REAC technology as optimizer of stallion spermatozoa liquid storage.

BACKGROUND: REAC technology (acronym for Radio Electric Asymmetric Conveyor) is a technology platform for neuro and bio modulation. It has already proven to optimize the ions fluxes at the molecular level and the molecular mechanisms driving cellular asymmetry and polarization. METHODS: This study was designed to verify whether this technology could extend spermatozoa life-span during liquid storage, while preserving their functions, DNA integrity and oxidative status. At 0, 24, 48, and 72 h. of storage at 4 °C, a battery of analyses was performed to assess spermatozoa viability, motility parameters, acrosome status, and DNA integrity during REAC treatment. Spermatozoa oxidative status was assessed by determining lipid peroxidation, the activity of superoxide dismutase (SOD), and the total antioxidant capacity. RESULTS: During liquid storage REAC treated spermatozoa, while not showing an increased viability nor motility compared to untreated ones, had a higher acrosome (p > 0.001) and DNA integrity (p > 0.01). Moreover, the analysis of the oxidative status indicated that the mean activity of the intracellular superoxide dismutase (SOD) was significantly higher in REAC treated spermatozoa compared to untreated controls (p < 0.05), while the intracellular concentration of malondialdehyde (MDA), an end product of lipid peroxidation, at the end of the REAC treatment was higher in untreated controls (p > 0.05). The REAC efficacy on spermatozoa oxidative status was also evidenced by the higher trolox equivalent antioxidant capacity (TEAC) found in both the cellular extract (p < 0.05) and the storage media of REAC treated spermatozoa compared to untreated controls (p < 0.0001). CONCLUSION: The present study demonstrated that REAC treatment during liquid storage preserves spermatozoa acrosome membrane and DNA integrity, likely due to the enhancement of sperm antioxidant defenses. These results open new perspective about the extending of spermatozoa functions in vitro and the clinical management of male infertility.

Postnatal pituitary and follicular activation: a revisited hypothesis in a sheep model.

The importance of postnatal pituitary activation as regards female reproductive development is not yet understood. By taking advantage of the experimental model developed in a previous study, i.e. ewe lambs expressing markedly different ovarian phenotypes at 50 days of age, we designed this study to determine whether differences found in ovarian status during the early prepubertal period are due to different patterns of postnatal pituitary activation, and to assess whether these differences have long lasting effects on subsequent reproductive performance. Results showed that ewe lambs with high antral follicle count (AFC) at 50 days of age had significantly lower plasma FSH concentrations and higher anti-Mullerian hormone (AMH) concentrations during the first 9 weeks of age compared with low AFC ewe lambs (P<0.0001). With a longitudinal experiment we showed that a high AFC in the early prepubertal period is associated with consistently higher AMH concentrations and numbers of antral follicles up to the postpubertal period, and with higher pregnancy rates in the first breeding season. In addition, the effect of age in decreasing AMH concentrations was more marked in the low AFC group. Results of the present study demonstrate that ewe lambs undergo different patterns of postnatal pituitary activation. A high AFC at 50 days of age indicates an advanced phase of ovarian maturation, which was accompanied by constantly higher AMH concentrations up to the postpubertal period, a greater ovarian response to FSH stimulation and by higher pregnancy rates at first mating, as compared with the low AFC group.

Melatonin deprival modifies follicular and corpus luteal growth dynamics in a sheep model.

This study assessed the effect of melatonin deprival on ovarian status and function in sheep. Experimental procedures were carried out within two consecutive breeding seasons. Animals were divided into two groups: pinealectomised (n=6) and sham-operated (n=6). The completeness of the pineal gland removal was confirmed by the plasma concentration of melatonin. Ovarian status was monitored by ovarian ultrasonography for 1 year to study reproductive seasonality. Follicular and corpus luteal growth dynamics were assessed during an induced oestrous cycle. As the effects of melatonin on the ovary may also be mediated by its antioxidant properties, plasma Trolox equivalent antioxidant capacity (TEAC) was determined monthly for 1 year. Pinealectomy significantly extended the breeding season (310±24.7 vs 217.5±24.7 days in controls; P<0.05). Both pinealectomised and sham-operated ewes showed a well-defined wave-like pattern of follicle dynamics; however, melatonin deficiency caused fewer waves during the oestrous cycle (4.3±0.2 vs 5.2±0.2; P<0.05), because waves were 1 day longer when compared with the controls (7.2±0.3 vs 6.1±0.3; P<0.05). The mean area of the corpora lutea (105.4±5.9 vs 65.4±5.9 mm(2); P<0.05) and plasma progesterone levels (7.1±0.7 vs 4.9±0.6 ng/ml; P<0.05) were significantly higher in sham-operated ewes compared with pinealectomised ewes. In addition, TEAC values were significantly lower in pinealectomised ewes compared with control ones. These data suggest that melatonin, besides exerting its well-known role in the synchronisation of seasonal reproductive fluctuations, influences the growth pattern of the follicles and the steroidogenic capacity of the corpus luteum.

In vitro maturation is slowed in prepubertal lamb oocytes: ultrastructural evidences.

BACKGROUND: In vitro maturation (IVM) of immature oocytes retrieved from unstimulated ovaries may avoid side effects connected to hyperstimulation during IVF procedures, including the risk of cancer recurrence. In humans, the scarce availability of immature oocytes limits morphological studies. The monovular ovine may represent an experimental model for IVM studies. METHODS: To assess if the scarce developmental competence of prepubertal oocytes (PO) is related to morphological changes we analyzed, by light and transmission electron microscopy, cumulus-oocyte-complexes (COCs) from lambs (30-40 days old) and sheep (4-6 years old) at sampling and after 7 h, 19 h, 24 h of IVM. Meiotic progression was determined at the same time points. RESULTS: At sampling, the germinal vesicle (GV) of PO was round and centrally or slightly eccentrically located, whereas in adult oocytes (AO) it was irregularly shaped and flattened against the oolemma. PO, differently from AO, showed numerous trans-zonal projections. Organelles, including cortical granules (CGs), were more abundant in AO. After 7 h, the percentage of AO that underwent GVBD-MI transition increased significantly. In PO, the oolemma was juxtaposed to the ZP; in AO, it showed several spikes in correspondence of cumulus cells (CC) endings. In PO, organelles and isolated CGs were scattered in the ooplasm. In AO, groups of CGs were also present under the oolemma. After 19 h, PO underwent GVBD-MI transition; their oolemma showed several spikes, with CC projections retracted and detached from the ZP. AO underwent MI-MII transition; their oolemma regained a round shape. CGs were located beneath the plasmalemma, arranged in multiple, continuous layers, sometime discontinuous in PO. After 24 h, both groups reached the MII-stage, characterized by a regular oolemma and by expanded CCs. PO showed CGs distributed discontinuously beneath the oolemma, while AO showed a continuous monolayer of CGs. CONCLUSIONS: Even if PO were able of reaching morphological maturation after 24 h of IVM, our ultrastructural analysis allowed detecting the presumptive sequence of cytoplasmic alterations connected with the delay of nuclear maturation, that might explain the reduced developmental competence of such oocytes. Data from the sheep model are of interest for zootechny, and provide an experimental basis for improving human IVM technology.

Predictive value of antral follicle count and anti-Müllerian hormone for follicle and oocyte developmental competence during the early prepubertal period in a sheep model.

Circulating anti-Müllerian hormone (AMH) and antral follicle count (AFC) are addressed as suitable markers of oocyte quantity and quality during adulthood. To investigate whether AFC and circulating AMH could predict follicle development and oocyte quality during the prepubertal period we used 40-day-old ewe lambs with high, intermediate and low AFC (≥30, 16-29 and≤15 follicles respectively). The analysis of the response to the exogenous FSH ovarian reserve test showed a positive correlation between AFC, AMH plasma levels, total follicle number and the number of large follicles (≥3mm) grown after exogenous FSH administration. The incorporation of abattoir-derived oocytes collected from ovaries with different AFC in an in vitro embryo production system showed that a high AFC can predict oocyte quality in prepubertal ovaries, reflecting an ovarian status suitable for follicular development. The histological quantification of the ovarian reserve evidenced that AFC was not predictive of differences in either the number of healthy follicles or the size of the primordial follicle pool in prepubertal ovaries. Further studies are needed to investigate the implication on the reproductive performance of the significant inter-individual differences found in the present study in AFC and circulating AMH in the early prepubertal period.

Fetal Growth and Osteogenesis Dynamics during Early Development in the Ovine Species.

Increased knowledge of the developmental processes during gestation could provide valuable information on potential alterations in embryonic/fetal development. We examined the development of ovine conceptus between the 20th and 70th day of gestation with three convergent analyses: (1) uterus ultrasound examination and measurement (eco) of crown-rump length (CRL) and biparietal diameter (BPD) of the conceptus; (2) direct measurement (vivo) of CRL and BPD of the conceptus outside the uterus (3) osteo-cartilage dynamics during development by differential staining. No significant differences were observed between eco and vivo measurements for CRL and BPD in all examined concepti. CRL and BPD, instead, showed a significant positive linear correlation with gestational age. The study of osteogenesis dynamics has demonstrated a completely cartilaginous ovine fetus at up to 35 days of gestation. The ossification begins in the skull (40th day) and is almost complete between the 65th and the 70th of pregnancy. Our study highlighted that CRL and BPD are accurate parameters for gestational age estimation in the first part of sheep pregnancy and provides an overview of osteochondral temporal dynamics. Furthermore, tibia ossification is a valid parameter to estimate fetal age by ultrasound.

Effect of aging on follicular function may be relieved by exogenous gonadotropin treatment in a sheep model.

The current study investigated hormonal and ovarian changes during physiological reproductive aging in Sarda ewes. In a first experiment, follicular and corpus luteum dynamics were compared during an induced oestrus cycle in aged (12-14 years) and young adult ewes (4-5 years). Oestrus cycle characteristics did not differ between the two experimental groups. However, follicular function during the follicular phase showed significant alterations in aged ewes, as determined by a lack of dominance effect and by lower mean values of circulating oestradiol (E(2)) and inhibin levels, compared with young adult ewes. In a second experiment, differences in follicle growth, hormonal milieu and oocyte quality in response to exogenous FSH administration were assessed in aged and adult ewes. No differences were recorded in ovarian response to FSH treatment between young adult and aged ewes, as evaluated by ultrasonographic data and circulating concentrations of LH, E(2) and inhibin-A. Although the total number of recovered oocytes was similar in the two age groups, the number of good quality oocytes selected for IVM was significantly lower in aged ewes compared with adult ones. Thereafter, no differences were recorded in cleavage rates, total blastocyst output, embryo developmental kinetic and quality between aged and adult groups. In conclusion, this study demonstrated that reproductive aging in sheep is associated with impaired follicle functionality and an increase in the proportion of oocytes showing morphological abnormalities. However interestingly, oocyte developmental competence in vitro and embryo cryotolerance were not affected by the aging process, when only good quality oocytes were chosen.

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 [...].

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<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.

Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep.

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily obtained from a slaughterhouse. Adult ovaries always bear several antral follicles, while in pre-pubertal donors the maximal numbers of oocytes are available at 4 weeks of age, when ovaries bear peak numbers of antral follicles. Thus, 4 weeks old lambs are considered good donors, even if the developmental competence of prepubertal oocytes is lower compared to their adult counterpart. Basic research and commercial applications would be boosted by the possibility of successfully cryopreserving vitrified oocytes obtained from both adult and prepubertal donors. The vitrification of oocyte collected from prepubertal donors would also allow shortening the generation interval and thus increasing the genetic gain in breeding programs. However, the loss of developmental potential after cryopreservation makes mammalian oocytes probably one of the most difficult cell types to cryopreserve. Among the available cryopreservation techniques, vitrification is widely applied to animal and human oocytes. Despite recent advancements in the technique, exposures to high concentrations of cryoprotective agents as well as chilling injury and osmotic stress still induce several structural and molecular alterations and reduce the developmental potential of mammalian oocytes. Here, we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming incubation period. Oocytes vitrified at the MII stage can indeed be fertilized following warming, but they need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, post-warming culture conditions and timing are crucial steps for the restoration of oocyte developmental potential, especially when oocyte are collected from juvenile donors.

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.

Molecular and Histological Evaluation of Sheep Ovarian Tissue Subjected to Lyophilization.

Cryopreservation is routinely used to preserve cells and tissues; however, long time storage brings many inconveniences including the use of liquid nitrogen. Freeze-drying could enable higher shelf-life stability at ambient temperatures and facilitate transport and storage. Currently, the possibility to freeze-dry reproductive tissues maintaining vitality and functions is still under optimization. Here, we lyophilized sheep ovarian tissue with a novel device named Darya and a new vitrification and drying protocol and assessed effects on tissue integrity and gene expression. The evaluation was performed immediately after lyophilization (Lio), after rehydration (LR0h) or after two hours of in vitro culture (IVC; LR2h). The tissue survived lyophilization procedures and maintained its general structure, including intact follicles at different stages of development, however morphological and cytoplasmic modifications were noticed. Lyophilization, rehydration and further IVC increasingly affected RNA integrity and caused progressive morphological alterations. Nevertheless, analysis of a panel of eight genes showed tissue survival and reaction to the different procedures by regulation of specific gene expression. Results show that sheep ovarian tissue can tolerate the applied vitrification and drying protocol and constitute a valid basis for further improvements of the procedures, with the ultimate goal of optimizing tissue viability after rehydration.

Capillary electrophoresis with laser-induced fluorescence detection for ATP quantification in spermatozoa and oocytes.

We describe a new capillary electrophoresis laser-induced fluorescence (CE-LIF) method for the quantification of adenosine 5'-triphosphate (ATP) in spermatozoa and oocytes. The optimization of the precapillary derivatization reaction between ATP and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) has been described. BODIPY-ATP conjugate was analysed in an uncoated fused silica capillary of 75 µm ID and 50 cm effective length using a 10 mmol/L tribasic sodium phosphate buffer, pH 11.5, at 22 kV in <5 min. A good reproducibility of intra- and inter-assay tests was obtained (CV = 4.55% and 7.14%, respectively). With respect to our previous CE-UV assay, the new method showed an improvement in sensitivity that was about 120-fold (limit of quantification, 0.15 vs 18 µmol/L). Method applicability was proven on the reproductive cells of several animal species (roosters, horses, sheep and goats). Due to the elevated sensitivity, the new assay allows the measurement of adenosine 5'-triphosphate levels from just 20 oocytes. Considering that ATP concentration in reproductive cells is related to the mitochondrial integrity after cryopreservation, the proposed method could be a useful tool in assisted reproductive technologies.

Involvement of E-cadherin in early in vitro development of adult and juvenile sheep embryos.

The oocyte-to-embryo transition in mammals depends on maternal proteins and transcripts, which accumulate during oocyte differentiation. The aim of the present study was to examine the role of the junctional proteins beta-catenin and E-cadherin during preimplantation in vitro embryo development in sheep, comparing the competence of adult and prepubertal oocytes. We analysed the concentration of beta-catenin and E-cadherin in immature and in vitro-matured oocytes. There was a significant increase in E-cadherin concentration after 24 h of in vitro maturation and this was lower in prepubertal oocytes than in adult ones. We therefore studied the expression and distribution of E-cadherin during the major transition from maternal to embryonic genome. E-cadherin distribution and localisation in sheep was age- and developmental-stage dependent and was related to developmental kinetics. In fact, in adults, the majority of embryos showed the proper distribution of E-cadherin just beneath the membrane surfaces of all blastomeres and the percentage of embryos with this distribution increased with the increase in cell number during development. On the contrary, and regardless of their developmental stage, the majority of prepubertal embryos showed an uneven distribution of the protein, often associated with the occurrence of cellular fragmentation. In conclusion, our results suggest that E-cadherin plays a pivotal role during preimplantation embryo growth in sheep and may be one of the possible cytoplasmic factors involved in the reduced developmental competence of prepubertal female gametes.

Morphological features and microtubular changes in vitrified ovine oocytes.

Cryobanking of oocytes collected from prepubertal donors may supply a virtually unlimited number of female gametes for both basic research and commercial applications. Prepubertal oocytes show some structural and functional limitations compared to the adult ones that may impair their ability to recover damages from cryopreservation. In oocytes, the meiotic spindle is acutely sensitive to temperature deviation, but capable of regeneration following cryopreservation. In the present work, we studied the effects of vitrification and post-warming incubation on the microtubular cytoskeleton and the tubulin post-translational modifications (tyrosination and acetylation) in prepubertal and adult oocytes. Obtained results showed that prepubertal oocytes are more affected by vitrification-induced injuries than adult ones. In fact, prepubertal oocytes showed more severe alterations of the meiotic spindle conformation and a higher percentage of parthenogenetic activation compared to adult ones. Moreover, in the adult oocytes the equilibrium between tyrosinated and acetylated α-tubulin was restored after 4 h of post-warming incubation. Diversely, in prepubertal oocytes the imbalance between tyrosinated and acetylated α-tubulin was increased during post-warming incubation. Our study shows that prepubertal oocytes react differently to the insults provoked by vitrification compared to adult oocytes, showing an impaired ability to recover from vitrification-induced injuries. In the evaluation of oocyte ability to recover from vitrification-induced injuries, tubulin post-translational modifications represent an important indicator for assessing oocyte quality.

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.

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 microl), 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.

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

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.

In vitro production and cryotolerance of prepubertal and adult goat blastocysts obtained from oocytes collected by laparoscopic oocyte-pick-up (LOPU) after FSH treatment.

This study compares the developmental capacity and cryotolerance of embryos produced from oocytes of stimulated prepubertal and adult Sarda goats. Twelve prepubertal and 13 adult goats were each given 110 and 175 IU FSH, respectively, and cumulus-oocyte complexes (COCs) were collected by laparoscopic oocyte-pick-up (LOPU). After in vitro maturation, fertilisation and culture (IVMFC), blastocysts were vitrified, warmed and blastocoel re-expansion and gene expression were evaluated. Prepubertal goats produced a higher COCs number than adults (mean +/- s.e.m., 89.67 +/- 5.74 and 26.69 +/- 3.66, respectively; P < 0.01). Lower developmental competence was demonstrated in the prepubertal oocytes as shown by a higher number of COCs discarded before IVM (21.1% and 14.7% for prepubertals and adults, respectively; P < 0.01) and IVF (23.4% v. 9.1%; P < 0.01) and by the lower cleavage (55.6% and 70.3%, respectively; P < 0.01) and blastocyst rates (24.2% and 33.9%, respectively; P < 0.05). Compared with the adult, prepubertal vitrified/warmed blastocysts showed significantly (P < 0.05) lower in vitro viability, as determined by the re-expansion rate (62.5% and 40.3%). No differences were observed in the time required for blastocoel re-expansion or in cyclin B1, E-cadherin, Na/K ATPase, HSP90beta and aquaporin 3 messenger RNA quantity. These results show that in vitro-produced embryos produced from prepubertal goat oocytes have a lower developmental rate and cryotolerance compared with their adult counterparts. However, we can assume that the quality of re-expanded embryos does not differ between the two groups.

Commercial human kits' applicability for the determination of biochemical parameters in sheep plasma.

Currently, several commercially available biochemical kits are validated for their use in human but not in animals. The purpose of this work is to demonstrate the applicability of human kits for alanine-aminotransferase, aspartato-aminotransferase, albumin, total protein, total cholesterol, and triglycerides in ovine plasma. Assays were validated according to international guidelines and stability was explored. Accuracy values were between 67 and 100%, and intra and interday precisions (%RSD) were <15% for all studied parameters. These results confirm the suitability of the studied human kits for their use in ovine plasma and they were used in plasma collected from pregnant ewes.