V. Longobardi

Effect of energy source during culture on in vitro embryo development, resistance to cryopreservation and sex ratio

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.

The influence of gamete co-incubation length on the in vitro fertility and sex ratio of bovine bulls with different penetration speed.

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<0.01). The differences in the kinetics among bulls did not reflect their in vitro fertility. The incidence of polyspermy was higher for faster penetrating bulls (36%, 24%, 16% and 4%, respectively for bulls A, B, C and D; p<0.01) and at longer co-incubation times (0%, 16%, 19%, 30% and 34%, respectively at 4, 8, 12, 16 and 20 h p.i.; p<0.01). The fertilizing ability of individual bulls may be improved by adapting the co-incubation length to their penetration speed. A sperm-oocyte co-incubation length of 8 h ensured the greatest blastocyst yields for the two faster penetrating bulls. On the contrary, 16 h co-incubation was required to increase (p<0.01) cleavage rate of the two slower bulls. Bulls with a faster kinetics did not alter the embryo sex ratio towards males. The female/male (F/M) ratios recorded were 2.1, 1.4, 1.2, 1.3 and 1.6, respectively at 4, 8, 12, 16 and 20 h p.i.

Osteopontin improves sperm capacitation and in vitro fertilization efficiency in buffalo (Bubalus bubalis)

The aim of this study was to evaluate the effect of osteopontin (OPN), an ubiquitous acid glycoprotein, on in vitro sperm capacitation and on in vitro embryo production (IVEP) efficiency in buffalo. In experiment 1, after swim-up separation the sperm were incubated in Tyrode albumin lactate pyruvate medium in the absence of capacitating agents (control), with the standard concentration of heparin (0.01 mM) and three different concentrations of OPN (0.1, 1, and 10 mcg/mL), both in the presence and absence of heparin, for 2 and 4 hours. Capacitation was assessed indirectly by estimating the percentage of acrosome-reacted sperm after incubation with lysophosphatidylcholine. In order to determine the effect of OPN, in the presence of heparin, on fertilization (Experiment 2) and in vitro embryo development (experiment 3), in vitro-matured buffalo oocytes were fertilized in the presence of 0, 0.1, 1, and 10 mcg/mL of OPN. After IVF, the presumptive zygotes were dezonated, fixed, stained, and then evaluated microscopically. At Days 5 and 7 of culture, the cleavage and blastocyst rates were evaluated, respectively. Two hours of treatment with OPN at the two higher concentrations (1 and 10 mcg/mL) promoted in vitro capacitation of buffalo sperm (experiment 1). A synergic action of OPN with heparin was also done for all OPN concentrations tested. At 4 hours incubation, all treatments, including heparin (20.4%), improved (P < 0.01) capacitation compared with the control (16.2%). Interestingly, the best results were reported in all groups treated with OPN + heparin (40.8%, 38.6%, and 33.8%, respectively; P < 0.01). The addition of OPN to the IVF medium had a positive influence on total penetration, synchronous pronuclei formation (experiment 2), and IVEP efficiency (experiment 3). In particular, the two lower concentrations of OPN (0.1 and 1 mcg/mL), compared with the control, gave higher synchronous pronuclei formation (73.5%, 75.0%, and 46.5%, respectively; P < 0.01) and cleavage rates (70.3%, 71.6%, and 59.3%, respectively; P < 0.01). Interestingly, the treatments also improved blastocyst yields (29.3%, 30.3%, and 19.4%, respectively; P < 0.01). In conclusion, these results indicate that adding OPN to the IVF system improves IVEP efficiency by enhancing in vitro sperm capacitation and blastocyst yields in buffalo.

164 CARNITINE IMPROVES POST-THAWING SPERM MOTILITY BY INCREASING ADENOSINE TRIPHOSPHATE CONTENT IN BUFFALO (Bubalus bubalis)

Semen cryopreservation plays a critical role for a wide application of both AI and in vitro embryo production in buffalo. In this species, spermatozoa are more susceptible to hazards during freezing and thawing than cattle spermatozoa, thus resulting in lower fertilizing potential (Andrabi et al. 2008 Anim. Reprod. Sci. 104, 427-433). Carnitine is a quaternary ammonium compound with antioxidant capacities, able to reduce the availability of lipids for peroxidation by transporting fatty acids into the mitochondria for β-oxidation to generate adenosine triphosphate (ATP) energy (Tanphaichitr and Leelahagul 1993 Nutrition 9, 246-54). It is known that cryopreservation processes decreases the intracellular concentration of carnitine in spermatozoa (Reyes-Moreno et al. 2000 J. Androl. 21, 876-86). In cattle, supplementation of semen extender with carnitine improves sperm motility and DNA integrity (Bucak et al. 2010 Cryobiology 61, 248-53). The aim of this study was to evaluate whether supplementation of semen extender with carnitine would increase ATP content in buffalo sperm and affect post-thawing motility. Eight ejaculates from 4 bulls were used for the trial. Each ejaculate was split into 3 equal aliquots and diluted at 37°C with BioXcell extender containing 0 (control), 2.5, and 7.5mM carnitine to a final concentration of 30×106 spermatozoa/mL. After 4h at 4°C, the straws were frozen in an automated system. At thawing, sperm motility was evaluated by phase contrast microscopy at 40× magnification (Gillan et al. 2008 Anim. Reprod. Sci. 103, 201-204). Adenosine triphosphate content was measured using a Colourimetric ATP Assay Kit (Biovision, Milpitas, CA, USA). Briefly, Percoll-separated spermatozoa were homogenised and then deproteinized using 10-kDa spin columns. Samples were incubated at RT for 30min and absorbance was measured at 570 nM in a microplate reader. Differences in sperm motility and ATP content among groups were analysed by ANOVA. Both concentrations of carnitine increased post-thawing sperm motility compared with the control (44.4±3.5, 53.1±3.9, and 52.5±3.6, respectively, with 0, 2.5, and 7.5mM carnitine; P<0.05). Interestingly, carnitine increased ATP content of buffalo frozen-thawed sperm in a dose-dependent manner (4.1±0.1, 5.3±0.1, and 8.2±0.4 nM×108 sperm, respectively, with 0, 2.5, and 7.5mM carnitine; P<0.01). In conclusion, the enrichment of semen extender with carnitine improved post-thawing motility of buffalo sperm by boosting mitochondrial ATP production, hence providing energy for use by spermatozoa.

51 CASPASE-3 INHIBITOR Z-VAD-FMK ENHANCES CRYOTOLERANCE OF IN VITRO-PRODUCED BOVINE PRE-IMPLANTATION EMBRYOS

In vitro-produced (IVP) bovine embryos are still less viable and resistant to cryopreservation than their in vivo counterparts. Cryopreservation induces cell degeneration through the apoptotic pathway in bovine oocytes and embryos (Men et al. 2003 Cryobiology 47, 73-81). Apoptosis can be prevented by inhibition of caspase activity, leading to improved cryosurvival in mammalian cells (Stroh et al. 2002 FASEB J. 16, 1651-3). Interestingly, cryotolerance of porcine embryos was improved by inhibiting apoptosis using a caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) during vitrification and subsequent culture (Men et al. 2006 Theriogenology 66, 2008-16). Aim of this work was to evaluate whether cryotolerance of bovine IVP embryos may be improved by using Z-VAD-FMK during cryopreservation and post-warming in vitro culture. Abattoir-derived bovine oocytes (n=753, over 4 replicates) were in vitro matured and fertilized according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-55). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium at 39°C with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed and blastocysts (except the hatched blastocysts) were randomly divided in 2 groups: vitrification and post-warming culture in presence (n=60) or absence (n=54) of 20µM Z-VAD-FMK. Vitrification was carried out by Cryotop in 16.5% ethylene glycol, 16.5% DMSO, and 0.5M sucrose (Rubessa et al. 2011 Theriogenology 76, 1347-55). Blastocysts were warmed in decreasing sucrose solutions (0.25M for 1min and 0.15M for 5min) and cultured for 2 days. Resistance to cryopreservation was evaluated by assessing the survival rate, based on morphological criteria and hatching rate after 48h culture. Furthermore, TUNEL staining was used to evaluate the total cell (TC) number and the apoptotic rate of vitrified blastocysts after 48-h post-warming culture. Differences between groups in survival and hatching rates after 48-h post-warming culture were analysed by Chi-squared test, whereas differences in TC number and in number and percentage of apoptotic cells were analysed by Students t-test. Inhibition of caspase activity induced by Z-VAD-FMK increased embryo cryotolerance, as indicated by higher survival (92.6v. 55.0%; P<0.01) and hatching rates (40.7v. 23.3%; P<0.05) after 48h of post-warming culture. Furthermore, Z-VAD-FMK decreased both the average number (7.1±0.6v. 4.2±0.3; P<0.01) and the percentage (6.3±0.6v. 3.0±0.2; P<0.01) of apoptotic cells in blastocysts. No differences were recorded in TC number between groups (on average, 128.90±1.6). These results suggest that addition of 20µM Z-VAD-FMK during vitrification/warming and post-warming culture significantly inhibits apoptosis (DNA fragmentation) and improves the cryotolerance of IVP bovine embryos.

Influence of γ-glutamyltransferase and alkaline phosphatase activity on in vitro fertilisation of bovine frozen/thawed semen

Abstract The aim of this work was to evaluate whether the residual amount of γ-glutamyl-transferase (GGT) and alkaline phosphatase (ALP) in bovine sperm after freezing/thawing is correlated with fertility parameters, including blastocyst rates after in vitro fertilisation (IVF). The enzyme activities were determined in both spermatozoa and supernatant after centrifugation. While ALP was only correlated with sperm viability, GGT activity was correlated with sperm motility (rs = .4; p < .05) both in sperm and supernatant. Interestingly, GGT activity was also correlated with cleavage (rs = .5; p < .05 and .8; p < .01, for sperm and supernatant respectively) and blastocyst (rs = .6 and .9, for sperm and supernatant respectively; p < .01) rates obtained after IVF. These results suggest that GGT could play an important role in the protection of sperm against oxidative stress and could be considered a reliable marker to assess frozen/thawed sperm quality in bovine.

88 ENRICHMENT OF CULTURE MEDIUM WITH CROCETIN IMPROVES IN VITRO EMBRYO DEVELOPMENT IN CATTLE

The high incidence of developmental failure of bovine in vitro-produced embryos is due to suboptimal culture conditions that induce oxidative stress. Indeed, increased oxidative stress is one of the main factors affecting in vitro mammalian embryo development, decreasing the viability of IVP embryos. It is known that saffron has a powerful antioxidant capacity, mainly due to its active components crocin and crocetin. The aim of this study was to evaluate whether enriching the in vitro culture medium with crocetin improves in vitro embryo production efficiency in cattle. The range of concentrations of crocetin was chosen after a preliminary dose response trial (322 total presumptive zygotes were cultured with 0, 1, 10, and 50 μM, over 2 replicates) that showed beneficial and deleterious effects, respectively, with the lowest and highest concentration compared with the control (36.6 ± 5.6, 57.4 ± 4.5, 46.4 ± 4.4, and 6.8 ± 3.7% blastocyst rates, respectively, with 0, 1, 10, and 50 μM; P < 0.01). Therefore, the range of concentrations to test was reduced. Abattoir-derived bovine oocytes (n = 832, over 4 replicates) were in vitro matured and fertilized according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium with 0 (control; n = 208), 1 μM (n = 208), 2.5 μM (n = 208), and 5 μM (n = 208), at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2. The embryos obtained by the end of culture (i.e. on Day 7 post-IVF) were scored for quality, based on morphological criteria, and for developmental stage, as previously described (Robertson and Nelson 2010, Manual of the IETS, 86–105). The percentages of total transferable embryos and grade 1 and 2 blastocysts were recorded. As the chronology of development is a reliable parameter to assess quality, the percentage of fast-developing embryos (i.e. hatched and expanded blastocysts) was also compared among groups. Differences among groups were analysed by ANOVA, and Tukey method was used as a post-hoc test. Data are presented as means ± s.d. The supplementation of crocetin during culture did not affect cleavage rate (74.9 ± 6.3, 76.4 ± 8.4, 81.4 ± 4.3, and 76.4 ± 8.4%, respectively, with 0, 1, 2.5, and 5 μM). However, post-fertilization embryo development improved with 1 µM crocetin compared with the control, both in terms of total embryo output (43.8 ± 4.4, 61.1 ± 5.2, 50.4 ± 6.7, and 53.3 ± 7.3%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.01) and grade 1 and 2 blastocysts (41.0 ± 3.6, 54.3 ± 5.4, 46.2 ± 6.7, and 49.4 ± 6.5%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.05), whereas no differences were observed among the other groups. Moreover, the percentage of fast developing embryos increased with 1 µM (P < 0.05) crocetin compared with the control, with no other differences recorded among groups (17.7 ± 5.8, 34.7 ± 5.7, 24.9 ± 5.1, and 28.7 ± 7.8%, respectively, with 0, 1, 2.5, and 5 μM). In conclusion, these results demonstrated a beneficial effect of low concentrations of crocetin (1 μM) during culture both on blastocyst yield and quality, as indicated by the improved chronology of embryo development.