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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., (Crown Copyright © 2021. Published by Elsevier B.V. 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.)

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.)

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.)

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.