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Successful cryopreservation of bison semen is fundamental for restoration of genetic diversity in Canada's wood bison. Conventional bovine semen extenders contain animal products, such as egg yolk and milk, which are undesirable because of biosecurity risks and undefined composition. In this study, we examined the efficacy of an exogenous protein-free extender containing cholesterol-cyclodextrin complex (CC) to cryopreserve bison semen. The study also provided an opportunity to determine the effectiveness of different ovulation synchronization protocols for fixed-time artificial insemination in bison. Semen was collected from wood bison bulls via electroejaculation and cryopreserved in either Tris-egg yolk-glycerol (called 'TEYG') extender or pretreated with CC (2 mg/mL semen) and diluted in Tris-glycerol (collectively called 'CC-TG') extender. Post-thaw sperm motion characteristics and in vitro fertilization of cattle oocytes confirmed that CC alone without egg yolk protected bison sperm during cryopreservation process. In the first fertility trial, however, no pregnancy was obtained following fixed-time artificial insemination of bison cows with CC-TG extender. In a follow-up trial, low concentration of CC (1 mg/mL semen) resulted in better post-thaw sperm motion characteristics, fertility rate, and birth of live calves following fixed-time artificial insemination. Results showed that 1 mg CC/mL semen completely replaced egg yolk in bison semen extender. In addition, both follicular ablation and steroid treatment protocols provided ovulation synchrony to permit successful application of fixed-time artificial insemination in bison. This is the first report on the birth of live bison calves following fixed-time artificial insemination using semen cryopreserved in a defined extender., Competing Interests: Declaration of competing interest The authors do not have a conflict of interest., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

The purpose of this study was to assess the viability and growth of follicles in ovarian tissues of cattle vitrified using two non-permeating cryoprotectants (sucrose and trehalose) and two cryodevices (cryovial and cryotop). Cortical slices (1-2 mm 3 ) from cattle ovaries (n = 5) were assigned to one of the 14 treatment groups. Cortical slices were vitrified in a TCM199 medium supplemented with ethylene glycol, DMSO, calf serum and either 0.5 M sucrose or trehalose, in cryovials or on cryotops. After warming, cortical slices were either fixed immediately for histology or grafted on a chorioallantoic membrane (CAM) of 10-day old chick embryos. Angiogenesis in ovarian tissues was determined. Viable and atretic preantral (primordial, primary and secondary) follicle densities were examined histologically. There was angiogenesis (chicken) in cortical slices grafted on the CAM by day 5 of culture, however, there was no difference for blood vessel densities when there was use of non-permeating cryoprotectants or cryodevices. Total, viable and atretic follicle densities did not differ (P > 0.05) with use of non-permeating cryoprotectants or cryodevices. The proportion of viable follicles was greater (P < 0.001) in fresh-control than CAM culture-control or vitrification groups. The inclusion of sucrose in the vitrification solution resulted in a larger number of atretic follicles than in the fresh-control group (P < 0.05). In summary, sucrose and trehalose, and cryotop and cryovial were equally suitable for vitrification of ovarian tissues of cattle. Vitrification of ovarian tissues of cattle with subsequent use of CAM culture adversely affected follicular development., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

Embryo cryopreservation is a major tool for conservation and propagation of genetically superior animals. However, it adversely affects the survival of embryos. The objective of this study was to determine the effects of cryopreservation technique (vitrification compared with slow freezing) and different seasons in which oocytes were obtained on the post-warming survival of in vitro produced (IVP) cattle morulae. In experiment 1, morulae (Day 6 post-IVF), obtained from abattoir-sourced oocytes during spring, summer, fall and winter over a period of 3.5 years, were subjected to either vitrification (n=271 morulae), slow freezing (n=281 morulae) or no freezing (control; n=249 morulae). After warming, the morulae were cultured to the expanded blastocyst stage (Day 8 post-IVF). Data were compared using Glimmix procedure in SAS(®). Blastocyst rate differed (P<0.05) among the treatments: unfrozen control (78±3.6%), vitrification (52±4.6%) and slow freezing (35±4.2%). The re-expansion of vitrified morulae upon warming was not correlated with subsequent blastocyst rate (r=-0.048; P>0.05). The morulae produced during fall season had lesser (P<0.05) cleavage and morula rates (67±1.6%; Day 2 post-IVF and 22±1.4%; Day 6 post-IVF, respectively) than all other seasons (74±1.1 and 30±1.2%, respectively). Blastocyst rate was the least (P<0.05) when oocytes were collected during the summer season in both control and slowly frozen groups. Blastocyst development rate did not change due to season in vitrification group (P>0.05). In conclusion, vitrification is a more desirable technique than slow freezing for cryopreservation of IVP cattle morulae. If the slow freezing method is employed, greater success can be achieved using oocytes collected in the winter and spring with a primary contributing factor being lesser morulae development if oocytes are collected in the fall and also the lesser blastocyst formation of cryopreserved morulae when oocytes are collected in the summer., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.

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 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]

Our objectives were to determine if feeding mature and yearling Angus bulls ergot alkaloids (from Claviceps purpurea) within the Canadian permissible limit (∼3 mg/kg) affect post-thaw sperm quality. In Experiment 1, mature Angus bulls were group-fed ergot alkaloids (∼1 and ∼2 mg/kg of daily dry matter intake, DMI; n = 8 and n = 6 bulls, respectively) for 61 d; semen was collected and cryopreserved bi-weekly, from 12 wk pre-exposure to 10 wk post-exposure. In Experiment 2, yearling Angus bulls (12–13 mo) were individually fed placebo or ergot alkaloids (3.4 mg/kg of DMI; n = 7 bulls/group) daily for 9 wk, with semen collected and cryopreserved once weekly, from 5 wk before to 9 wk after exposure. All frozen semen was assessed 0 and 2 h post-thaw. In Experiment 1, post-thaw total and progressive sperm motilities decreased (P ≤ 0.05) from pre-exposure to exposure period, then returned to pre-exposure level. Likewise, during exposure, VAP and VSL decreased (P ≤ 0.01) at 0 h compared to pre-exposure and subsequently returned. Live sperm with intact acrosomes at 2 h post-thaw was affected by ergot (P = 0.01). Medium mitochondrial membrane potential increased (P ≤ 0.01) during exposure compared to pre-exposure and subsequently decreased. In Experiment 2, total and progressive sperm motilities at 0 and 2 h increased (P ≤ 0.01) throughout the study. During post-exposure, VCL, VAP and VSL at 0 h increased (P ≤ 0.01) whereas VSL at 2 h increased (P ≤ 0.01) from pre-exposure to exposure to post-exposure. Live sperm with intact acrosomes increased (P ≤ 0.01) at both 0 and 2 h during post-exposure. Medium mitochondrial membrane potential increased (P ≤ 0.01) from pre-exposure to exposure, followed by a slight decrease post-exposure. Mature Angus bulls partially supported our hypothesis, with only transient effects of ergot on sperm motilities and velocities. Post-thaw sperm characteristics in yearling bulls underwent expected age-related improvements, with any effects of ergot alkaloids potentially masked by sexual maturation. Overall, results partially supported our hypotheses that ergot has no detectable adverse effect on post-thaw sperm characteristics in mature and yearling bulls. • Low-dose ergot had transient effects on post-thaw sperm parameters in mature bulls. • Ergot effect on post-thaw sperm parameters in yearling bulls was masked due to age. • Several post-thaw sperm characters were affected during ergot exposure period. • Current Canadian limits of ergot in feed are safe for post-thaw bull semen quality. [ABSTRACT FROM AUTHOR]

• Cholesterol-cyclodextrin was a suitable alternative for egg yolk in bovine semen extender. • High concentrations of exogenous cholesterol (≥ 2 mg CC/mL) adversely impacted fertility. • Pregnancy rate up to 60 % was achieved following fixed-time artificial insemination. Egg yolk, a major semen extender constituent, lacks a defined composition, therefore, there are biosecurity concerns with use of egg yolk. Cryopreservation of bull semen without inclusion of animal protein in the semen extender, therefore, is an important consideration. Cholesterol may be delivered and incorporated into the sperm plasma membrane by cyclodextrins to protect sperm during cryopreservation. The aim of this study was to determine suitability of a cholesterol-cyclodextrin semen extender, without inclusion of egg yolk, for cryopreservation of bull semen. Bull semen was collected and cryopreserved in either egg yolk or with inclusions of three different concentrations of cholesterol-cyclodextrin complex (0.5, 1 or 2 mg/mL semen) in Tris-glycerol (TG) extender. Sperm motion characteristics examined using the computer-assisted sperm analysis, and plasma membrane and acrosome integrity examined using flow cytometry, were similar for all extenders. The inclusion of the greatest concentration of cholesterol-cyclodextrin complex (2 mg/mL semen) followed by dilution in TG extender resulted in lesser pregnancy rates (P < 0.05). There was a pregnancy rate of as great as 56 % when sperm cryopreserved in 0.5 mg/mL cholesterol-cyclodextrin Tris-glycerol extender were used for artificial insemination following imposing of a hormonal treatment regimen for synchrony of timing of ovarian functions among cows for conducting fixed-time artificial insemination (FTAI). Results indicate cholesterol-cyclodextrin Tris-glycerol extender, with a chemically defined composition and without inclusion of egg yolk, may be used to cryopreserve bull sperm with there being acceptable pregnancy rates when this semen is used for FTAI. [ABSTRACT FROM AUTHOR]