Bovine somatic cell nuclear transfer using mitomycin C-mediated chemical oocyte enucleation.

SummaryChemical oocyte enucleation holds the potential to ease somatic cell nuclear transfer (SCNT), although high enucleation rates remain limited to micromanipulation-based approaches. Therefore, this study aimed to test mitomycin C (MMC) for use in bovine functional chemical oocyte enucleation. Incubation of denuded eggs in 10 µg ml-1 MMC for different periods did not affect most maturation rates (0.5 h: 85.78%A, 1.0 h: 72.77%B, 1.5 h: 83.87%A, and 2.0 h: 82.05%A) in comparison with non-treated controls (CTL; 85.77%A). Parthenogenetic development arrest by MMC was efficient at cleavage (CTL: 72.93%A, 0.5 h: 64.92%A,B, 1.0 h: 60.39%B,C, 1.5 h: 66.35%A,B, and 2.0 h: 53.84%C) and blastocyst stages (CTL: 33.94%A, 0.5 h: 7.58%B, 1.0 h: 2.47%C, 1.5 h: 0.46%C, and 2.0 h: 0.51%C). Blastocysts were obtained after nuclear transfer (NT) using MMC enucleation [NT(MMC): 4.54%B] but at lower rates than for the SCNT control [NT(CTL): 26.31%A]. The removal of the meiotic spindle after MMC incubation fully restored SCNT blastocyst development [NT(MMC+SR): 24.74%A]. Early pregnancies were obtained by the transfer of NT(MMC) and NT(MMC+SR) blastocysts to synchronized recipients. In conclusion, MMC leads to functional chemical oocyte enucleation during SCNT and further suggests its potential for application towards technical improvements.

Skin Microbiota in Obese Women at Risk for Surgical Site Infection After Cesarean Delivery.

The obesity pandemic in the obstetrical population plus increased frequency of Cesarean delivery (CD) has increased vulnerability to surgical site infection (SSI). Here we characterized the microbiome at the site of skin incision before and after CD. Skin and relevant surgical sites were sampled before and after surgical antisepsis from obese (n = 31) and non-obese (n = 27) pregnant women. We quantified bacterial biomass by qPCR, microbial community composition by 16sRNA sequencing, assigned operational taxonomic units, and stained skin biopsies from incision for bacteria and biofilms. In obese women, incision site harbors significantly higher bacterial biomass of lower diversity. Phylum Firmicutes predominated over Actinobacteria, with phylotypes Clostridales and Bacteroidales over commensal Staphylococcus and Propionbacterium spp. Skin dysbiosis increased post-surgical prep and at end of surgery. Biofilms were identified post-prep in the majority (73%) of skin biopsies. At end of surgery, incision had significant gains in bacterial DNA and diversity, and obese women shared more genera with vagina and surgeon's glove in CD. Our findings suggest microbiota at incision differs between obese and non-obese pregnant women, and changes throughout CD. An interaction between vaginal and cutaneous dysbiosis at the incision site may explain the a priori increased risk for SSI among obese pregnant women.

Comparison of the Lund and Browder table to computed tomography scan three-dimensional surface area measurement for a pediatric cohort.

BACKGROUND: Treating burns effectively requires accurately assessing the percentage of the total body surface area (%TBSA) affected by burns. Current methods for estimating %TBSA, such as Lund and Browder (L&B) tables, rely on historic body statistics. An increasingly obese population has been blamed for increasing errors in %TBSA estimates. However, this assumption has not been experimentally validated. We hypothesized that errors in %TBSA estimates using L&B were due to differences in the physical proportions of today's children compared with children in the early 1940s when the chart was developed and that these differences would appear as body mass index (BMI)-associated systematic errors in the L&B values versus actual body surface areas. MATERIALS AND METHODS: We measured the TBSA of human pediatric cadavers using computed tomography scans. Subjects ranged from 9 mo to 15 y in age. We chose outliers of the BMI distribution (from the 31st percentile at the low through the 99th percentile at the high). We examined surface area proportions corresponding to L&B regions. RESULTS: Measured regional proportions based on computed tomography scans were in reasonable agreement with L&B, even with subjects in the tails of the BMI range. The largest deviation was 3.4%, significantly less than the error seen in real-world %TBSA estimates. CONCLUSIONS: While today's population is more obese than those studied by L&B, their body region proportions scale surprisingly well. The primary error in %TBSA estimation is not due to changing physical proportions of today's children and may instead lie in the application of the L&B table.

Investigating skin-to-skin care patterns with extremely preterm infants in the NICU and their effect on early cognitive and communication performance: a retrospective cohort study.

OBJECTIVES: The primary objective of the study was to investigate how patterns of skin-to-skin care might impact infant early cognitive and communication performance. DESIGN: This was a retrospective cohort study. SETTING: This study took place in a level-IV all-referral neonatal intensive care unit in the Midwest USA specialising in the care of extremely preterm infants. PARTICIPANTS: Data were collected from the electronic medical records of all extremely preterm infants (gestational age <27 weeks) admitted to the unit during 2010-2011 and who completed 6-month and 12-month developmental assessments in the follow-up clinic (n=97). OUTCOME MEASURES: Outcome measures included the cognitive and communication subscales of the Bayley Scales of Infant Development, Third Edition (Bayley-III); and skin-to-skin patterns including: total hours of maternal and paternal participation throughout hospitalisation, total duration in weeks and frequency (hours per week). ANALYSIS: Extracted data were analysed through a multistep process of logistic regressions, t-tests, χ2 tests and Fisher's exact tests followed with exploratory network analysis using novel visual analytic software. RESULTS: Infants who received above the sample median in total hours, weekly frequency and total hours from mothers and fathers of skin-to-skin care were more likely to score ≥80 on the cognitive and communication scales of the Bayley-III. However, the results were not statistically significant (p>0.05). Mothers provided the majority of skin-to-skin care with a sharp decline at 30 weeks corrected age, regardless of when extremely preterm infants were admitted. Additional exploratory network analysis suggests that medical and skin-to-skin factors play a parallel, non-synergistic role in contributing to early cognitive and communication performance as assessed through the Bayley-III. CONCLUSIONS: This study suggests an association between early and frequent skin-to-skin care with extremely preterm infants and early cognitive and communication performance.

MoFlow: visualizing conformational changes in molecules as molecular flow improves understanding.

BACKGROUND: Current visualizations of molecular motion use a Timeline-analogous representation that conveys "first the molecule was shaped like this, then like this...". This scheme is orthogonal to the Pathline-like human understanding of motion "this part of the molecule moved from here to here along this path". We present MoFlow, a system for visualizing molecular motion using a Pathline-analogous representation. RESULTS: The MoFlow system produces high-quality renderings of molecular motion as atom pathlines, as well as interactive WebGL visualizations, and 3D printable models. In a preliminary user study, MoFlow representations are shown to be superior to canonical representations for conveying molecular motion. CONCLUSIONS: Pathline-based representations of molecular motion are more easily understood than timeline representations. Pathline representations provide other advantages because they represent motion directly, rather than representing structure with inferred motion.

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues.

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional units (e.g., domains). Traditional consensus-building models fail to account for interpositional dependencies - functionally required covariation of residues that tend to appear simultaneously throughout evolution and across the phylogentic tree. These relationships can reveal important clues about the processes of protein folding, thermostability, and the formation of functional sites, which in turn can be used to inform the engineering of synthetic proteins. Unfortunately, these relationships essentially form sub-motifs which cannot be predicted by simple "majority rule" or even HMM-based consensus models, and the result can be a biologically invalid "consensus" which is not only never seen in nature but is less viable than any extant protein. We have developed a visual analytics tool, StickWRLD, which creates an interactive 3D representation of a protein alignment and clearly displays covarying residues. The user has the ability to pan and zoom, as well as dynamically change the statistical threshold underlying the identification of covariants. StickWRLD has previously been successfully used to identify functionally-required covarying residues in proteins such as Adenylate Kinase and in DNA sequences such as endonuclease target sites.

Morphological characterization and conservation of bovine spermatogenic cells by refrigeration at 4°C and freezing using different cryoprotective molecules.

The objectives of this study were study a practical method to characterize bovine spermatogenic cells and test the efficiency cells conservation by refrigeration at 4°C and cryopreservation in different solutions using two cooling curves. Cellular identification was performing by analysis of shape, size and morphology, associated with nucleus positioning and nuclear-cytoplasm ratio (NCR). Cellular samples were kept at 4°C for a period of 96 h in refrigeration solution and every 24h plasma membrane and DNA integrity were evaluated. Cryopreservation of cells was carried out using solutions containing 10% Dimethyl sulfoxide, 5% Dimethylformamide, 7% Glycerol and 7% Ethylene glycol, using a controlled and non-controlled cooling curve. Results of cellular characterization demonstrated that spermatocytes II presented a cylindrical shape, NCR of 1:1.5 and diameter ranging from 14.5 to 17.5 µm. Round spermatids presented diameter ranging from 7.6 to 13.4 µm, acrosomal cap and NCR of 1:2. Elongation and elongated spermatids showed to marked divergence in shape. There was a daily significant loss of viability of cooled cells until third day of storage, however they presented 72.77±5.16% viability after 4 days of storage at 4°C. There was no difference among the cryoprotectant solutions and cooling curves. In conclusion we demonstrated that association of microscopes and staining was a practical method to identify bovine spermatogenic cells. Furthermore, refrigeration at 4°C is an important strategy to preserve over 70% of viable cells after 4 days and cryopreservation, regardless of cryoprotectant solution or cooling curve used, can maintain over 50% of cells viable.

Understanding the sequence requirements of protein families: insights from the BioVis 2013 contests.

INTRODUCTION: In 2011, the BioVis symposium of the IEEE VisWeek conferences inaugurated a new variety of data analysis contest. Aimed at fostering collaborations between computational scientists and biologists, the BioVis contest provided real data from biological domains with emerging visualization needs, in the hope that novel approaches would result in powerful new tools for the community. In 2011 and 2012 the theme of these contests was expression Quantitative Trait Locus analysis, within and across tissues respectively. In 2013 the topic was updated to protein sequence and mutation visualization. METHODS: The contest was framed in the context of a real protein with numerous mutations that had lost function, and the question posed "what minimal set of changes would you propose to rescue function, or how could you support a biologist attempting to answer that question?". The data was grounded in actual experimental results in triosephosphate isomerase(TIM) enzymes. Seven teams composed of 36 individuals submitted entries with proposed solutions and approaches to the challenge. Their contributions ranged from careful analysis of the visualization and analytical requirements for the problem through integration of existing tools for analyzing the context and consequences of protein mutations, to completely new tools addressing the problem. RESULTS: Judges found valuable and novel contributions in each of the entries, including interesting ways to hierarchicalize the protein into domains of informational interaction, tools for simultaneously understanding both sequential and spatial order, and approaches for conveying some types of inter-residue dependencies. In this manuscript we document the problem presented to the contestants, summarize the biological contributions of their entries, and suggest opportunities that this work has highlighted for even more improved tools in the future.

Vitrification of bovine oocytes at different meiotic stages using the Cryotop method: assessment of morphological, molecular and functional patterns.

This study aimed to investigate the functional, morphological and molecular patterns of bovine oocytes vitrified at different times during in vitro maturation (IVM). Four groups of oocytes were used: non-vitrified control oocytes (CG), oocytes vitrified at 0 h (V0), oocytes vitrified after 8 h of IVM (V8) and oocytes vitrified after 22 h of IVM (V22). After vitrification, the oocytes were warmed and then returned to the incubator to complete a total of 24h of IVM. To evaluate the effect of vitrification, the nuclear maturation and fertilization rates were assessed by lacmoid staining and ultrastructural electron microscopy. The cleavage and blastocyst rates were evaluated at D2, D7 and D8. The expression levels of CASP3, TP53, HDAC2, SUV39H1 and DNMT1 were investigated by RT-qPCR. The nuclear maturation, oocyte fertilization, cleavage and blastocyst rates were higher (P < 0.05) in the CG group (80%; 81.3%; 88.5%; and 35.8%) than in the V0 (44%; 44.6%; 22.7%; and 2.6%), V8 (50%; 63%; 21.5%; and 2.2%) and V22 (55.5%; 66.9%; 24.1%; and 4.6%) groups. Ultrastructural analysis revealed significant damage within the cytoplasm of all vitrified groups, but more severe degeneration was observed in the V22 group. The gene expression profiles were not affected by vitrification (P > 0.05). In conclusion, cytoplasm degeneration seems to be the most severe form of damage caused by vitrification. The use of the Cryotop method for vitrification severely reduces bovine oocyte viability regardless of whether it is performed at GV, GVBD or MII stage.

Vitrification of bovine blastocysts pretreated with sublethal hydrostatic pressure stress: evaluation of post-thaw in vitro development and gene expression.

Sublethal stress treatment has been reported to enhance gametes' performance in subsequent procedures, such as cryopreservation. The aim of the present study was to evaluate the effect of different equilibration times between the termination of a sublethal hydrostatic pressure (HP) stress treatment and the initiation of vitrification on the post-thaw survival, continued in vitro development, hatching rate and gene expression of selected candidate genes of in vitro-produced (IVP) expanded bovine blastocysts. Day 7 IVP blastocysts were subjected to 600 bar pressure for 60 min at 32°C. Immediately after pressure treatment (HP0h) or after 1 or 2h incubation (HP1h and HP2h groups, respectively), embryos were either vitrified and warmed using the open pulled straw method, followed by 72 h in vitro culture or were stored at -80°C until gene expression analysis. Re-expansion and hatching rates after vitrification-warming were significantly (P<0.05) higher in the HP0h (88 and 76%, respectively) and HP1h (90 and 75%, respectively) groups than in the untreated (82 and 63%, respectively) and HP2h groups (79 and 70%, respectively). Moreover, the HP1h group showed further improvement in the speed of re-expansion and resumption of normal in vitro development. Cumulative analysis of all genes (SC4MOL, HSP1A1A, SOD2 and GPX4) revealed a similar pattern of expression, with a tendency for peak transcript abundance 1h after HP treatment. Application of HP stress treatment was found to be efficient in increasing the in vitro developmental competence of vitrified bovine embryos.

Allele-specific expression of the MAOA gene and X chromosome inactivation in in vitro produced bovine embryos.

During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre-implantation bovine embryo development by characterizing the allele-specific expression pattern of the X chromosome-linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4-, 8- to 16-cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT-PCR-RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele-specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4-, 8- to 16-cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele-specific expression of an X-linked gene that is subject to XCI in in vitro bovine embryos from the 4-cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production.

Effect of follicular wave synchronization on in vitro embryo production in heifers.

Aiming to achieve the ideal time of ovum pick-up (OPU) for in vitro embryo production (IVP) in crossbred heifers, two Latin square design studies investigated the effect of ovarian follicular wave synchronization with estradiol benzoate (EB) and progestins. For each experiment, crossbred heifers stage of estrous cycle was synchronized either with a norgestomet ear implant (Experiment 1) or a progesterone intravaginal device (Experiment 2) for 7d, followed by the administration of 150microg d-cloprostenol. On Day 7, all follicles >3mm in diameter were aspirated and implants/devices were replaced by new ones. Afterwards, implant/device replacement was conducted every 14d. Each experiment had three treatment groups. In Experiment 1 (n=12), heifers in Group 2X had their follicles aspirated twice a week and those in Groups 1X and 1X-EB were submitted to OPU once a week for a period of 28d. Heifers from Group 1X-EB also received 2mg EB i.m. immediately after each OPU session. In Experiment 2 (n=11), animals from Group 0EB did not receive EB while heifers in Groups 2EB and 5EB received 2 and 5mg of EB respectively, immediately after OPU. The OPU sessions were performed once weekly for 28d. Therefore, in both experiments, four OPU sessions were performed in heifers aspirated once a week and in Experiment 1, eight OPU sessions were done in heifers aspirated twice a week. Additionally, during the 7-d period following follicular aspiration, ovarian ultrasonography examinations were conducted to measure diameter of the largest follicle and blood samples were collected for FSH quantification by RIA. In Experiment 1, all viable oocytes recovered were in vitro matured and fertilized. Results indicated that while progestin and EB altered follicular wave patterns, this treatment did not prevent establishment of follicular dominance on the ovaries of heifers during OPU at 7-d intervals. Furthermore, the proposed stage of follicular wave synchronization strategies did not improve the number and quality of the recovered oocytes, or the number of in vitro produced embryos.

Changes in gene expression profiles of bovine embryos produced in vitro, by natural ovulation, or hormonal superstimulation.

Embryos produced by hormonal superstimulation have been used as an in vivo control in most published research on embryo gene expression. However, it is not known if this is the most appropriate control for gene expression profile studies. We compared the expression of GRB-10, IGF-II, IGF-IIR, MnSOD, GPX-4, catalase, BAX, and interferon-tau genes, in embryos produced in vivo by hormonal superovulation (SOV), by in vitro fertilization (IVF) or in vivo without any hormonal stimulus (NOV). GRB-10 was less expressed in NOV than IVF embryos, whereas no differences were found for the other genes. The genes related to stress response were then grouped and compared; the sum of expression of MnSOD, GPX-4, and catalase genes tended to be greater in IVF than NOV embryos. A correlation analysis was performed; we found a distinct behavior for NOV embryos when compared with SOV and IVF in the expression of GRB-10, IGF-II and IGF-IIR genes. However, the behavior of these genes was similar in SOV and IVF embryos. We conclude that ovarian hormonal stimulation can affect embryos by altering gene expression. Although this conclusion was based on investigation of only a few genes, we suggest that SOV embryos should be used with caution as a control in gene expression studies.

Recovery, cryopreservation and fertilization potential of bovine spermatozoa obtained from epididymides stored at 5 degrees C by different periods of time.

The objetive of the present study was to evaluate the effect of the interval between animal's death and sperm recovery on the freezability and fertilizing ability of spermatozoa from bull epididymides stored for different periods of time. Testis from 25 bulls were collected at the abattoir 2h after the slaughter. In the laboratory spermatozoa from one epididymis were recovered and analysed for motility. The remaining epididymis was stored for 24h (G24), 48h (G48) and 72h (G72) at 5 degrees C. At the end of each time period, spermatozoa were recuperated and cryopreserved in Tris-egg yolk and glycerol. Pre-freeze and post-thaw sperm samples were taken to assess total and progressive motility, concentration, membrane integrity and acrosome integrity. For evaluation of fertilizing ability, in each time period five straws of each bull were thawed, pooled and used for in vitro embryo production. The results showed that after 48h of storage there was a decline in total motility, which did not change until 72h. Progressive motility, plasma membrane and acrosome integrity were not affected by any of the storage periods. Conversely, all sperm parameters, except progressive motility, were reduced after cryopreservation. Embryo production was less (P<0.05) in the treatments than in the reference group. However, there was no differences (P>0.05) in blastoycst rate among experimental groups. Considering all the embryos produced by epididymal spermatozoa a greater proportion of female embryos was observed, which was similar to the reference embryos. The shift observed on sex ratio toward female for those two groups was also observed when they were compared with the expected 1:1 ratio (P<0.05). The results showed the possibility to produced in vitro embryos using cryopreseved spermatozoa from epididymides and stored for long period of time at 5 degrees C. These procedures became an important tool for animal preservation when the sperm cells cannot be cryopreserved immediately after the animal's death.

Effect of Percoll volume, duration and force of centrifugation, on in vitro production and sex ratio of bovine embryos.

The objective was to evaluate the effect of Percoll volume, and duration and force of centrifugation on sperm quality characteristics, embryo development, and sex ratio of in vitro-produced (IVP) bovine embryos. Frozen-thawed semen from four bulls were submitted to three Percoll procedures: T1-4 mL of Percoll, centrifuged for 20 min at 700 g; T2-800 microL of Percoll, centrifuged for 20 min at 700 g; and T3-800 microL of Percoll, centrifuged for 5 min at 5,000 g. Sperm total motility, morphology and integrity of the sperm acrosome, membrane and chromatin were determined before and after Percoll treatment, and semen was used for in vitro fertilization (IVF) of in vitro-matured oocytes. All Percoll methods increased the proportion of motile sperm (P<0.05). There were no significant effects of treatment for any sperm characteristic; however, for every end point, there were significant differences among bulls. Similarly, rates of cleavage and blastocyst formation were not affected by the Percoll procedure (P>0.05), but were affected by sire (P<0.05). Sex ratio was similar among treatments for Bulls 2 and 3, whereas semen from Bull 1 processed by T1 yielded a greater percentage of male embryos. However, when only treatments were considered, independent of bulls, the proportion of male:female embryos did not differ significantly from an expected 1:1 ratio. In conclusion, decreasing Percoll volume, reducing duration of centrifugation, and using a higher force of centrifugation did not significantly affect sperm quality, embryo development, or sex ratio of in vitro-produced bovine embryos.

Placentation in cloned cattle: structure and microvascular architecture.

To elucidate the morphological differences between placentas from normal and cloned cattle pregnancies reaching term, the umbilical cord, placentomes and interplacentomal region of the fetal membranes were examined macroscopically as well as by light and scanning electron microscopy. In pregnancies established by somatic nucleus transfer (NT), the umbilical cord and fetal membranes were edematous. Placentomal fusion was common, resulting in increased size and a decreased number of placentomes. Extensive areas of the chorioallantoic membrane were devoid of placentomes. An increased number of functional or accessory microcotyledons (<1 cm) were present at the maternally oriented surface of fetal membranes. Extensive areas of extravasated maternal blood were present within the placentomes and in the interplacentomal region. The crypts on the caruncular surface were dilated and accommodated complexes of more than one primary villus, as opposed to a single villus in non-cloned placentae. Scanning electron microscopy of blood vessel casts revealed that there was also more than one stem artery per villous tree and that the ramification of the vessels failed to form dense complexes of capillary loops and sinusoidal dilations as in normal pregnancies. At the materno-fetal interface, however, the trophoblast and uterine epithelium had normal histology. In conclusion, the NT placentas had a range of pathomorphological changes; this was likely associated with the poor clinical outcome of NT pregnancies.

The use of the acridine orange test and the TUNEL assay to assess the integrity of freeze-dried bovine spermatozoa DNA.

The ability to detect nuclear damage is an important tool for the development of sperm preservation methods. We used the acridine orange test (AOT) and the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) assay to assess the DNA status of sperm cells preserved with different lyophilization media. The AOT did not detect any differences between different lyophilization media. However, differences in DNA integrity were observed among treatments with the TUNEL assay, suggesting that TUNEL is a more sensitive method to evaluate sperm DNA. The use of TCM 199 and 10% FCS as a lyophilization medium resulted in 14% of the cells with DNA fragmentation in TUNEL test. The AOT indicated only 4% of the cells with chromatin damage, with this same treatment, with no significant differences when compared to the other treatments. The degree of DNA fragmentation was negatively related to fertilizing potential, as sperm DNA damage was inversely correlated with pro-nucleus formation. The TUNEL assay was found to be an efficient method to detect DNA damage in sperm, and it could be used as a tool to predict male fertility.

Cryopreservation of epididymal bovine spermatozoa from dead animals and its uses in vitro embryo production.

The present study aimed to evaluate viability and in vitro fertilizing ability of cryopreserved epididymal spermatozoa obtained from dead animals. To collect spermatozoa, epididymides from three males (Bulls A1, A2 and A3) were collected at a local slaughterhouse. As a reference ejaculate from a bull with known in vitro fertility, was used. Sperm characteristics (motility, chromatin and acrosome integrity) were evaluated before and after cryopreservation. Then, frozen spermatozoa from all animals were used for in vitro fertilization. Cleavage and blastocyst rates at 48 h (day 2) and 168 h (day 7) post in vitro insemination, for bull A1 (82.1 and 38.6%) and A2 (80.7 and 33.8%) were similar (P>0.05) to the reference bull (88.9 and 57.2%). Bull A3 had the lesser cleavage (42.0%) and blastocyst (26.1%) rates. The results showed that epididymal spermatozoa from dead animals can be successfully cryopreserved and used in vitro production of embryos.

Effects of freeze-drying on cytology, ultrastructure, DNA fragmentation, and fertilizing ability of bovine sperm.

Freeze-drying sperm is an alternative to cryopreservation. Although sperm from various species has been freeze-dried, there are few reports for bovine sperm. The primary objective of this study was to evaluate the protective effect of various freeze-drying media on the structural and functional components of bovine sperm. The media tested were composed of TCM 199 with Hanks salts supplemented with 10% fetal calf serum (FCS) and TCM 199 with Hanks salts supplemented with 10% FCS and 0.2 M trehalose and EGTA solution. The efficiency of each medium on the preservation of freeze-dried sperm structures was evaluated with conventional and electron microscopy, DNA integrity was analyzed by a TUNEL assay, and fertilizing ability of lyophilized sperm was determined with ICSI. Although the plasma membrane was damaged in all media tested, mitochondria were similarly preserved in all freeze-drying treatments. The acrosome was best preserved in the media that contained trehalose (other treatments also conserved this structure). In contrast, media containing EGTA or trehalose most effectively preserved the nuclei in freeze-dried sperm, with only 2 and 5%, respectively, of cells with fragmented DNA. Furthermore, sperm conserved with these media also had higher (P<0.05) rates of sperm head decondensation (32.5 and 27.5%), pronucleus formation (37.5 and 45.0%) and blastocyst formation (19.4 and 18.3%) than medium supplemented with FCS (15.0, 20.0 and 10.2%, respectively). In conclusion, media with EGTA and trehalose adequately protected bovine sperm during freeze-drying by preserving the viability of their nuclei.

The use of the acridine orange test and the TUNEL assay to assess the integrity of freeze-dried bovine spermatozoa DNA

The ability to detect nuclear damage is an important tool for the development of sperm preservation methods. We used the acridine orange test (AOT) and the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) assay to assess the DNA status of sperm cells preserved with different lyophilization media. The AOT did not detect any differences between different lyophilization media. However, differences in DNA integrity were observed among treatments with the TUNEL assay, suggesting that TUNEL is a more sensitive method to evaluate sperm DNA. The use of TCM 199 and 10% FCS as a lyophilization medium resulted in 14% of the cells with DNA fragmentation in TUNEL test. The AOT indicated only 4% of the cells with chromatin damage, with this same treatment, with no significant differences when compared to the other treatments. The degree of DNA fragmentation was negatively related to fertilizing potential, as sperm DNA damage was inversely correlated with pro-nucleus formation. The TUNEL assay was found to be an efficient method to detect DNA damage in sperm, and it could be used as a tool to predict male fertility