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Diverse strategie sono state sviluppate per minimizzare i danni apoptotici indotti dalla crioconservazione. In particolare, sono stati testati diversi inibitori dell’apoptosi nel tentativo di preservare l’integrità delle membrane senza alterarne il potenziale, ridurre la frammentazione del DNA in seguito all’insulto del congelamento e modulare l’attività delle caspasi bloccando il segnale apoptotico. Pertanto, lo scopo della tesi è stato quello di individuare delle possibili strategie di prevenzione dell’apoptosi indotta dal congelamento, mediante inibizione delle caspasi, al fine migliorare la criotolleranza degli embrioni e dei gameti (oociti e spermatozoi) nella specie bovina. In particolare, è stato investigato l’effetto dell’inibitore delle caspasi Z-VAD-FMK all’interno dei terreni di vitrificazione, scongelamento e coltura per gli embrioni e per gli oociti e dei terreni di congelamento e incubazione per gli spermatozoi al fine di ridurre l’apoptosi e i danni da congelamento, migliorando la criotolleranza post-scongelamento degli embrioni e dei gameti bovini. Lo scopo dell’esperimento 1 è stato quello di valutare l'effetto dell’aggiunta dell’inibitore Z-VAD-FMK (20µM) prima, durante e dopo la vitrificazione sulla criotolleranza degli embrioni bovini prodotti in vitro. A tale fine sono state valutate la sopravvivenza, la progressione allo sviluppo e le percentuali di schiusa delle blastocisti vitrificate dopo 24 e 48 ore di coltura di post-riscaldamento, nonché il numero di cellule embrionali e la loro distribuzione tra trofectoderma e nodo embrionale. Inoltre l’influenza dell’inibitore sull’apoptosi è stata valutata mediante misurazione della frammentazione del DNA e dell’attività della caspasi 3. Lo scopo dell’esperimento 2, è stato quello di studiare gli effetti dell’inibitore Z-VAD-FMK (20µM), prima, durante e dopo la vitrificazione sulla criotolleranza degli oociti bovini maturati in vitro. L’efficacia dell’inibitore sulla prevenzione dell’apoptosi è stata valutata mediante stima della frammentazione del DNA, dell’attività caspasica, del potenziale di membrana mitocondriale, nonché della vitalità e della competenza allo sviluppo embrionale dopo fecondazione in vitro. L’esperimento 3 si è, infine, prefisso l’obiettivo di studiare l’influenza di un trattamento con l’inibitore Z-VAD-FMK (20 e 100 µM) prima, durante e dopo il congelamento sulla criotolleranza degli spermatozoi bovini. L’efficacia del trattamento è stata verificata su diversi parametri indicativi di fertilità spermatica, quali la motilità, la vitalità, l'integrità di membrana, la frammentazione del DNA, l’attività della Caspasi 3 ed il potenziale di membrana mitocondriale. I risultati dell’ esperimento 1 hanno dimostrato che il trattamento con l'inibitore della caspasi Z-VAD-FMK, migliora la criotolleranza degli embrioni bovini prodotti in vitro, prevenendo l'apoptosi indotta da crioconservazione come dimostrato dalle maggiori percentuali di sopravvivenza (76.1 vs 51.1%, P

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.

The aim of this study was to evaluate the treatment of bovine semen with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), before or after freezing on semen quality. After the initial assessment, sperm from 4 bulls were pooled (Experiment 1) and cryopreserved in BioXcell containing 0, 20 and 100 μM Z-VAD-FMK. After thawing semen viability, motility, membrane integrity, as well as DNA fragmentation and ΔΨm were evaluated. In Experiment 2, bovine frozen/thawed sperm were incubated for 1 hr with 0, 20 and 100 µM Z-VAD-FMK before assessing the semen quality. The treatment with Z -VAD-FMK before cryopreservation improved post-thawing sperm motility compared to the control group (p < .05), while no differences were recorded in sperm viability and membrane integrity among groups (on average 86.8 ± 1.5 and 69.1 ± 1.4, respectively). Interestingly, at the highest concentration, DNA fragmentation decreased (p < .05), while the percentage of spermatozoa with high ΔΨm increased (p < .05). The results of Experiment 2 showed that 1-hr treatment with Z-VAD-FMK did not affect sperm motility and viability (on average 63.4 ± 5.8 and 83.7.1 ± 1.2, respectively). However, Z-VAD-FMK improved sperm membrane integrity (p < .05) and at the highest concentration tested decreased the proportion of sperm showing DNA fragmentation (p < .05). No differences were recorded in the percentage of spermatozoa with high ΔΨm (on average 57.0 ± 11.4). In conclusion, the treatment with 100 µM of the caspase inhibitor Z-VAD-FMK before freezing increased bovine sperm mass motility and ΔΨm, while decreasing sperm DNA fragmentation. Treatment of semen after thawing with 100 µM Z-VAD-FMK improved sperm membrane integrity and reduced DNA fragmentation.

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.

The aim of this study was to investigate the effect of cholesterol-loaded cyclodextrins (CLC) on motility, viability, capacitation status, and in vivo fertility of buffalo frozen-thawed sperm. After the initial semen assessment, buffalo sperm were diluted in BULLXcell extender containing 0- (control), 1.5-, and 3-mg/mL CLC and cryopreserved. At thawing, sperm motility was evaluated by phase contrast microscopy, and viability-capacitation status was assessed by Hoechst 33258-chlortetracycline (CTC) assay. Capacitation status was also evaluated by an indirect immunofluorescence assay to localize phosphotyrosine-containing proteins. Moreover, buffaloes were artificial inseminated to assess the in vivo-fertilizing potential of CLC-treated semen. No differences among control, 1.5-, and 3-mg/mL CLC-treated groups were recorded in both sperm motility (66.5 ± 5.6, 68.8 ± 4.8, and 68.8 ± 4.8, respectively) and viability (86.5 ± 1.9, 87.6 ± 1.5, 88.4 ± 2.3, respectively). However, the extender supplementation with CLC significantly reduced sperm cryocapacitation. Indeed, CLC treatment decreased (P < 0.01) the proportion of sperm showing the CTC pattern B (capacitated sperm) compared with the control (69.6 ± 3.4, 37.8 ± 1.5, and 51.3 ± 4.7, respectively, with 0, 1.5-, and 3-mg/mL CLC; P < 0.01). Furthermore, the percentage of sperm displaying tyrosine-phosphorylated pattern EA (i.e. high capacitation level) was reduced (P < 0.01) in both CLC-treated groups (10.8 ± 3.3 and 5.6 ± 1.6, respectively, with 1.5- and 3-mg/mL CLC) compared with the control (37.3 ± 6.9), reaching values similar to those recorded in fresh semen (11.0 ± 3.5). In addition, treating sperm with 3-mg/mL CLC increased (P < 0.01) the percentage of nonfluorescent (pattern NF), i.e., non-capacitated sperm (41.8 ± 3.6) compared with fresh semen (11.0 ± 6.9). No differences were recorded in pregnancy rates at 60 days post-artificial insemination among control, 1.5- and 3-mg/mL CLC groups (59.7%, 65.6%, and 56.9%, respectively). In conclusion, CLC treatment of buffalo sperm strongly decreases sperm cryocapacitation damages, without affecting the in vivo fertilizing capability.

The aim of this work was to assess the effect of supplementation of bovine culture medium with the natural antioxidant crocetin on in vitro blastocyst development and quality. This was evaluated as cryotolerance, apoptosis index, and total cells number and allocation. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid medium, supplemented with 0, 1, 2.5, and 5 μM crocetin (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed and the blastocysts were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO, and 0.5 M sucrose. Finally, blastocysts produced on Day 8 in the absence (control) and presence of 1 μM crocetin were used for terminal deoxynucleotidyl transferase–mediated dUTP nick end labelling and differential staining to evaluate, respectively, the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm (TE) lineages (experiment 2). Embryo development was higher in the 1 μM crocetin group compared to the control, both in terms of total embryo output (37.7 ± 4.2%, 52.9 ± 6.3%, 40.9 ± 7.6%, and 42.4 ± 8.7%, respectively, with 0, 1, 2.5, and 5 μM; P

The aim of this study was to evaluate whether supplementation of bovine culture medium with the natural antioxidant L-ergothioneine (LE), improves in vitro blastocyst development and quality, assessed as resistance to cryopreservation, total cells number, cellular differentiation, and apoptosis index. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid with 0, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM of LE (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On the basis of the results of this dose-response trial, the range of concentrations to test was reduced in experiment 2, in which presumptive zygotes were cultured with 0, 0.05 mM, and 0.1 mM of LE. On Day 7, embryo yields were assessed, and the blastocysts (BL) were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO and 0.5 M sucrose. Finally, BL produced on Day 8 in the absence (control) and presence of 0.1 mM LE were used for transferase-mediated dUTP nick end labeling and differential staining to evaluate, respectively the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm lineages (experiment 3). Despite similar blastocyst yields, supplementation of culture medium with 0.1 mM LE improved the cryotolerance of in vitro-produced (IVP) embryos compared to the control group, as indicated by higher (P < 0.05) hatching rates recorded after 48-hour post-warming culture (48.5%, 50.0%, and 63.8%, respectively with 0, 0.05, and 0.1 mM LE). Interestingly, when embryos were cultured in the presence of 0.1 mM LE, the percentage of BL with the most physiological ICM:total cells ratio (20%-40%) increased (85.1 vs. 66.0%, P < 0.05), confirming a beneficial effect on embryo quality. Furthermore, 0.1 mM LE decreased (P < 0.01) both the average number (4.3 ± 0.2 vs. 9.1 ± 0.3) and the proportion (3.6 ± 0.3 vs. 8.1 ± 0.5) of apoptotic cells in BL compared to the control. In conclusion, the enrichment of bovine culture medium with 0.1 mM LE improves embryo quality, as indicated by the improved cryotolerance, the lower apoptotic rate, and the higher percentage of BL with the most physiological ICM:total cells ratio.