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Abstract Background The present studies evaluated the effects of cryoprotectants, the vitrification procedure and time in the warming solution containing sucrose on cleavage and embryo development of immature (GV stage) bovine cumulus-oocyte complexes (COCs). Methods Two experiments were conducted. In Experiment 1, COCs (n = 420) were randomly assigned to four groups: 1) Control group: no treatment; 2) VS1 group: COCs were exposed to vitrification solution 1 (VS1) containing 7.5% ethylene glycol [EG] + 7.5% dimethyl sulfoxide [DMSO] + 20% calf serum [CS] in TCM-199 at 37 C for 5 min; 3) VS1 + VS2 group: COCs were exposed to VS1 for 5 min followed by VS2 (15% EG + 15% DMSO + 17.1% sucrose + 20% CS) at 37 C for 45–60 sec; and 4) Vitrified group: COCs were exposed to VS1 and VS2, loaded on cryotops, vitrified in liquid nitrogen and then warmed in TCM-199 + 17.1% sucrose + 20% CS at 37 C for 1 min. In Experiment 2, COCs (n = 581) were assigned to the same groups, but those in VS1, VS1 + VS2 and Vitrified groups were sub-divided and exposed to the warming solution for either 1 or 5 min. After treatment and/or warming, all COCs in both experiments underwent in vitro maturation, in vitro fertilization and in vitro culture. Results Cleavage and blastocyst rates did not differ among Control, VS1 and VS1 + VS2 groups in either experiment. In Experiment 2, there was no effect of time in the warming solution. However, both cleavage and blastocyst rates were lower (P Conclusions The permeating cryoprotectants (EG and DMSO) present in VS1 and VS2 solutions and the time in the warming solution containing sucrose had no adverse effects on cleavage and blastocyst rates of immature bovine COCs. However, cleavage rate and early embryo development were reduced following the vitrification and warming.

Sugars are commonly supplemented into vitrification solution to dehydrate cells in order to reduce the formation of fatal intracellular ice crystals. Natural honey is a mixture of 25 sugars (mainly fructose and glucose) that have different biological and pharmacological benefits. The present study was designed to determine if honey can be used as a nonpermeating cryoprotectant in vitrification of bovine oocytes. In the first experiment, denuded-MII oocytes were exposed to 0.25, 0.5, 1.0, 1.5 or 2.0 M of honey or sucrose. Natural honey and sucrose caused similar ooplasm dehydration. A significant relationship existed between time and ooplasm volume change (P < 0.05), during dehydration and rehydration phases, in both honey and sucrose solutions. In the second experiment, the immature cumulus-oocyte complexes (COCs) were vitrified in an EG/DMSO-based vitrification solution containing honey (0.5, 1 or 1.5 M) or sucrose (0.5 M) as a gold standard. The vitrified-warmed COCs were matured in vitro and evaluated for nuclear maturation. The maturation (MII) rate was greater in nonvitrified control (81%) than vitrified groups (54%, P < 0.05). In the third experiment, COCs were either remained nonvitrified (control) or vitrified in 1.0 M honey or 0.5 M sucrose, followed by IVM, IVF and IVC (for 9 days). Cleavage rate was greater in control (74%) than in vitrified groups (47%, P < 0.05), without significant difference between sugars. Blastocyst rate was 34, 13 and 3% in control, honey and sucrose groups respectively (P < 0.05). In conclusion, natural honey acted as a nonpermeating cryoprotectant in vitrification solution and improved the embryonic development in vitrified bovine COCs. [ABSTRACT FROM AUTHOR]

Two experiments were done using a two‐by‐two design to determine the effects of season and superstimulatory protocol on embryo production in wood bison. In Experiment 1 (in vivo‐derived embryos), ovarian superstimulation was induced in female bison during the ovulatory and anovulatory seasons with either two or three doses of FSH given every‐other‐day (FSH × 2 vs. FSH × 3, respectively). Bison were given hCG to induce ovulation, inseminated 12 and 24 hr after hCG, and embryos were collected 8 days after hCG (n = 10 bison/group). In Experiment 2 (in vitro embryo production), ovarian superstimulation was induced in female bison during the ovulatory and anovulatory seasons with two doses of FSH, and in vivo maturation of the cumulus–oocyte complexes (COC) was induced with hCG at either 48 or 72 hr after the last dose of FSH. COC were collected 34 hr after hCG, and expanded COC were used for in vitro fertilization and culture. In Experiment 1, the number of follicles ≥9 mm, the proportion of follicles that ovulated, the number of CL, and the total number of ova/embryos collected did not differ between seasons or treatment groups, but the number of transferable embryos was greater (p <.05) in the ovulatory season. In Experiment 2, no differences were detected between seasons or treatment groups for any end point. The number of transferable embryos produced per bison was greatest (p <.05) using in vitro fertilization and was unaffected by season (1.5 ± 0.2 and 1.1 ± 0.3 during anovulatory and ovulatory seasons, respectively), in contrast to in vivo embryo production which was affected by season (0.1 ± 0.01 and 0.7 ± 0.2 during anovulatory and ovulatory seasons, respectively). Results demonstrate that transferable embryos can be produced throughout the year in wood bison by both in vivo and in vitro techniques, but the efficiency of embryo production of in vivo‐derived embryos is significantly lower during the anovulatory season. [ABSTRACT FROM AUTHOR]

Egg yolk is a common ingredient of mammalian semen extender to protect sperm against initial cold shock. However, egg yolk has biosecurity risks. Our main objectives were to cryopreserve bull semen without egg yolk using exogenous cholesterol and to study the protective role of glycerol in egg yolk-free semen extender. Other objectives were to compare protein profiles and in vitro fertilization potential of bull sperm frozen with and without egg yolk. In first experiment, semen was either diluted in conventional tris-egg yolk glycerol (TEYG control) extender or first treated with cholesterol-cyclodextrin complex (CC, 2 mg/ml semen) followed by dilution in egg yolk-free tris-glycerol (TG) extender (collectively called as "CC+TG") at 22°C or 4°C, and frozen. Post-thaw sperm motion characteristics were similar between CC+TG and TEYG control extenders, and temperature of glycerol addition. In second experiment, semen was frozen in CC+TG extender varying in glycerol concentration (7 to 0%; v/v). Post-thaw sperm quality decreased with the decline in glycerol concentration in TG extender, even higher concentration of CC complex (3 or 4 mg/ml semen) could not protect sperm in the absence of glycerol in TG extender. In third experiment, SDS electrophoresis of proteins from fresh sperm and sperm frozen in CC+TG, and TEYG control extenders was conducted. Protein profiles in fresh sperm and CC+TG frozen sperm were almost similar. Egg yolk proteins bound tightly with sperm plasma membrane. In fourth experiment, in vitro fertilization potentials of sperm frozen in TEYG control and CC+TG extenders were tested. Cleavage and blastocyst rates of semen frozen in CC+TG and TEYG control extenders were similar. In conclusion, cholesterol-cyclodextrin replaced egg yolk from the semen extender; glycerol remained essential for egg yolk-free sperm cryopreservation; and CC+TG extender did not modify sperm plasma membrane CC+TG whereas egg yolk extender changed the plasma membrane composition of bull sperm. [ABSTRACT FROM AUTHOR]

Cryopreservation is known for its marked deleterious effects on embryonic health. Bovine compact morulae were vitrified or slow-frozen, and post-warm morulae were cultured to the expanded blastocyst stage. Blastocysts developed from vitrified and slow-frozen morulae were subjected to microarray analysis and compared with blastocysts developed from unfrozen control morulae for differential gene expression. Morula to blastocyst conversion rate was higher (P < 0.05) in control (72%) and vitrified (77%) than in slow-frozen (34%) morulae. Total 20 genes were upregulated and 44 genes were downregulated in blastocysts developed from vitrified morulae (fold change ≥ ± 2, P < 0.05) in comparison with blastocysts developed from control morulae. In blastocysts developed from slow-frozen morulae, 102 genes were upregulated and 63 genes were downregulated (fold change ≥ ± 1.5, P < 0.05). Blastocysts developed from vitrified morulae exhibited significant changes in gene expression mainly involving embryo implantation (PTGS2, CALB1), lipid peroxidation and reactive oxygen species generation (HSD3B1, AKR1B1, APOA1) and cell differentiation (KRT19, CLDN23). However, blastocysts developed from slow-frozen morulae showed changes in the expression of genes related to cell signaling (SPP1), cell structure and differentiation (DCLK2, JAM2 and VIM), and lipid metabolism (PLA2R1 and SMPD3). In silico comparison between blastocysts developed form vitrified and slow-frozen morulae revealed similar changes in gene expression as between blastocysts developed from vitrified and control morulae. In conclusion, blastocysts developed form vitrified morulae demonstrated better post-warming survival than blastocysts developed from slow-frozen morulae but their gene expression related to lipid metabolism, steroidogenesis, cell differentiation and placentation changed significantly (≥ 2 fold). Slow freezing method killed more morulae than vitrification but those which survived up to blastocyst stage did not express ≥ 2 fold change in their gene expression as compared with blastocysts from control morulae. [ABSTRACT FROM AUTHOR]

This study examines whether incorporating cholesterol-loaded methyl-β-cyclodextrin (CLC) in the bovine oocyte plasma membrane improves oocyte tolerance to vitrification. In vitro matured oocytes were incubated with 2 mg/ml BODIPY-labeled CLC for different time intervals in FCS or PVA supplemented medium or exposed to different CLC concentrations to examine the subcellular localization of cholesterol by confocal microscopy live-cell imaging. Subsequently, the effects of optimized CLC concentrations and incubation times prior to vitrification on early embryo development were assessed. Then, we evaluated the effects of pretreatment with 2 mg/ml CLC for 30 min before the vitrification of immature (GV) and in vitro matured (MII) oocytes on developmental competence and gene expression. Our results indicate a high plasma membrane labeling intensity after 30 min of incubation with 2 mg/ml CLC for 30 min, regardless of the holding medium used. When oocytes were incubated with 1 mg/ml, 2 mg/ml and 3 mg/ml of CLC, intense labeling was observed at the plasma membrane after 40, 30 and 20 min, respectively. CLC pre-treatment before the vitrification of bovine oocytes did not affect subsequent cleavage and embryo development rates irrespective of CLC concentrations, incubation times or meiotic stage. However, pretreatment seems to improve the quality of embryos derived from vitrified oocytes, mainly when oocytes were vitrified at the GV stage. [ABSTRACT FROM AUTHOR]

The objectives of this study were to confirm the relationship of apoptosis-associated membrane and nuclear changes in bull spermatozoa with field fertility, to predict the fertility of beef bulls used for natural breeding and to study the role of DNA-nicked spermatozoa in early embryonic development. In Experiment 1, the relationship between fertility and different sperm populations identified by the Annexin V/propidium iodide (PI) and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assays was determined. Bull fertility was related to live (P,0.05) and necrotic (P,0.01) and DNA-nicked (P,0.001) spermatozoa. In Experiment 2, the percentage of DNA-nicked spermatozoa was determined in 15 beef bulls used for natural breeding and their fertility potential was determined using a regression model developed in Experiment 1.The predicted fertility deviation of beef bulls ranged from 7.3 to 2.4. In Experiment 3, the effect of DNA-nicked spermatozoa on in vitro cleavage and blastocyst rates was evaluated, using 30 000 or 300 000 spermatozoa per droplet. Cleavage rate was adversely affected (P,0.05) by DNA-nicked spermatozoa, regardless of sperm concentration. Blastocyst rate was lower (P,0.05) in high DNA-nicked spermatozoa at the lower sperm concentration. In conclusion, the incidence of DNA-nicked spermatozoa is a useful marker to predict a bull's fertility potential. DNA-nicked spermatozoa showed adverse effects on early embryonic development. [ABSTRACT FROM AUTHOR]

This study was designed to compare the kinetics of sperm survival in different types of bull semen. Fresh ejaculates from four bulls were pooled, diluted in Tris-citric acid-egg yolk-glycerol extender, cooled to 4°C, frozen in LN2 and thawed at 37°C. Fresh, diluted, cooled and frozen-thawed semen were incubated at 37°C, and evaluated at 0, 2, 4, 6, 12 and 24 h after the beginning of incubation. In Experiment 1, progressive sperm motility, normal acrosomes and plasma membrane integrity and asymmetry were determined. In Experiment 2, generation of superoxide anion (…) along with plasma membrane permeability and generation of hydrogen peroxide (…) along with plasma membrane integrity were assessed. In Experiment 1, frozen-thawed semen had shorter survival times for progressive sperm motility, and spermatozoa with intact plasma membranes and acrosomes (IPM-IACR) as compared with other types of semen (P < 0.05). Fresh spermatozoa underwent a necrotic pathway, diluted and cooled spermatozoa underwent an apoptosis-like pathway and frozen-thawed spermatozoa underwent both necrotic and apoptosis-like pathways. In Experiment 2, spermatozoa in all four types of semen exhibited … generation and increased plasma membrane permeability, and became necrotic without H[sub 2]O[sub 2] generation during incubation (P < 0.05). In conclusion, frozen-thawed semen had shorter sperm longevity, which has important implications relating to the timing of artificial insemination. Different types of semen followed different death pathways. During incubation, spermatozoa in all types of semen generated O[sub 2][sup *-], which increased the permeability and compromised the integrity of the plasma membrane. Sperm survival is important for the fertilisation of oocytes. The kinetics of progressive sperm motility, acrosomes, plasma membrane and generation of reactive oxygen species were determined in vitro over 24 h. Bull sperm longevity was poor in frozen-thawed semen. Different types of spermatozoa followed different death pathways. All types of spermatozoa generated superoxide anions during incubation, which adversely affected their plasma membranes. [ABSTRACT FROM AUTHOR]

Vitrification of bovine cumulus-oocyte complexes (COCs) is not as successful as bovine embryos, due to oocyte's complex structure and chilling sensitivity. Synchrotron X-ray diffraction (SXRD), a powerful method to study crystal structure and phase changes, was used to detect the glass or ice formation in water, tissue culture medium (TCM)-199, vitrification solution 2 (VS2), and vitrified bovine COCs and morulae. Data revealed Debye's rings and peaks associated with the hexagonal ice crystals at 3.897, 3.635, 3.427, 2.610, 2.241, 1.912 and 1.878 Å in both water and TCM-199, whereas VS2 showed amorphous (glassy) appearance, at 102K (−171°C). An additional peak of sodium phosphate monobasic hydrate (NaH2PO4.H2O) crystals was observed at 2.064 Å in TCM-199 only. All ice and NaH2PO4.H2O peaks were detected in the non-vitrified (control) and vitrified COCs, except two ice peaks (3.145 and 2.655 Å) were absent in the vitrified COCs. The intensities of majority of ice peaks did not differ between the non-vitrified and vitrified COCs. The non-vitrified bovine morulae in TCM-199 demonstrated all ice- and NaH2PO4.H2O-associated Debye's rings and peaks, found in TCM-199 alone. There was no Debye's ring present in the vitrified morulae. In conclusion, SXRD is a powerful method to confirm the vitrifiability of a solution and to detect the glass or ice formation in vitrified cells and tissues. The vitrified bovine COCs exhibited the hexagonal ice crystals instead of glass formation whereas the bovine morulae underwent a typical vitrification. [ABSTRACT FROM AUTHOR]

Two experiments were done to test the hypothesis that morphologic characteristics of wood bison cumulus-oocyte complexes (COC) are reflective of the ability of the oocyte to develop to an advanced embryonic stage after in vitro maturation, fertilization and culture, and to determine the effect of prolonging the interval from the end of superstimulation treatment to oocyte collection (FSH starvation period). Experiments were done during the anovulatory season. In Experiment 1, ovarian superstimulation was induced in 10 bison with two doses of FSH given at 48 h intervals beginning at the time of follicular wave emergence. COC were collected 3 days (72 h) after the last dose of FSH by follicular aspiration and classified as compact, expanded or denuded. The COC were matured in vitro for 24 h before fertilization in vitro (Day 0). Embryo development was assessed on Days 3, 7 and 8. The blastocyst rate was 7/34, 2/10 and 0/3 in COC classified as compact, expanded and denuded, respectively; however, only compact COC resulted in embryos that reached the expanded blastocyst stage. In Experiment 2, COC were collected at either 3 or 4 days (72 or 96 h) after the last dose of FSH (n = 16 bison/group) to determine the effect of the duration of FSH starvation on oocyte competence. The COC were classified as compact good (>3 layers of cumulus cells), compact regular (1–3 layers of cumulus cells), expanded or denuded, and then matured, fertilized and cultured in vitro . Although follicles were larger (P < 0.05) in the 4-day FSH starvation group, there was no effect of starvation period on the distribution of COC morphology; overall, 112/194 (57.7%) were compact, 29/194 (26.3%) were expanded, 39/194 (20.1%) were denuded, and 14/194 (7.2%) were degenerated (P < 0.05). Similarly, there was no effect of starvation period on embryo development. Compact good COC had the highest cleavage (88%) and blastocyst rates (54%; P < 0.05), followed by compact regular COC at 73% and 25%, respectively. Expanded and denuded COC had low cleavage (40% vs. 59%, respectively) and blastocyst rates (5% vs. 8%, respectively). We conclude that morphologic characteristics of wood bison COC are reflective of the ability of the oocyte to develop into an embryo in vitro . Importantly, oocytes collected from superstimulated bison during the anovulatory season were competent to develop to the blastocyst stage following in vitro maturation, fertilization and culture. [ABSTRACT FROM AUTHOR]

Experiments were conducted in wood bison to determine the effect of additional maturation time on embryo development of in vivo matured oocytes. In experiment 1, cumulus-oocyte complexes (COC) were collected 30 hours after hCG treatment in superstimulated wood bison, and expanded COC were fertilized immediately or after 4 hours of additional in vitro maturation. Embryo development was assessed on Days 3, 7, and 8 (Day 0 = day of fertilization). No difference in cleavage rate was detected (55.3% vs. 60.5%, P = 0.82), but the Day 8 blastocyst rate was higher after an additional 4 hours of in vitro maturation time (44.7 vs. 18.4%, P = 0.03). In experiment 2, COC were collected at either 30 hours or 34 hours after hCG treatment. Expanded COC from the 30 hours group were fertilized after 4 hours of in vitro maturation, whereas those from the 34 hours group were fertilized immediately. A higher cleavage rate (74.3 vs. 57.0%) and blastocyst rate (54.1 vs. 37.2%) were found in the 34 hours group versus the 30 hours group (P < 0.05). In conclusion, an additional short period of in vitro maturation, or an extended period of in vivo maturation are beneficial for in vitro embryo production in wood bison. [ABSTRACT FROM AUTHOR]

The objectives of this study were to confirm the relationship of apoptosis-associated membrane and nuclear changes in bull spermatozoa with field fertility, to predict the fertility of beef bulls used for natural breeding and to study the role of DNA-nicked spermatozoa in early embryonic development. In Experiment 1, the relationship between fertility and different sperm populations identified by the Annexin V/propidium iodide (PI) and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assays was determined. Bull fertility was related to live (PPPin vitro cleavage and blastocyst rates was evaluated, using 30000 or 300000 spermatozoa per droplet. Cleavage rate was adversely affected (PP<0.05) in high DNA-nicked spermatozoa at the lower sperm concentration. In conclusion, the incidence of DNA-nicked spermatozoa is a useful marker to predict a bull's fertility potential. DNA-nicked spermatozoa showed adverse effects on early embryonic development.

Early estimation of bull fertility is highly desirable for the conservation of male genetics of endangered species and for the exploitation of genetically superior sires in artificial insemination programs. The present work was conducted as a proof-of-principle study to identify fertility-associated metabolites in dairy bull seminal plasma and blood serum using proton nuclear magnetic resonance ((1)H NMR). Semen and blood samples were collected from high- and low-fertility breeding bulls (n = 5 each), stationed at Semex, Guelph, Canada. NMR spectra of serum and seminal plasma were recorded at a resonance frequency of 500.13 MHz on a Bruker Avance-500 spectrometer equipped with an inverse triple resonance probe (TXI, 5 mm). Spectra were phased manually, baseline corrected, and calibrated against 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid at 0.0 parts per million (ppm). Spectra were converted to an appropriate format for analysis using Prometab software running within MATLAB. Principal component analysis was used to examine intrinsic variation in the NMR data set, and to identify trends and to exclude outliers. Partial least square-discriminant analysis was performed to identify the significant features between fertility groups. The fertility-associated metabolites with variable importance in projections (VIP) scores >2 were citrate (2.50 ppm), tryptamine/taurine (3.34-3.38 ppm), isoleucine (0.74 ppm), and leucine (0.78 ppm) in the seminal plasma; and isoleucine (1.14 ppm), asparagine (2.90-2.94 ppm), glycogen (3.98 ppm), and citrulline (1.54 ppm) in the serum. These metabolites showed identifiable peaks, and thus can be used as biomarkers of fertility in breeding bulls., (© 2015 Wiley Periodicals, Inc.)

The preservation of the female portion of livestock genetics has become an international priority; however, in situ conservation strategies are extremely expensive. Therefore, efforts are increasingly focusing on the development of a reliable cryopreservation method for oocytes, in order to establish ova banks. Slow freezing, a common method for cryopreservation of oocytes, causes osmotic shock (solution effect) and intracellular ice crystallization leading to cell damage. Vitrification is an alternative method for cryopreservation in which cells are exposed to a higher concentration of cryoprotectants and frozen with an ultra rapid freezing velocity, resulting in an ice crystal free, solid glass-like structure. Presently, vitrification is a popular method for cryopreservation of embryos. However, vitrification of oocytes is still challenging due to their complex structure and sensitivity to chilling.

Assessing semen quality is crucially important for the exploitation of genetically superior sires in an artificial insemination (AI) program. In this study, we compare modern and conventional techniques to estimate bovine sperm concentration and membrane integrity. First, the NucleoCounter SP-100 was validated for sperm concentration and provided statistically reliable and repeatable estimates among aliquots and replicates of 25 fresh ejaculates. Sperm concentrations in 78 ejaculates were then determined with hemacytometer, flow cytometer, and NucleoCounter SP-100 and were significantly correlated (P < .001), with regression coefficients among these 3 techniques close to 1 (P < .01). However, the sperm concentration determined by hemacytometer was lower (P < .01) than by flow cytometer and NucleoCounter SP-100. Forty frozen-thawed semen samples were then assessed for sperm concentration and membrane integrity with hemacytometer, flow cytometer and NucleoCounter SP-100. Significant relationships were found for sperm concentration determined by hemacytometer and NucleoCounter SP-100 and for sperm membrane integrity determined by flow cytometer and NucleoCounter SP-100 (P < .01). Finally, the standard curves of sperm concentrations in 6 spectrophotometers, comparing optical density against counts drawn by hemacytometer and NucleoCounter SP-100 (n = 94 fresh ejaculates) showed different (P < .01) intercepts and regression coefficients (linear, quadratic, cubic). It was calculated that a breeding station can improve its production potential by 13% with the use of NucleoCounter SP-100 instead of hemacytometer for calibration of spectrophotometers. Flow cytometer and NucleoCounter SP-100 can be used with equal confidence to estimate sperm concentration and membrane integrity in domestic animals and human semen.