrated in media with only FCS (s/s). After warming, the number of dead cells in the p/p group was significantly (P<0.05) lower than in all other groups. In experiment 2, the [35S]methionine uptake by embryonic cells of the s/p group was significantly (P<0.01) higher than in other groups. The incorporation of labelled methionine into newly synthesised proteins was significantly lower in the p/p group (P<0.01) than in all other groups. No differences in the newly synthesised proteins were observed between groups. In conclusion, these results suggest that it is possible to replace serum with defined macromolecules in vitrification and warming/rehydration media for in vitro derived ovine blastocysts but this leads to a decrease in viability and a reduction in protein synthesis after warming.