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ABSTRACT: In buffalo, in vitro embryo production (IVEP) technology is the best tool to improve the genetic merit through the maternal lineage. A major limitation of IVEP technology in buffalo species is the poor cryotolerance of the embryos, likely due to their high lipid content (Gasparrini 2002 Theriogenology 57, 237-256). It was previously demonstrated that supplementing bovine culture media with L-carnitine, a cofactor of β-oxidation, improves in vitro embryo development (Sutton-McDowall et al. 2012 Theriogenology 77, 1632-1641). The aim of this work was to evaluate whether L-carnitine supplementation during in vitro culture (IVC) improves blastocyst development and cryotolerance of in vitro produced buffalo embryos. After a preliminary dose response trial, we selected the concentration of 0.25mM for the experiment. Cumulus-oocytes complexes (n=288, over 4 replicates), recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures (Gasparrini et al. 2006 Theriogenology 65, 275-287). On Day 1 (Day 0=IVF), zygotes were cultured in SOF supplemented with 8mgmL(-1) BSA, in the absence (control, n=143) or presence of 0.25mM L-carnitine (n=145). In vitro culture was carried out at 38.5°C under 5% CO(2), 7% O(2), and 88% N(2). Cleavage rate was evaluated on Day 5, when the cleaved embryos were transferred into fresh medium for further 2 days. On Day 7 after IVF, embryo outcome was assessed and all the embryos were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide (DMSO), and 0.5M sucrose (De Rosa et al. 2007 Ital. J. Anim. Sci. 6(Suppl 2), 747-750). The resistance to cryopreservation was evaluated by assessing the survival rate, on the basis of morphological criteria, after 24h culture. Data were analyzed by chi-square test. No differences were found in cleavage rates between the control (81.5%) and the L-carnitine group (78.8%). The blastocyst yields (calculated in relation to the cleaved embryos) were not significantly influenced by the L-carnitine treatment (40.2 and 52.9%, in the control and the L-carnitine groups, respectively). However, buffalo embryos cultured in the presence of L-carnitine showed an increased resistance to cryopreservation, as indicated by the higher survival rates recorded after 24h culture (78.7 and 96.4%, in the control and the L-carnitine groups, respectively; P<0.01). In conclusion, these results demonstrated that L-carnitine supplementation of culture medium improves the resistance to cryopreservation of in vitro produced buffalo embryos. We speculate that the increased cryotolerance observed in the presence of L-carnitine may be due to a better utilization of the endogenous lipid stores, resulting in improved embryo quality.
Reproduction Fertility and Development 12/2012; 25(1):214. · 2.11 Impact Factor
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ABSTRACT: Despite the great improvement of in vitro embryo production (IVEP) efficiency recorded over the years in cattle, the in vitro produced (IVP) embryos are still less viable and resistant to cryopreservation than their in vivo counterparts. One of the major factor impairing in vitro embryo development is oxidative stress. Resveratrol is an important antioxidant polyphenolic compound found in several vegetal sources, that contributes to red wine's beneficial effects on the prevention of human cardiovascular disease. Recently, the interest in resveratrol has increased exponentially following the major findings that this molecule has positive effects on cancer chemoprevention, cardioprotection, inflammatory processes, several aspects of metabolism, leading to increased lifespan of various organisms from yeasts to vertebrates (Pirola et al. 2008IUBMB Life 60, 323-332). A positive effect of resveratrol on in vitro embryonic development was demonstrated in swine (Lee et al. 2010 J. Reprod. Dev. 56, 330-335). The aim of this study was to evaluate whether supplementation of culture medium with resveratrol improves in vitro blastocyst development and the embryo resistance to cryopreservation in cattle. A preliminary dose response trial indicated that the optimal concentration in the range tested (from 0.5 to 10µM) was 0.5µM, with evident toxic effects at concentration higher than 5µM. Abattoir-derived oocytes (n=581, over 5 replicates) were matured and fertilized in vitro according to our standard procedure (Rubessa et al. 2011 Theriogenology 76, 1347-1355). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium, supplemented with 5% bovine serum, in the absence (control, n=271) or presence of 0.5µM resveratrol (n=310) at 39°C under humidified air with 5% CO(2), 7% O(2), and 88% N(2). On Day 7 (IVF=Day 0), embryo yields were assessed and the blastocysts (except the hatched blastocysts) were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide (DMSO), and 0.5M sucrose (Rubessa et al. 2011). The resistance to cryopreservation was evaluated by assessing the survival rate, on the basis of morphological criteria, and hatching rate after 48h culture. Data were analyzed by chi-square test. Resveratrol supplementation during culture did not affect either cleavage (69.1 v. 72.0%, in the control and resveratrol groups, respectively) or blastocyst yields (38.3 v. 36.3%, in the control and resveratrol groups, respectively). However, treatment with resveratrol increased the cryotolerance of IVP embryos, as indicated by higher survival rates (74.7 v. 88.4%, in the control and resveratrol groups, respectively; P<0.05) and hatching rates (35.1 v. 53.8%, in the control and resveratrol groups, respectively; P=0.06) at 48h. In conclusion, these results demonstrated that resveratrol supplementation during culture improves the quality, and hence the resistance to cryopreservation, of IVP bovine embryos.
Reproduction Fertility and Development 12/2012; 25(1):213-4. · 2.11 Impact Factor
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ABSTRACT: Most systems for producing bovine embryos in vitro use glucose as an energy source despite putative toxic effects. Glucose has a selective embryotoxicity towards female embryos, due to the higher expression of the X-linked glucose-6-phosphate dehydrogenase gene (Kimura et al. 2005 Mol. Reprod. Dev. 72, 201-207). Recently, the replacement of glucose with citrate and myo-inositol in SOF medium supplemented with 5% bovine serum (BS) increased the percentage of female embryos (Rubessa et al. 2011 Theriogenology 76, 1347-1355). Serum also affects the sex ratio of in vitro-produced (IVP) bovine embryos, favoring the male gender (Gutierrez-Adan et al. 2001 Theriogenology 55, 1117-1126). The aim of this work was to evaluate the effect of glucose replacement with myo-inositol during in vitro culture, in the presence of either BS or BSA, on bovine embryo sex ratio. Abattoir-derived oocytes (n=1164, over 4 replicates) were matured and fertilized in vitro as previously described (Rubessa et al. 2011). After 20 to 22h of gametes co-incubation, zygotes were denuded and cultured for 7 days in SOF with: group A) 0.34mM trisodium citrate+2.77mM myo-inositol+5% BS (n=287); group B) 0.34mM tri-sodium citrate+2.77mM myo-inositol+8mgmL(-1) BSA(n=290); group C) 1.5mM glucose+5% BS (n=302) and group D) 1.5mM glucose+8mgmL(-1) BSA (n=285). Representative samples of blastocysts produced in each group (n=96, 58, 99, and 70, respectively in groups A, B, C, and D) were sexed by PCR as previously described (Rubessa et al. 2011). Differences among groups in blastocyst yields were analyzed by ANOVA. The percentages of female embryos were analyzed by chi-square test. Blastocyst rates in group C were lower (28.1%) than those recorded in groups A, B, and D (35.9, 41.0 and 36.1%, respectively; P<0.01). A higher (P<0.05) percentage of female embryos was observed in group A (61.5%) compared to group C (45.5%), with intermediate values in groups B (51.7%) and D (60.0%). Therefore, the replacement of glucose with citrate and myo-inositol favored the development of female embryos in the presence of BS but was ineffective in the presence of BSA. Furthermore, when glucose was the energy source, a tendency to greater incidence of female embryos was observed when the medium was supplemented with BSA rather than BS (P=0.06). As a small amount of glucose is present in the BS, we hypothesize an additional glucose-dependent toxic effect on female embryos in group C. However, we cannot rule out that other factors present in the BS may interact with the energy source, playing a role in determining the sex ratio. Furthermore, the shift in sex ratio in favor of males or females embryo can be due to a better development of embryo of one sex, or to the delayed development or degeneration of embryos of the other sex. In conclusion, these results suggest that manipulating the metabolic profile of the embryos during culture may have an impact on both blastocyst production and sex ratio.
Reproduction Fertility and Development 12/2012; 25(1):211. · 2.11 Impact Factor
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ABSTRACT: 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.
Reproduction in Domestic Animals 04/2011; 46(6):1090-7. · 1.36 Impact Factor
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ABSTRACT: The current knowledge on metabolism and glucose utilisation of preimplantation bovine and ovine embryos suggest the reduction of glucose concentration during early culture. On the contrary, it has been demonstrated that glucose is absolutely required for in vitro culture of buffalo embryos, as indicated by the poor efficiency recorded in the absence of this substrate during early embryonic development (Monaco et al. 2006 Reprod. Domest. Anim. 41, 332). However, complete removal of glucose from culture medium throughout pre-elongation development is unlikely to benefit the embryo because glucose plays other roles including ribose and NADPH production through the pentose-phosphate pathway. Therefore, the aim of this study was to investigate the effect of reducing glucose concentration up to 0.15mM (1/10 compared to the standard concentration in SOF) on embryo development in buffalo. In order to evaluate the role of this substrate during development, glucose was reduced at different stages of embryo culture. Cumulus-oocyte complexes (n=573, over 4 replicates), recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures (Gasparrini et al. 2006, Theriogenology, 65, 275-287). On day 1 (Day 0=IVF), zygotes were cultured in SOF with group A) 1.5mM glucose (standard concentration in SOF) throughout culture (control); group B) 1.5mM glucose for early culture (Day 1 to Day 4) and 0.15mM glucose for late culture (Day 4 to Day 7); group C) 0.15mM glucose throughout culture; and group D) 0.15mM glucose for early culture and 1.5mM glucose for subsequent culture. In vitro culture was carried out at 38.5°C under 5% CO(2), 7% O(2), and 88% N(2). Cleavage rate was evaluated on Day 4, and blastocyst yield, in relation to cleaved embryos, was recorded on Day 7. Differences among groups in blastocyst rate were analysed by chi-square test. The reduction of glucose concentration did not affect cleavage rate (73.7 v. 65.1%, respectively, for Groups A-B and C-D). Nevertheless, blastocyst rates significantly decreased when glucose was reduced throughout culture (Group C: 10.1%; P<0.01) and to a limited degree during early culture (Group D: 17.2%; P<0.05) compared with the control (Group A: 38.3%). On the contrary, a decreased glucose concentration during late culture did not reduce embryo development (Group B: 35.18%). This finding indicates that energy requirements of buffalo embryos during IVC are different from those of sheep and cattle, which show a significant rise in glucose uptake just around compaction, i.e. during late culture (Thompson et al. 1991 Reprod. Fertil. Dev. 3, 571-576; Thompson et al. 1996 J. Reprod. Fertil. 106, 299-306). In conclusion, in buffalo, unlike sheep and cattle, glucose is more critical for early embryo development than for post-compaction development, suggesting the importance of developing other strategies for optimizing in vitro embryo production efficiency.
Reproduction Fertility and Development 01/2011; 23(1):168. · 2.11 Impact Factor
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ABSTRACT: Most systems for producing mammalian embryos in vitro use glucose as an energy source in the media despite putative toxic effects (Schini and Bavister 1988 Biol. Reprod. 39, 1183-1192; Takahashi and First 1992 Theriogenology 37, 963-978). Currently there is a tendency to identify other suitable energy sources in an attempt to replace glucose from culture media. Glyceraldehyde-3-phosphate (G3P), a glucose-derived high-energy compound, is the end product of the energy-consuming phase of glycolysis that enters the pay-off phase of the pathway characterised by a net gain of energy. The aim of this study was to determine whether G3P is a valid energy source for supporting in vitro embryo development in cattle. Abattoir-derived oocytes (n=832, over 4 replicates) were matured in vitro in TCM-199 with 15% bovine serum (BS), 0.5μgmL(-1) FSH, 5μgmL(-1) LH, 0.8mML-glutamine, and 50mgmL(-1) gentamicin. Mature COC were fertilized in Tyrode's modified medium, with 30mgmL(-1) heparin, 30mM penicillamine, 15mM hypotaurine, 0.15mM epinephrine, and 1% BS. Both IVM and IVF were carried out at 39°C and 5% CO(2) in air. After 20 to 22h of gamete co-incubation, presumptive zygotes were denuded and cultured in SOF containing either 1.5mM glucose (control group) or G3P at 3 different concentrations (0.125, 0.5, and 1.5mM). It is worth specifying that in the 3 G3P-supplemented groups small amounts of glucose were left (0.15mM) because it is known that a complete removal would affect embryo development by interfering with ribose synthesis through the pentose-phosphate pathway. In vitro culture was carried out at 39°C under humidified air with 5% CO(2), 7% O(2), and 88% N(2) in air for 7 days, when the percentages of tight morulae-blastocysts (TMBL) and superior quality blastocysts (BL) were recorded. Differences in embryo yields among groups were analysed by chi-square test. Supplementation of IVC medium with 1.5mM G3P reduced (P<0.01) TMBL (5.0%) and BL (5.0%) rates compared with all other groups, indicating a toxic effect. However, when G3P was added at lower concentrations, no differences in TMBL (37.3 and 26.1, respectively, with 0.125 and 0.5mM G3P) and in BL rates (35.3 and 25.5%, respectively, with 0.125 and 0.5mM G3P) were observed compared with the control (32.7% TMBL and 31.4% BL, respectively). Within G3P-treated groups, the higher embryo yields were recorded with 0.125mM compared with 0.5mM (P<0.05) and 1.5mM (P<0.01). Interestingly, embryos produced with G3P at the lower concentrations (0.125 and 0.5mM) seemed to show a faster development compared with the control. In conclusion, these results demonstrated that G3P is a valid energy source for bovine preimplantation embryos and, hence, that G3P supplementation of IVC medium may be a suitable option for reducing glucose concentration in the media. However, further studies are needed to investigate lower concentrations of G3P and to better evaluate embryo viability.
Reproduction Fertility and Development 01/2011; 23(1):167. · 2.11 Impact Factor
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ABSTRACT: Removal of cumulus cells is necessary for several technologies such as vitrification, intracytoplasmic sperm injection, and nuclear transfer. However, it is known that the presence of cumulus cells during IVF of buffalo oocytes is fundamental for fertilization and embryo development (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335-342; Nandi et al. 1998 Theriogenology 50, 1251-1262). The aim of this work was to evaluate whether co-culture with intact bovine cumulus-oocyte complexes (COC) during IVF would restore the developmental competence of denuded buffalo oocytes. Due to the scarce availability of buffalo ovaries, the somatic support was provided by bovine cumulus cells. Abattoir-derived COC were matured in vitro according to our standard procedures (Gasparrini et al. 2006, Theriogenology, 65, 275-287) and randomly distributed in 3 fertilization groups: 1) a control group of COC (n=122), 2) a negative control of denuded oocytes (DO; n=119), and 3) DO co-cultured with in vitro matured bovine COC (DO+COC; n=103) in a 1:1 ratio (3 bovine COC+3 denuded buffalo oocytes/50μL drop). Fertilization was carried out with frozen-thawed spermatozoa from a tested bull in TALP medium supplemented by 0.2mM penicillamine, 0.1mM hypotaurine, and 0.01mM heparin at 38.5°C under a controlled gas atmosphere of 5% CO(2) in humidified air. After fertilization the zygotes were cultured in SOF medium including essential and nonessential amino acids and 8mgmL(-1) BSA, at 38.5°C under humidified 5% CO(2), 7% O(2), and 88% N(2), up to the blastocyst stage. On Day 5 and on Day 7 (Day 0=IVF) cleavage and blastocyst rates were respectively recorded. Data were analysed by chi-square test. As expected, cleavage and blastocyst rates were lower (P<0.01) in DO (36.1 and 9.2%, respectively) compared with the control (67.2 and 27.1%, respectively). However, co-culture during IVF (DO+COC) significantly increased (P<0.01) both parameters compared with DO, giving cleavage (70.9%) and blastocyst (27.2%) rates similar to the control. The results of this study demonstrated that co-culture with bovine intact COC during IVF of buffalo denuded oocytes completely restores their fertilizing capability and blastocyst developmental competence. We conclude that this may be a suitable strategy for preserving the developmental competence of oocytes devolved to technologies, such as oocyte vitrification, that require cumulus removal.
Reproduction Fertility and Development 01/2011; 23(1):167. · 2.11 Impact Factor
