Differences in the kinetic of the first meiotic division and in active mitochondrial distribution between prepubertal and adult oocytes mirror differences in their developmental competence in a sheep model

Our aim is to verify if oocyte developmental potential is related to the timing of meiotic progression and to mitochondrial distribution and activity using prepubertal and adult oocytes as models of low and high developmental capacity respectively. Prepubertal and adult oocytes were incorporated in an in vitro maturation system to determine meiotic and developmental competence and to assess at different time points kinetic of meiotic maturation, 2D protein electrophoresis patterns, ATP content and mitochondria distribution. Maturation and fertilization rates did not differ between prepubertal and adult oocytes (95.1% vs 96.7% and 66.73% vs 70.62% respectively for prepubertal and adult oocytes). Compared to adults, prepubertal oocytes showed higher parthenogenesis (17.38% vs 2.08% respectively in prepubertals and adults; P<0.01) and polispermy (14.30% vs 2.21% respectively in prepubertals and adults; P<0.01), lower cleavage rates (60.00% vs 67.08% respectively in prepubertals and adults; P<0.05) and blastocyst output (11.94% vs 34.% respectively in prepubertals and adults; P<0.01). Prepubertal oocytes reached MI stage 1 hr later than adults and this delay grows as the first meiotic division proceeds. Simultaneously, the protein pattern was altered since in prepubertal oocytes it fluctuates, dropping and rising to levels similar to adults only at 24 hrs. In prepubertal oocytes ATP rise is delayed and did not reach levels comparable to adult ones. CLSM observations revealed that at MII, in the majority of prepubertal oocytes, the active mitochondria are homogenously distributed, while in adults they are aggregated in big clusters. Our work demonstrates that mitochondria and their functional aggregation during maturation play an active role to provide energy in terms of ATP. The oocyte ATP content determines the timing of the meiotic cycle and the acquisition of developmental competence. Taken together our data suggest that oocytes with low developmental competence have a slowed down energetic metabolism which delays later development

Ejaculate collection efficiency and post-thaw semen quality in wild-caught Griffon vultures from the Sardinian population

This study aimed to test the feasibility of a programme of semen collection and cryopreservation in Griffon vultures. Four wild-caught individuals kept in captivity because of unrecoverable traumas were used. Semen collection attempts were made twice a week during three consecutive reproductive seasons (December – March) using the abdominal massage method. Ejaculation was successfully induced between late January and late February. Semen collection efficiency was rather low (27.9%) and it did not vary among individuals (p > 0.05). No differences were found in ejaculate volumes (12.5 +/- 9.1 μl), spermatozoa concentration (28.4 +/- 30.9 million cells/ml) and viability (61.3 +/- 13.9%) among the 4 vultures. ATP values differed among the four vultures (p < 0.001); B showed higher nucleotide concentration than both C and D, while it did not differ form A, whose values were higher compared with D. After freezing and thawing, semen in vitro viability, DNA integrity and ATP intracellular concentration were determined. Spermatozoa viability after thawing did not differ among the four individuals (52.6 +/- 5.8 in A, 53.4 +/- 4.6 in B, 50.4 +/- 3.2 in C, 42.5 +/- 2.7 in D), but it decreased significantly compared to fresh semen (p < 0.05). During 4 hrs in vitro culture, spermatozoa collected from B maintained over time a higher viability in vitro when compared to A, C and D. As evaluated by the comet assay method, DNA fragmentation after freezing and thawing did not differ in the 4 vultures. ATP concentration in frozen/thawed semen was significantly lower than in fresh semen (p < 0.0001). This study indicates that semen cryopreservation can be considered as a useful tool in the conservation of Griffon vulture genetic resources, but further studies are needed to optimize this technique

Morphofunctional aspects of in vitro matured oocytes from prepubertal and adult sheep

The aim of this study was to analyze the morphofunctional aspects of prepubertal and adult sheep oocytes subjected to in vitro maturation (IVM). The structural and ultrastructural morphology was examined by light and transmission electron microscopy (LM and TEM) while the metabolic competence, as determined by the distribution of active mitochondria, was assessed by confocal microscopy (CM). Cumulus-oocyte complexes underwent IVM in standard condition for 24 h. Half of the mature metaphase II (MII) stage oocytes were processed for LM and TEM observations. The other half was subjected to immunostaining with MitoTracker Red (to stain mitochondria with functionally active membrane potential), SNARF-1 (a pH sensitive fluoroprobe), Hoechst 33342 (to label DNA) and analyzed by CM. Immature germinal vesicle (GV) stage oocytes, retrieved at 0 h IVM, were used as controls. By LM and TEM all the oocytes were regularly rounded, covered by microvilli and surrounded by an intact zona pellucida. Numerous rounded, oval or hooded mitochondria appeared either isolated or grouped in the ooplasm. The GV was usually rounded in prepubertal oocytes. In the adult, the GV often appeared flattened against the oolemma, with a crescent-shaped outline (an early sign of meiotic resumption). Scattered cortical granules (CGs) were rarely found in the ooplasm of both prepubertal and adult GV oocytes. After 24 h of IVM, CGs became abundant and distributed in a single row under the oolemma, particularly in adult oocytes. CM showed a homogeneous fine-to-granular mitochondrial distribution in prepubertal GV and MII oocytes. In adult GV oocytes, the mitochondrial distribution pattern was granular while in MII oocytes mitochondria were arranged in heterogeneous clusters. Thus, both prepubertal and adult oocytes completed maturation after 24 h in culture and showed an overall good preservation after IVM. However, the diverse distribution patterns of mitochondria in prepubertal oocytes reflect their low developmental competence

Semen molecular and cellular features: these parameters can reliably predict subsequent ART outcome in a goat model

Currently, the assessment of sperm function in a raw or processed semen sample is not able to reliably predict sperm ability to withstand freezing and thawing procedures and in vivo fertility and/or assisted reproductive biotechnologies (ART) outcome. The aim of the present study was to investigate which parameters among a battery of analyses could predict subsequent spermatozoa in vitro fertilization ability and hence blastocyst output in a goat model. Ejaculates were obtained by artificial vagina from 3 adult goats (Capra hircus) aged 2 years (A, B and C). In order to assess the predictive value of viability, computer assisted sperm analyzer (CASA) motility parameters and ATP intracellular concentration before and after thawing and of DNA integrity after thawing on subsequent embryo output after an in vitro fertility test, a logistic regression analysis was used. Individual differences in semen parameters were evident for semen viability after thawing and DNA integrity. Results of IVF test showed that spermatozoa collected from A and B lead to higher cleavage rates (0 < 0.01) and blastocysts output (p < 0.05) compared with C. Logistic regression analysis model explained a deviance of 72% (p < 0.0001), directly related with the mean percentage of rapid spermatozoa in fresh semen (p < 0.01), semen viability after thawing (p < 0.01), and with two of the three comet parameters considered, i.e tail DNA percentage and comet length (p < 0.0001). DNA integrity alone had a high predictive value on IVF outcome with frozen/thawed semen (deviance explained: 57%). The model proposed here represents one of the many possible ways to explain differences found in embryo output following IVF with different semen donors and may represent a useful tool to select the most suitable donors for semen cryopreservation

New Challenges in Cryopreservation: A Reproductive Perspective

Cryopreservation is a fundamental procedure to preserve the structure and function of cells and tissues by storing them at low temperatures for long periods [...

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

[EN] 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% ethylene glycol + 16.5% dimethyl sulphoxide) supplemented with 1% of SuperCool X-1000 and 1% SuperCool Z-1000 (Ice Blockers) or 20% 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% and 85.9%), cleaveage rates (21.4% and 19.6%) and blastocyst development rates (4.8% and 4.5%) 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.This work was supported by funds from D’ajudes per a la realització de projectes precompetitius D’I+D per a equips (GVPRE/2008/206).Marco Jiménez, F.; Berlinguer, F.; Leoni, GG.; Succu, S.; Naitana, S. (2012). Effect of "ice blockers" in solutions for vitrification of in vitro matured ovine oocytes. Cryo Letters. 33(1):41-44. http://hdl.handle.net/10251/65509S414433

Seasonal Effect on Developmental Competence, Oxidative Status and Tubulin Assessment of Prepubertal Ovine Oocyte

The reproductive seasonality of domestic animals is often manipulated in order to have more reproductive periods for commercial purposes related to the production of milk and meat. It is scientifically proven that such an alteration of the reproductive activity in sheep entails a deterioration in oocyte quality, leading to an inability to generate embryos. Since oocytes obtained from prepubertal ewes can be incorporated into an in vitro embryo production system and considering that their quality is crucial to the success of in vitro procedures, the aim of this work was to investigate the effect of seasons on the quality of prepubertal ovine oocytes collected in autumn and spring. Ovaries were collected from a local slaughterhouse from 30–40-day-old suckling lambs during both seasons. Following 24 h of in vitro maturation, oocytes developmental competence, reactive oxygen species (ROS) intracellular levels, and mitochondrial activity were evaluated, and a tubulin assessment was performed. The results on embryo production, as a percentage of first divisions and number of blastocysts obtained, were significantly higher in oocytes collected in the spring. Mitochondrial activity in oocytes was higher, and ROS production significantly lower, in spring than in autumn. Tubulin PTMs (tyrosinated and acetylated α-tubulin) showed a higher immunoreactivity in oocytes collected in spring compared with autumn sampling. Our data showed that seasons may affect the developmental competence, energetic status, and tubulin assessment of oocytes recovered from prepubertal ewes. Therefore, special care should be taken when choosing the period of the year for prepuberal ovine oocytes collection aimed at in vitro embryo reproduction programs

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

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 &lt; 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 &lt; 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

Soybean lecithin–based extender preserves spermatozoa membrane integrity and fertilizing potential during goat semen cryopreservation

Soybean lecithin may represent a suitable alternative to egg yolk for semen cryopreservation in livestock species. However, additional studies are needed to elucidate its effects on spermatozoa functional properties. Semen collected from five Sarda bucks was cryopreserved in Tris-based extender and glycerol (4% v:v) with different supplementations. In a preliminary experiment, different soybean lecithin concentrations were tested (1%–6% wt/vol) and results in terms of viability, percentages of progressive motile and rapid spermatozoa, and DNA integrity after thawing showed that the most effective concentration was 1%. In the second experiment, semen was frozen in a Tris-based extender with no supplementation (EXT), with 1% lecithin (EXT LC), and 20% egg yolk (EXT EY). The effectiveness of these extenders was also compared with a commercial extender. The EXT EY led to the highest viability and motility parameters after freezing and thawing (P &lt; 0.0001). No significant differences were observed in intracellular ATP concentrations. Additional molecular features revealed that sperm functionality was affected in EXT EY, as demonstrated by lower DNA and acrosome integrity (P &lt; 0.05), and higher lipid peroxidation compared with spermatozoa cryopreserved in EXT LC (P &lt; 0.0001). Results obtained in the heterologous in vitro fertilization test showed that EXT LC better preserved spermatozoa functionality, as demonstrated by the higher fertilization rates compared with the other media (66.2 ± 4.5% for EXT LC vs. 32.7 ± 4.5%, 38.7 ± 4.5%, 39.6 ± 5.2% for EXT, EXT EY, and commercial extender; P &lt; 0.01). The present study demonstrated that lecithin can be considered as a suitable alternative to egg yolk in goat semen cryopreservation, because it ensures higher fertilization rates and a better protection from membrane damage by cold shock

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 &lt; 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 &lt; 0.05) and cleavage rates (58% TRH vs 39.8% CTR; p &lt; 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 &lt; 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 &lt; 0.05), and enhanced in particular the number of blastocysts that developed on the day 6th of culture (28.6% vs 17% CTR; p &lt; 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