Induction of oxidative stress does not increase the cryotolerance of vitrified embryos.

Short-term treatment of mammalian oocytes with different stressors induces stress tolerance of embryos derived from these oocytes. The aims of this study were to evaluate effects on embryo development when there was treatment of oocyte complexes (COCs) used to derive the embryos with hydrogen peroxide (H2O2).The COCs were not incubated with H2O2: control (0 µM), or were incubated with 25, 50, 75, or 100 µM concentrations of H2O2 for 1 h prior to in vitro fertilization, and presumptive zygotes were cultured until day 7. Blastocysts at day 7 of development derived from H2O2-treated (25 µM treatment concentration) COCs were vitrified. Percentage of embryos undergoing cleavage was not affected by any treatment, while percentage of embryos developing to the blastocyst stage was less when there was treatment of COCs with 100 µM of H2O2. Embryo quality was less when COCs used to derive blastocysts were treated with 50, 75, or 100 µM concentrations of H2O2. There were lesser relative abundances of some mRNA transcripts of interest in blastocysts when there was treatment of COCs with H2O2. After vitrification, there were no differences in embryo re-expansion and hatching rates compared with fresh and vitrified blastocysts of the control group and those derived from COCs treated with 25 µM H2O2. In conclusion, treatment of COCs used to derive blastocysts with H2O2 does not induce stress tolerance in vitrified embryos of cattle; however, the viability of these blastocysts is similar to those of the control group.

Gradient sperm selection for reproductive techniques in cattle: Is Isolate a suitable replacement for Percoll?

In assisted reproductive techniques, it is essential to perform a sperm selection to obtain spermatozoa with high motility and membrane integrity for in vitro fertilisation (IVF) and high-DNA integrity for intracytoplasmic sperm injection (ICSI). In this study, we evaluated whether Isolate® was a suitable substitute for Percoll® for assisted reproductive techniques. Commercial cryopreserved bovine semen was used after selection in both gradients, and plasma and acrosome membrane integrity, reactive oxygen species (ROS) levels, DNA integrity and mitochondrial membrane potential (ΔΨm) were assessed by flow cytometry. Motility parameters were also evaluated by CASA system. A similar percentage of spermatozoa with intact plasma membrane, acrosome integrity and high ΔΨm was observed in both sperm selection methods, but only Percoll® showed higher percentage of spermatozoa with intact plasma and acrosome membrane compared to the post-thawing group. No differences were observed in the motility, ROS, DNA fragmentation and on the in vitro embryo production in all experimental groups. In conclusion, the selection of bovine spermatozoa with Isolate® generates spermatozoa with similar quality parameters and embryonic development compared to Percoll® providing a suitable alternative sperm selection method for assisted reproductive techniques in this species.

Effect of sperm pretreatment with glutathione and membrane destabilizing agents lysolecithin and Triton X-100, on the efficiency of bovine intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an assisted reproduction tool with several applications. Its effectiveness in bovines is lower than that in other species, mainly because of difficulties in the decondensation of the sperm nucleus after injection, and the presence of the acrosome and the plasma membrane which remain intact in this procedure. In this study, we assessed the effect of lysolecithin (LL) and Triton X-100 (TX), in combination with glutathione (GSH) as sperm pretreatments prior to ICSI. The GSH-LL and GSH-TX groups showed 0% of spermatozoa with intact membrane (SYBR 14+/PI), in comparison with the control (63.3%) and GSH (65.7%) groups. The proportions of spermatozoa with damaged acrosome membrane in the GSH-LL, GSH-TX, GSH and control groups were 46%, 35.9%, 10.5% and 7.5%, respectively. Sperm chromatin decondensation analysis showed that the groups incubated for 3 hr with GSH presented greater decondensation (p < .05). Although fertilization was improved in all treatment groups evaluated, no differences were observed in the cleavage rate 72 hr after activation in the GSH (73.7%), GSH-LL (80.2%) and GSH-TX (77.8%) groups compared to the control (66.3%), neither in the blastocyst rate on day 8 (24.0%, 26.2%, 27.1% and 28.4% for the control, GSH, GSH-LL and GSH-TX groups, respectively). No differences were also observed in the total number of cells in all groups. In conclusion, although these sperm treatments promoted nuclear decondensation and induced plasma membrane disruption, these effects were not sufficient to improve bovine embryonic development after ICSI.

Decrease in bovine in vitro embryo production efficiency during winter season in a warm-summer Mediterranean climate.

Retrospective analysis of monthly embryo production from December 2011 to May 2015 and its correlation with meteorological data in our geographic zone was made. We had observed that in certain time of the year, in vitro blastocyst production decreases. Accordingly, was examined the association between blastocyst production and climatological parameters. Cleavage rates correlate positively with blastocyst rates (p < .05). Significant differences in cleavage rates between autumn and summer (79.8%; 71.5%), and between winter and autumn (71.8%; 79.8%), were found. Blastocyst production had lower efficiency in June (9 ± 12%) and July (4.9 ± 5.7%), which coincides with winter season. In contrast, higher embryo production was obtained in February (22.2 ± 9.7%), March (22.9 ± 14%) and September (25.2 ± 6.6%), which coincides with autumn and spring season. Similarly, embryo production correlates with meteorological parameters: blastocyst production positively correlates with sunshine hours, maximum temperature and average temperature. Similarly, blastocyst production inversely correlates with total precipitation and days >1 mm precipitation (p < .05). There is a significant decrease in bovine in vitro embryo production efficiency during winter season in our warm-summer Mediterranean climate zone. It remains to be investigated the direct effect of environmental factors on oocyte quality and its impact on in vitro production efficiency.

Effect of short-term exposure of cumulus-oocyte complex to 3-morpholinosydnonimine on in vitro embryo development and gene expression in cattle.

Short-term exposure of gametes to different types of stress might induce stress tolerance in mammalian embryos. The aim of this study was to evaluate the effect of short-term exposure of bovine mature cumulus-oocyte complex (COC) to 3-morpholinosydnonimine (SIN-1) on subsequent in vitro embryo development, embryo quality and relative gene expression. Matured COCs were incubated with SIN-1 (0, 0.1, 1, 10 and 100 µM SIN-1) for 1 hr before in vitro fertilization and zygotes were cultured until Day 7. The cleavage rate at 72 hr did not show any differences among groups. However, the blastocyst rate on Day 7 decreased with all treatments evaluated, with the embryos generated with 10 µM SIN-1 showing the lowest embryo production rate. Embryo quality analysis did not show any differences in total cell number (TCN) or inner cell mass (ICM) among groups. Relative gene expression analysis showed a downregulation of eNOS expression and an upregulation of nNOS expression in all treatments evaluated compared to the control group. Also, a downregulation was observed in some treatments: SOD2 at 0.1 µM; SOD1 at 0.1 and 100 µM; PRDX5 at 0.1, 10 and 100 µM; and NANOG at 10 and 100 µM; and an upregulation of CDX2 expression was observed at 100 µM. The other genes (OCT4, HIF1A, HSPA1A, BCL2A and iNOS) did not show any differences in the relative gene expression. These results suggest that the short-term exposure of mature bovine COCs to SIN-1 does not induce stress tolerance and has no beneficial effect on bovine in vitro embryo production.

Methyl-ß-Cyclodextrin Improves Sperm Capacitation Status Assessed by Flow Cytometry Analysis and Zona Pellucida-Binding Ability of Frozen/Thawed Bovine Spermatozoa.

Mammalian sperm undergo a series of biochemical transformations in the female reproductive tract that are collectively known as capacitation. Cyclodextrins added to the sperm culture medium have been described to induce in vitro sperm capacitation, enabling its use in protein-free media. However, the additive capacitating effect of methyl-ß-cyclodextrin (MßCD) in the medium containing bovine serum albumin (BSA) is unknown in the bovine species. In this study, we evaluated the effects of incubating frozen-thawed bovine spermatozoa in a BSA-containing medium supplemented with MßCD on different sperm quality and functional parameters. Sperm viability decreased with the addition of MßCD in a dose-dependent manner (p < 0.05), and DNA damage could be observed but only with the highest concentration of MßCD. However, pre-incubation of spermatozoa in MßCD-supplemented medium improved the capacitation status as assessed by the increase in plasma membrane fluidity, intracellular calcium concentration, induced acrosome reactivity and zona pellucida (ZP)-binding ability (p < 0.05). Thus, we conclude that MßCD supplementation is able to enhance the capacitation status of frozen-thawed bovine spermatozoa cultured in capacitation medium containing BSA and could result in a valid strategy for its application on artificial reproductive technologies such as in vitro fertilization or intracytoplasmic sperm injection.

Effects of short-term exposure of mature oocytes to sodium nitroprusside on in vitro embryo production and gene expression in bovine.

Stress tolerance can be induced in embryos by oocyte exposure to hydrostatic pressure, osmotic agents, heat shock, or reactive oxygen species. This study assessed the effects of exposing bovine oocytes to a nitric oxide (NO) donor, sodium nitroprusside (SNP), on subsequent in vitro embryo production, embryo quality and the expression of genes involved in NO production (iNOS, eNOS, and nNOS), stress tolerance (HSP70 and HSP90), oxidative stress (HIF1A and PRDX5), and apoptosis (BCL2A1). In vitro mature oocytes were incubated with SNP (control, 10(-6) M, 10(-5) M, and 10(-4) M) for 1 hour before in vitro fertilization, and cultured until Day 7. Cleavage and blastocyst rates were recorded. Next, embryo quality (ratio of inner cell mass to total cell number) and relative gene expression of iNOS, eNOS, nNOS, HSP70, HSP90, HIF1A, PRDX5, and BCL2A1 were determined in expanded blastocysts. Cleavage rates were significantly lower for 10(-4) M SNP compared with the control and 10(-5) M SNP treatments (77 ± 7.1%, 82 ± 8.4%, and 84.9 ± 4.1%, respectively). Total blastocyst rates were lower in the 10(-4) M SNP group relative to the control group (26.2 ± 4.9% and 34.1 ± 7.8%, respectively). Embryo quality was similar among the groups. However, our relative gene expression analysis revealed the downregulation of endothelial oxide nitric synthase messenger RNA in expanded blastocysts in all the treatment groups compared with the control treatment. These results suggest that the short-term exposure of mature bovine oocytes to a NO donor does not induce their stress tolerance and has no beneficial effect on the in vitro embryo production of bovine embryos.

Activation treatment of recipient oocytes affects the subsequent development and ploidy of bovine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos.

We evaluated the potential effect of anisomycin, an antibiotic produced by Streptomyces griseolus, on the parthenogenetic activation of bovine oocytes and reconstructed somatic-cell nuclear transfer (SCNT) embryos. A higher cleavage and blastocyst rate were achieved with anisomycin (70.3% and 27.8%) and 6-dimethylaminopurine (DMAP) (73.3% and 30.2%), relative to oocytes parthenogenetically activated with cycloheximide (CHX) (54.1% and 20.2%). In reconstructed SCNT embryos, a greater proportion of embryos reached the blastocyst stage after anisomycin (32.2%) compared to DMAP (22.3%) and CHX (23.5%) treatment. Furthermore, the quality of embryos-assessed by the total number of cells and the inner cell mass-to-total-cell ratio-was higher with anisomycin (166.2 ± 6.9 and 26.9 ± 1.9) compared to DMAP (135.0 ± 8.7 and 39.4 ± 3.5) and CHX (149.1 ± 8.4 and 36.3 ± 2.5), while a lower percentage of chromosomal abnormalities was observed with anisomycin compared to DMAP and CHX treatments, both in parthenotes (though not significant) and in SCNT embryos (P < 0.05). Therefore, anisomycin can enhance the in vitro developmental potential in parthenotes and reconstructed SCNT embryos, specifically improving the quality of SCNT embryos and decreasing the abnormal ploidy of parthenotes and SCNT embryos compared to the traditional protocols of chemical activation with DMAP and CHX. These results may have important implications for the success of reproductive technologies, including SCNT and intracytoplasmic sperm injection, in different mammalian species.

N-acetyl-L-cysteine pre-treatment protects cryopreserved bovine spermatozoa from reactive oxygen species without compromising the in vitro developmental potential of intracytoplasmic sperm injection embryos.

Excess of reactive oxygen species (ROS) on in vitro embryo production systems negatively affects the quality and developmental potential of embryos, as result of a decreased sperm quality and increased DNA fragmentation. This issue is of major importance in assisted fertilisation procedures such as intracytoplasmic sperm injection (ICSI), because this technique does not allow the natural selection of competent spermatozoa, and therefore, DNA-damaged spermatozoa might be used to fertilise an egg. The aim of this study was to investigate a new strategy to prevent the potential deleterious effect of ROS on cryopreserved bovine spermatozoa. We evaluated the effect of a sperm pre-treatment with different concentrations of N-acetyl-L-cysteine (NAC) on ROS production, viability and DNA fragmentation and assessed the effect of this treatment on the in vitro developmental potential and quality of embryos generated by ICSI. The results show a strong scavenging effect of 1 and 10 mm NAC after exposure of spermatozoa to a ROS inducer, without compromising the viability and DNA integrity. Importantly, in vitro developmental potential and quality of embryos generated by ICSI with spermatozoa treated with NAC were not affected, confirming the feasibility of using this treatment before an ICSI cycle.

Supplementation of IVF medium with melatonin: effect on sperm functionality and in vitro produced bovine embryos.

Gamete co-incubation generates high free radical levels surrounding growing zygotes which may impair subsequent embryo viability. Melatonin eliminates a wide variety of free radicals; hence, we tried to improve in vitro embryo production by adding melatonin to in vitro fertilisation (IVF) media in high (Exp. 1) and low concentrations (Exp. 2), and we evaluated its effect on bull sperm function during IVF co-incubation time (Exp. 3). In Experiment 1, we supplemented IVF media culture with 0.01, 0.1 and 1 mmol of melatonin, along with a no melatonin control group. In Experiment 2, melatonin levels were reduced to 10, 100 and 1000 nmol, with a no melatonin control group. In Experiment 3, spermatozoa were incubated in IVF media with melatonin (as Exp. 2) and functional parameters were analysed at 0, 4 and 18 h. In Experiment 1, only 1 mmol melatonin showed lesser blastocyst rates than control (C: 23.2 ± 6.7% versus 1 mmol: 2.0 ± 1.7%). In Experiment 2, no statistical differences were found in cleavage percentage, blastocyst percentage and total cell count for any melatonin treatment. In Experiment 3, sperm samples with 1000 nmol melatonin had a significantly higher wobbler (WOB) coefficient, a lower percentage of intact acrosomes, a lower percentage of viable spermatozoa with ROS, greater DNA fragmentation and higher DNA oxidation than controls. Total fluorescence intensity for ROS at 10 nmol melatonin was significantly greater than controls (P < 0.05). IVF media with 1 mmol melatonin is deleterious for embryo development, and in lower concentrations, it modulated sperm functionality, but had no effects on embryo production.

Effect of mitochondrial calcium uniporter blocking on human spermatozoa.

Calcium (Ca(2+) ) regulates a number of essential processes in spermatozoa. Ca(2+) is taken up by mitochondria via the mitochondrial calcium uniporter (mCU). Oxygen-bridged dinuclear ruthenium amine complex (Ru360) has been used to study mCU because it is a potent and specific inhibitor of this channel. In bovine spermatozoa, it has been demonstrated that mitochondrial calcium uptake inhibition adversely affects the capacitation process. It has been demonstrated in human spermatozoa that mCU blocking, through Ru360, prevents apoptosis; however, the contribution of the mCU to normal human sperm function has not been studied. Therefore, the aim of this study was to evaluate the effect of mCU blocking on human sperm function. Spermatozoa obtained from apparently healthy donors were incubated with 5 and 10 µm Ru360 for 4 h at 37 °C. Viability was assessed using propidium iodide staining; motility was determined by computer-aided sperm analysis, adenosine triphosphate (ATP) levels using a luminescence-based method, mitochondrial membrane potential (ΔΨm) using JC-1 staining and reactive oxygen species (ROS) production using dihydroethidium dye. Our results show that mCU blocking significantly reduced total sperm motility and ATP levels without affecting sperm viability, ΔΨm and ROS production. In conclusion, mCU contributes to the maintenance of sperm motility and ATP levels in human spermatozoa.

Effect of sperm pretreatment with sodium hydroxide and dithiothreitol on the efficiency of bovine intracytoplasmic sperm injection.

The efficiency of intracytoplasmic sperm injection (ICSI) in bovines is lower than in other species due, in part, to a lack of optimal conditions for its implementation; this has hindered the achievement of high rates of embryonic development and the birth of live offspring. The aim of the present study was to evaluate the effects of pretreatment of bovine spermatozoa with NaOH and dithiothreitol (DTT) on the viability, plasma membrane integrity, DNA fragmentation and in vitro developmental potential of embryos generated by ICSI. Following pretreatment of spermatozoa with 5 mM DTT for 20 min and a low concentration of NaOH (1 mM for 60 min), there were fewer live and acrosome reacted spermatozoa (44% and 34%, respectively) than in the control group without treatment (82%). Spermatozoa subjected to higher alkali concentrations (10-50 mM) were mostly dead and reacted. However, pronuclear formation, cleavage, blastocyst rate and embryo quality did not differ between these pretreatment groups and the untreated control group. In conclusion, we have described, for the first time, the effects of NaOH treatment on bovine spermatozoa and subsequent in vitro embryonic development after ICSI, and have demonstrated that pretreatment of bovine spermatozoa with NaOH or DTT is not necessary for an appropriate in vitro embryo development in this species.

Effect of different sequential and two-step culture systems on the development, quality, and RNA expression profile of bovine blastocysts produced in vitro.

In the present study, we examined the effect of two-step and sequential culture systems on the development, quality, and gene expression profile of bovine embryos generated by in vitro fertilization. Presumptive zygotes were randomly allocated to four culture treatments: (1) KSOM + 0.4% BSA for 3 days, and then KSOM + 5% FBS to day 7 (K-K/FBS); (2) KSOM + 0.1% BSA for 3 days, and then SOF + 5% FBS to day 7 (K-S/FBS); (3) KSOM + 0.1% BSA for 3 days, and then SOF + 0.8% BSA to day 7 (K-S/BSA); and (4) KSOM + 0.4% BSA for 3 days, and then KSOM + 0.8% BSA to day 7 (K-K/BSA). Culture medium had no effect on cleavage rate. However, a significant difference (P < 0.01) was observed with the two-step culture systems, yielding higher rate of blastocysts (37 and 32% for K-K/FBS and K-K/BSA, respectively) compared to sequential culture systems (26 and 28% for K-S/FBS and K-S/BSA, respectively). Embryos cultured in sequential K-S/FBS developed slowly, had a lower hatching rate, fewer cells, and a higher apoptosis rate compared to other treatments. Gene expression analysis showed alterations of DNMT1, OCT-4, and SOD2 in embryos cultured in sequential K-S/FBS and SOD1 in embryos cultured in two-step K-K/BSA. In conclusion, in vitro culture systems may have an impact not just in the developmental potential and quality of the generated embryos but also in the gene expression profile, which suggests that changes in the culture medium composition can modulate global gene expression.

Molecular analysis of a 444 bp fragment of the bovine leukaemia virus gp51 env gene reveals a high frequency of non-silent point mutations and suggests the presence of two subgroups of BLV in Chile.

With the aim of achieve a better understanding of the epidemiology and distribution of bovine leukaemia virus (BLV) infection in Chile, we assessed the suitability of using DNA isolated from the leukocyte fraction of bulk milk samples to carry out PCR-RFLP and DNA sequence analysis. The env fragment of BLV was successfully amplified from 33 serologically positive bulk milk samples collected from different geographical areas in the south of Chile. Restriction analysis allowed to classify 17 isolates within the Australian subgroup and 16 within the Belgium subgroup. DNA sequence and multiple alignment analysis of eight Chilean isolates showed a significantly higher frequency of single and double nucleotide substitutions. Most of these mutations were non-silent, resulting in changes at the protein level in several important epitopes of gp51. The Chilean sequences and 59 BLV env sequences available at GenBank, were subjected to a phylogenetic analysis, resulting in four different clusters. The groups identified were not related to those previously defined by restriction analysis. Chilean isolates were included in two different clusters and were genetically not related to isolates collected from neighbouring countries. Considering our results we can conclude: (i) bulk milk samples are suitable to identify the presence of BLV allowing epidemiological and genetic studies to be conducted on large geographical areas; (ii) at least four different genetic groups of BLV were identified by phylogenetic analysis, with Chilean isolates included in two different sub clusters.

Overexpression of Raidd cDNA inhibits differentiation of mouse preadipocytes.

RAIDD (RIP-associated ICH-1 homologous protein with a death domain) is an adaptor molecule that mediates the action of cysteine proteases involved in apoptosis. To study the possibility of a novel system of cell ablation mediated by RAIDD, a preadipocyte cell line (3T3L1) was stably transfected with a plasmid containing the murine Raidd cDNA under the control of the adipocyte specific promoter aP2. Instead of the expected apoptosis, a blockage to differentiation upon hormonal induction was observed as judged by an absence of lipid accumulation, a lack of expression of adipocyte-specific genes and a fibroblastic appearance. Proliferation rate of Raidd-transfected clones remained unaffected. Overexpression of Raidd cDNA in 3T3L1 cell therefore inhibited differentiation, suggesting that Raidd plays a role in controlling differentiation of mouse preadipocytes and, perhaps, in other cell types, in addition to its established role in apoptosis.

Inducible ablation of adipocytes in adult transgenic mice expressing the E. coli nitroreductase gene.

We describe the use of an enzyme prodrug system based on E. coli nitroreductase (NTR) to achieve the specific ablation of adipose tissue. Transgenic mice expressing the NTR gene specifically in the adipose tissue were generated using the adipocyte specific promoter aP2. After treatment with the prodrug CB1954 these mice showed extensive cell depletion in all fat depots; this was directly correlated to both the dose of prodrug and the levels of NTR expression. Higher doses of CB1954 resulted in complete disappearance of visible adipose stores in some transgenic mice. These mice exhibited an impaired ability to thermoregulate body temperature. Lower doses of CB1954 resulted in a partial reduction of the adipose tissue leaving non-expressing cells that escape ablation. These animals show normal levels of blood glucose and triglycerides but have reduced leptin levels. After 30 days they were able to regenerate the fat depots and leptin levels returned to normal but, interestingly, no NTR-expressing cells were detectable. The present model provides a new approach to manipulate the number of adipocytes at different stages of mouse development and provides a new system for the study of fat metabolism especially in abnormal conditions such as obesity and its modulation through manipulation of the target cell population.