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A paper describing the differentiation of swine adipose-derived stem cells into osteoblasts.

Vitrification induces ultrastructural and structural damages in different oocyte structures such as membrane, zona pellucida (ZP) and cytoplasm.1 Recently Raman Microspectroscopy (RMS) has been used to assess the changes caused by vitrification/ warming of in vitro matured ovine oocytes.2 RMS is a noninvasive technique for studying the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. Aim of the present study was to investigate the structural modifications of ZP and cytoplasm of vitrified/warmed in vitro matured bovine oocytes by RMS. Abattoir derived in vitro matured oocytes (n=171) were denuded and divided in three experimental groups: control untreated oocytes (CTR), oocytes only exposed to vitrification/warming solutions (CPA) and vitrified/warmed oocytes (VITRI). Oocytes were exposed/vitrified in 20% EG + 20% of DMSO and 0.5 M sucrose and warmed into decreasing concentrations of sucrose (1.25 M-0.3 M). RMS analysis was carried out on CTR matured oocytes (after 22h) and on CPA and VITRI oocytes at different incubation times (0,1,2,3 and 4h) after exposure/warming. The analysis of the large spectral date set, acquired by RMS were performed by Principal Component Analysis (PCA). Our experimental outcomes suggest that vitrification induce a transformation of the protein secondary structure from the α-helices to the b-sheet form in the ZP, while lipids tend to assume a more ordered configuration. Both effects induce a mechanical stiffening of ZP, which could explain the reduced fertility of vitrified oocytes with respect to the untreated ones. Intriguingly, these transformations present a certain degree of reversibility, which renders vitrified oocytes more similar to untreated cells after 2h of warming at room temperature.

In mammals, the hormone prolactin (PRL) is best known for its role in the regulation of lactation (Lee et al. 2007). PRL affects multiple reproductive and metabolic functions through its receptors (PRLR), characterized by the ability to activate Janus kinase 2 and signal transducer and activator of transcription (Fleenor et al. 2006). PRL is an anterior pituitary peptide hormone involved in many endocrine activities essential for reproductive success (Vaclavicek et al. 2006). Several polymorphic sites have been detected within PRL and PRLR genes, and significant associations among their variants with milk production traits have been described in dairy cattle (Alfonso et al. 2012; Zhang et al. 2008). PRL (EMBL EF054878) and PRLR (EMBL GQ339914) genes consist of 5 and 9 exons, coding for a protein of 219 and 581 amino acids, respectively. A partial sequencing of PRL exon 5 and PRLR exon 9 performed on 10 Italian Mediterranean river buffaloes allowed us to identify 1 SNP for PRL (C108→T, numbering from the 1st nt of exon 5) and 7 SNPs for PRLR (G128→A, C374→T, G517→A, C520→A, C622→T, G626→A and T707→C, numbering from the 1st nt of exon 9). The PRL SNP is silent (Leu202) as well as 626 (Ala494) and 707 (Asp521) PRLR SNPs. The other 5 PRLR SNPs are responsible of the following amino acid changes: His328→Arg, Ala410→Val, Asp458→Asn, Gln459→Lys and Thr493→Met. 574 buffaloes of several farms in Salerno province (South Italy) were genotyped in out-sourcing (http://kbioscience.co.uk) for 108 PRL and 128 PRLR SNPs. The genotype distribution of investigated population was 10 C/C, 122 C/T and 442 T/T (frequency of T allele 0.87) for PRL and 9 G/G, 108 G/A and 457 A/A for PRLR (frequency of A allele 0.89). For both SNPs, the investigated population was in Hardy–Weinberg equilibrium. Such polymorphisms could represent useful genetic markers for association studies with quali-quantitative milk characteristics, but further studies are needed to evaluate their potential use.

Most media employed for producing bovine embryos in vitro include glucose as an energy source despite putative toxic effects. The aim of this work was to evaluate whether replacing glucose with myo-inositol and citrate during IVC improves in vitro embryo development and resistance to cryopreservation. Abattoir-derived oocytes were matured and fertilized in vitro using standard procedures. After 20–22 h of gametes co-incubation, zygotes were denuded and cultured in SOF containing either 1.5 mM glucose or 2.77 mM myo-inositol and 0.34 mM citrate for 7 days. Embryos were first incubated in 7.5 % EG and 7.5 % dimethyl sulfoxide (DMSO) for 3 min, then transferred into 16.5 % EG and 16.5 % DMSO and 0.5 M sucrose for 25 sec before being loaded into the cryotop. Warming was carried out by immerging the cryotop into a 0.25 M sucrose solution and by transferring the embryos into a 0.15 M sucrose for 5 min. Vitrified-warmed embryos were then cultured in vitro for further 24 hr, after which the embryo survival rate was recorded. Data were analyzed by Chi-Square test. The results of this study showed that myo-inositol-citrate increased blastocyst yield (37.4 vs 29.5 %, respectively; P

The objectives of this work were to evaluate whether the sperm penetration speed is correlated to the in vitro fertility and whether adapting the gamete co-incubation length to the kinetics of the bull improves in vitro fertility and affects the sex ratio. In vitro matured oocytes were co-incubated with spermatozoa from four different bulls (A-D). At various post-insemination (p.i.) times (4, 8, 12, 16 and 20 h), samples of oocytes were fixed and stained with DAPI for nuclei examination, while the remaining ones were transferred into culture to evaluate embryo development. The blastocysts produced were sexed by PCR. Two bulls (A and B) had faster kinetics than the others (C and D), as shown by the higher penetration rates recorded at 4 h p.i. (43%, 30%, 11% and 6%, respectively for bulls A, B, C and D; p

Negli ultimi anni, un numero sempre maggiore di aziende zootecniche ha iniziato ad avvalersi delle biotecnologie riproduttive, perché così facendo possono incrementare le performance produttive e indirizzare la produzione verso una qualità spesso impensabile, ottenendo nel contempo la riduzione dei rischi di trasmissione di malattie, tutto nell’ottica di una risposta più adeguata alle richieste di un mercato sempre più specifico. Pertanto prove sperimentali differenti sono state condotte al fine di migliorare, l’efficienza della fecondazione in vitro e la resa nella coltura embrionale in vitro nelle specie bovina ed in quella bufalina L’insieme dei risultati ha dimostrato l’esistenza di evidenti differenze nei fabbisogni metabolici degli embrioni bufalini rispetto a quelli bovini, di cui bisogna tener conto per ottimizzare il sistema in questa specie.

Although oocyte cryopreservation has great potentials in the field of reproductive technologies, it still is an open challenge in the majority of domestic animals and little is known on the biochemical transformation induced by this process in the different cellular compartments. Raman micro-spectroscopy allows the non-invasive evaluation of the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. The aim of this work was to assess the biochemical modifications of both the zona pellucida and cytoplasm of vitrified/warmed in vitro matured bovine oocytes at different post-warming times. By taking advantage of Principal Component Analysis, we were able to shed light on the biochemical transformation induced by the cryogenic treatment, also pointing out the specific role of cryoprotective agents (CPs). Our results suggest that vitrification induces a transformation of the protein secondary structure from the α-helices to the β-sheet form, while lipids tend to assume a more packed configuration in the zona pellucida. Both modifications result in a mechanical hardening of this cellular compartment, which could account for the reduced fertility rates of vitrified oocytes. Furthermore, biochemical modifications were observed at the cytoplasmic level in the protein secondary structure, with α-helices loss, suggesting cold protein denaturation. In addition, a decrease of lipid unsaturation was found in vitrified oocytes, suggesting oxidative damages. Interestingly, most modifications were not observed in oocytes exposed to CPs, suggesting that they do not severely affect the biochemical architecture of the oocyte. Nevertheless, in oocytes exposed to CPs decreased developmental competence and increased reactive oxygen species production were observed compared to the control. A more severe reduction of cleavage and blastocyst rates after in vitro fertilization was obtained from vitrified oocytes. Our experimental outcomes also suggest a certain degree of reversibility of the induced transformations, which renders vitrified oocytes more similar to untreated cells after 2 h warming.

Preliminary experiments carried out in our laboratory indicated that—by fertilizing oocytes matured in vitro for 24–32 and 48 h—the resulting blastocyst rates decreased from 22.4 % (24 h), to 14.0 % (32 h) to ‘0’ % (48 h). The aim of this study was investigate upon the variation in the incidence of chromosomal abnormalities occurring in bovine oocytes matured in vitro for prolonged periods of time, i.e. from 24 to 32 to 48 h. Abattoir-derived oocytes were matured in vitro using standard procedures, for 24–32 and 48 h. After incubation, the COCs were treated with ialuronidase (3mg/ml) to eliminate the cumulus cells, swelled in hypotonic (KCl, 0.075 M) for 5–10 min, fixed individually on microscope slides with Carnoy fixative, air dried and stained with 5 % Giemsa. Conventional karyotypes were prepared from 50 matured oocytes for each time of maturation, providing the following results: chromosomal abnormalities, including unreduced diploid metaphases, hypo-haploidy and hypo-diploidy, increased from 12 % at 24 h, to 20 % at 32 h, to 36 % at 48 h. The Chi-square test (with Yates corrections) showed significant differences (P>0.01) in the rate of chromosome abnormalities from 24 to 48 h of maturation as well as from 32 to 48 h, whereas the differences between 24 and 32 h were not significant. These results confirm previous data and provide further evidence that bovine oocytes to be used in IVF programs should not be matured in vitro longer than 24–32 h.

The female gametes are more susceptible to chromosome segregation errors during meiosis I division and therefore they are the major contributors to the embryo aneuploidies. The evaluation of aneuploidies in bovine oocytes is useful for monitoring the reproductive health of this species and FISH is the main method employed for this purpose. To date only 2–3 chromosomes were simultaneously investigated for aneuploidy detection in cattle oocytes. In this work we propose a multi-color ZOO-FISH by the simultaneous detection of six specific chromosome painting probes on secondary oocytes matured in vitro. Standard procedures were employed for 24 h in vitro oocytes maturation, whereas specific autosomal probes were prepared by microdissection and DOP-PCR using river buffalo mitosis (2n=50). Probes were labelled with spectrum-green and -orange in a second DOP-PCR. Three sequential rounds of FISH were achieved for the same slides. Each round was realized using two probes simultaneously hybridized on MII oocytes with the corresponding first polar bodies (I pb). Slides were counterstained with DAPI in antifade. Digital images were captured in gray-scale and pseudo-colored by the software. Six specific probes, painting 3 out of 5 sub-metacentric river buffalo chromosomes (BBU 1p, 1q, 3p, 3q, 4p and 4q) were sequentially hybridized on BTA secondary oocytes with the corresponding (I pb). The different colors of the probes allowed the identification of six cattle chromosomes (BTA 1, 5, 8, 19, 27 and 28) both on MII and polar bodies evidencing no abnormalities for the investigated cells, but confirming their potential use for aneuploidy detection in bovine oocytes. This result opens further opportunity of investigation for clinical cytogenetic applications also in the other species with difficult CGH karyotype.

Excess thymidine is capable of synchronizing lymphocyte cultures in vitro by acting with a feed-back mechanism during the S phase of the cell cycle (Harper 2005 Methods Mol. Biol. 296, 157-166). The possibility to synchronize the embryonic growth can be a good strategy for future epigenetic studies. The present study was undertaken to test whether excess thymidine could also synchronize in vitro development of bovine embryos. Abattoir-derived cumulus-oocyte complexes (COC) of the Agerolese breed of cattle were matured in vitro using standard procedures. After maturation, COCs were transferred in drops of 300µL of IVF-TALP (25/drop) and covered with mineral oil. Frozen sperm from a bull were selected by centrifugation on a Percoll discontinuous gradient (45 to 80%). The pellet was diluted in IVF medium and added to the COC at the concentration of 1×10(6)spermmL(-1). After 18-20h of gametes co-incubation, presumptive zygotes were denuded and cultured in SOF medium containing different concentrations of thymidine (0, 300, 600, 1200, 2400µgmL(-1), final concentrations). The day after (Day 2) presumptive zygotes were washed four times in fresh SOF, classified morphologically under a stereomicroscope as not divided (n.d.), 2 cells, 3-8 cells, and 9-32 cells and cultured in standard SOF at 39°C in a humidified mixture of 5% CO(2),7% O(2), and 88% N(2). On Day 3, the embryos were again examined for the growth stage in relation to the synchronization effects. On Day 7, the embryos were evaluated for the final growth efficiency (cleavage stage and blastocyst formation). The experiment was replicated 4 times (except the 2400µgmL(-1) condition, which was replicated 2 times because of its clear toxic effect). Data were analyzed by ANOVA test. At Day 2, there were no differences between groups whatever the concentration (n.d.-2 cells: 59.7±11.2, 52.9±26.3, 56.1±14.6, 66.0±2.8, 38.0±11.3; 3-8 cells : 40.3±11.2, 47.1±26.3, 43.9±14.6, 34.0±2.8, 59.9±9.5; respectively for 300, 600, 1200, 2400, and control), while differences at Day 3 and 7 are shown in Table 1. The dosage of 300µgmL(-1) slowed embryo development without altering the developmental rate, whereas the other dosages were somewhat toxic to the zygotes, affecting the final percentage of blastocysts.

The aim of this work was to evaluate whether minimizing the glucose concentration during culture or replacing the hexose with other energy substrates and/or embryotrophic compounds would affect the in vitro development, the resistance to cryopreservation and the sex ratio of bovine embryos. In vitro matured and fertilized oocytes were randomly assigned to 4 groups for in vitro culture, that differed in the energy substrates included: group A) 1.5 mM glucose, as in standard SOF; group B) 0.15 mM glucose; group C) 0.125 mM G3P, in the presence of 0.15 mM glucose and group D) 0.34 mM citrate, in combination with 2.77 mM myo-inositol. Blastocysts were evaluated on day 7, then vitrified by cryotop in 16.5% DMSO, 16.5% EG and 0.5 M sucrose and warmed in decreasing concentration of sucrose (0.25 to 0.15 M sucrose). The survival rates were assessed after 24 h in vitro culture. Finally, the blastocysts produced were sexed by PCR. An increased blastocyst rate was recorded in groups B, C and D, i.e., when glucose concentration was reduced, compared to group A (28.2, 41.0, 35.7 and 35.8, respectively in groups A, B, C and D; P < 0.01). However, the embryos cultured in group D showed the slowest developmental speed, indicated by the lowest percentage of advanced stage-embryos (expanded and hatched blastocysts) out of the total blastocysts (56.1, 45.8, 56.9 and 31.8 %, respectively in groups A, B, C and D; P < 0.01). Furthermore, survival rates after 24 h culture of vitrified-warmed blastocysts also decreased in group D (73.3, 73.1, 71.4 and 58.4%, respectively in groups A, B, C and D; P < 0.01). Interestingly, in group D a higher percentage of female embryos was obtained compared to group A, with intermediate values in groups B and C (45.6, 53.4, 50.0 and 61.5%, respectively in groups A, B, C and D; P < 0.05). In conclusion, it was demonstrated that the energy substrate during in vitro culture affects both the production and the viability of blastocysts. Furthermore, manipulating the metabolic profile of embryos during in vitro culture may have an impact on sex ratio.