Raman microspectroscopy as a non-invasive tool to assess the vitrification-induced changes of ovine oocyte zona pellucida.

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 ß-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.

In vivo and in vitro fertilizing capacity of cryopreserved European mouflon [Ovis gmelini musimon] spermatozoa used to restore genetically rare and isolated populations.

European mouflon sheep are an endangered species of ovidae residing primarily in the mountenous habitat of the islands of Sardinia and Corsica. The purpose of this study was to assess the fertilizing capacity of cryopreserved European mouflon spermatozoa after AI in synchronized mouflon and domestic ewes and after IVF in in vitro matured mouflon and domestic ewe oocytes collected by OPU technique. Domestic ram (Ovis aries) spermatozoa served as control. Semen was collected by artificial vagina from three mouflons and three domestic rams during the breeding season and was cryopreserved. At thawing, no significant differences in sperm viability were found between the wild and the domestic species (53.1 +/- 4.6% versus 56.0 +/- 4.7%) whereas the percentage of acrosome-intact sperm was lower in mouflon (55.5 +/- 4.6%) than in ram semen (62.7 +/- 3.1%; P < 0.05). Lambing rate did not differ between synchronized mouflon and domestic ewes (5/11 versus 8/12) after 150 and 156 days of pregnancy, respectively. After two OPU sessions, 87 and 132 oocytes were collected from three hyperstimulated mouflon and three domestic ewes. Cryopreserved/thawed semen was inseminated with an endoscope into the uterus of corresponding species during the non-breeding season. The oocytes were matured and fertilized in vitro; 61/73 mouflon and 81/101 domestic ewe oocytes were found to be fertilized. From these, we obtained 6/61 and 17/81 blastocysts. After vitrification and thawing, the hatching rate showed no significant difference between mouflon and sheep blastocysts (4/6 versus 14/17). In conclusion, our data showed that cryopreserved mouflon spermatozoa can be successfully used to carry out a genuine and complete program of genetic restoration in small and isolated groups of European mouflons.

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.

Superoxide dismutase affects the viability of thawed European mouflon (Ovis g. musimon) semen and the heterologous fertilization using both IVF and intracytoplasmatic sperm injection.

This study evaluated the effects of superoxide dismutase (SOD) on viability and acrosome integrity of European mouflon spermatozoa after cryopreservation and on the fertilization rates of sheep oocytes after i.v.f. or intracytoplasmatic sperm injection (i.c.s.i.). Frozen semen was thawed and washed with synthetic oviduct fluid supplemented with 0.6% bovine serum albumin. After centrifugation, the spermatozoa pellet was split into two culture systems: (i) without SOD; and (ii) in the presence of 1500 IU mL(-1) SOD. Sperm viability and acrosome integrity were evaluated simultaneously, immediately after thawing and after 3, 6 and 9 h of culture (5% CO2, 39 degrees C, 90% humidity), by incubating sperm with propidium iodide and fluorescein isothiocyanate-labelled Pisum sativum agglutinin. At the same time, sperm were assessed for motility using a standard scoring system (independent operators' observation of sperm) that graded degree of motility (i.e. 1 = immotile to 10 = maximum motility, as observed at the moment of thawing). For i.v.f., frozen-thawed semen derived from the two culture systems was placed in culture together with in vitro-matured sheep oocytes. For i.c.s.i., semen derived from the same culture systems as that for i.v.f. was used, and incubated for 1 h under standard conditions. The results showed a marked difference (P < 0.01) between the percentages of live spermatozoa in medium with SOD and those obtained in medium alone, after 3, 6 and 9 h of culture. The percentages of intact acrosome spermatozoa were higher in medium with SOD after 6 h (P = 0.05) of culture. Spermatozoa motility decreased significantly in SOD containing medium at 3 and 6 h of culture compared with motility in control medium. Fertilization rates were significantly lower in medium with SOD than in medium alone, whereas in the i.c.s.i. system fertilization rates were significantly higher in the presence of SOD. The results indicate that the addition of SOD to the culture media enhances the viability rates and the acrosome integrity of cryopreserved mouflon spermatozoa.

The effect of okadaic acid on meiotic maturation of canine oocytes of different size.

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% P<0.001%) 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 and >120 µm groups (P<0.001) compared to control group, but a significantly higher proportion of the oocytes>120 µm pre-incubated with OA progressed to MII (51.3% P<0.001). 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.

Resumption of metabolic activity of vitrified/warmed ovine embryos.

To evaluate resumption of metabolic activity of vitrified ovine embryos during a short time immediately after warming, blastocysts collected from superovulated Sarda ewes were incubated with (35)S-methionine. In vitrified/warmed embryo groups the protein secretion significantly (P < 0.05) increased from 0 to 24 hr of culture, reaching significantly (P < 0.01) higher activity at 18-24 hr and dropping to values similar to the control nonvitrified embryo group at 29-35 hr. Within the control group at 29-35 hr there was a significantly (P < 0.01) higher level of protein secretion compared to the other interval times. The electrophoretic pattern showed a 48-50 kDa secreted protein identified as urokinase-type plasminogen activator (PA). The caseinolytic assay of PA activity showed a course similar to protein secretion in both vitrified and control groups. During 29-35 hr of culture, we did not observe any improvement in PA activity as seen for secreted proteins. At this time, we observed the secretion of a new 20 kDa protein that was not present in vitrified/warmed embryos. Analysis of BrDU incorporation in newly synthesised DNA showed a significant (P < 0.01) improvement in positive cell number from 3 to 9 hr after warming, reaching a value similar to that of the control group at 12 hr of culture. Our results suggest that vitrified/warmed embryos require 9-12 hr of culture to complete resumption of DNA synthesis and 29-35 hr to re-acquire the full capacity of protein secretion but not the qualitative secretion pattern.

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.