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There is considerable interest in breeding programs to "rescue" semen with poor post-thaw fertility from bulls known as "bad freezers". We hypothesized that there may be an interaction between the post-thaw recovery of sperm and the efficacy of antioxidant addition to extenders. The current study assesses the effects of antioxidant additives hydroxytyrosol (HT) and resveratrol (RSV) on the post-thaw semen parameters in two groups of bulls classified as either high or low cryotolerant (i.e., "good" and "bad" freezers). Semen samples were collected from 40 bulls and processed in the extenders containing different concentrations of HT (experiment 1; 0, 25 and 50 µM) and RSV (experiment 2; 0.0, 0.01, 0.1 and 1 mM). In experiment 1, bulls in the low cryotolerance group had a significant improvement in post-thaw recovery at 25 µM and 50 µM (P < 0.05). These improvements were observed in motility and several cellular parameters. However, post-thaw semen quality in the high cryotolerance group was not significantly affected by the HT addition. In experiment 2, although RSV did not have any positive impact in high cryotolerance group, post-thaw recovery in the low cryotolerance bulls was significantly improved in 0.1 mM RSV. Oxidative stress markers in either high or low cryotolerance groups were not significantly changed by RSV addition (P > 0.05). It can be concluded that addition of optimized concentrations of HT and RSV to the extender could be a strategy for improving the post-thaw semen, especially in bulls with high genetic merit but low initial cryotolerance., Competing Interests: Conflict of interest There is no conflict of interest to declare., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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.)

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 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 24h 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 (O2(•)) along with plasma membrane permeability and generation of hydrogen peroxide (H2O2) 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 O2(•-) generation and increased plasma membrane permeability, and became necrotic without H2O2 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 O2(•-), which increased the permeability and compromised the integrity of the plasma membrane.

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.)

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.

The present study was conducted to detect sperm apoptosis in fresh and frozen semen and to determine its relationship with bull fertility. Three ejaculates were collected from five breeding bulls with different fertility levels and were cryopreserved using standard methods. Two flow cytometric methods were employed to measure apoptosis: an assay for phosphatidylserine (PS) translocation across the plasma membranes using fluorescein-labeled Annexin V and propidium iodide (PI), and an assay for nicked DNA using bromodeoxyuridine (BrdU), terminal deoxynucleotidyl transferase, and fluorescein-labeled anti-BrdU monoclonal antibody. Both assays showed that fresh sperm contained 10%-20% apoptotic sperm. Significant differences in the percentage of apoptotic sperm were observed among the bulls. Cryopreservation induced translocation of PS to the outer leaflet of the plasma membrane and caused most of the necrotic cells in fresh sperm to disintegrate. Bull fertility was significantly related to the percentage of necrotic or viable sperm in fresh semen as detected by the Annexin V/PI assay, to the number of apoptotic sperm in fresh semen as detected by the TUNEL assay, and to the level of chromatin or DNA condensation as detected by PI staining. The present study suggests that the presence of apoptotic spermatozoa in fresh semen could be one of the reasons for poor fertility in breeding bulls.

Canadian standards allow ≤3000 μg ergot alkaloids/kg cattle feed. A concentration-response relationship was hypothesized between ergot in feed and reductions in plasma prolactin, sperm motility, sperm function, and increase in sperm abnormalities. The study consisted of pre-treatment (12 weeks), treatment (9 weeks), and post-treatment periods (10 weeks). Adult bulls were fed 1113 (n = 8; low ergot group) or 2227 (n = 6; high) μg/kg of dry matter intake. Endpoints were measured every two weeks. Ejaculates were analyzed for sperm concentration, total and progressive motility, plasma membrane and acrosome integrity, mitochondrial membrane potential and sperm abnormalities. Data were analyzed by repeated measures MIXED PROC in SAS. Average outside ambient temperature during the pre-treatment, treatment, and post-treatment periods was −13 (−31 to 1), 0.5 (−18 to 19), and 21 (13–28) °C. Plasma prolactin decreased markedly during treatment (−52.4%; Experimental period p < 0.01). Rectal temperature increased during the treatment and post-treatment periods (EP p < 0.01) but was within the normal physiological range. Bull weight increased during the study (EP p < 0.01). Scrotal circumference in low ergot group increased during treatment (+0.8 cm; Tx∗EP p = 0.05). Progressive motility in high ergot group decreased during treatment (−7%; Tx∗EP p = 0.05), however, semen volume and sperm concentrations were unaffected (p ≥ 0.11). Live sperm with high and medium MMP decreased during treatment (−1.4 and −3.7%; EP p < 0.01). Results suggest that feeding ≤2227 μg ergot alkaloids/kg has only minor effects on adult bull semen quality. • Ergot alkaloid feeding of up to 2227 μg/kg/day for 9 weeks transiently reduced plasma prolactin concentration in adult bulls. • Ergot alkaloid feeding has no effect on sperm concentration, total motility, or ejaculate volume. • Subtle effects on progressive motility and mitochondrial membrane potential were observed in bulls receiving 2227 μg/kg. • Breeding soundness in adult bulls is not expected to be affected at current Canadian standards. • Feeding ≤2227 μg ergot alkaloids/kg has only minor effects on adult bull semen quality. [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]

Our objective was to determine whether feeding yearling bulls with the higher recommended Canadian limit of ergot alkaloids (∼3 mg/kg dry matter intake, DMI) would affect sperm characteristics and plasma prolactin concentrations. Aberdeen Angus bulls (12–13 mo old, n = 7/group) allocated by blocking for sperm concentration and body weight, were fed placebo or ergot alkaloids in gelatin capsules (60 μg/kg body weight daily, 3.4 mg/kg of DMI) for 9 wk. Semen samples were collected weekly by electroejaculation and examined with a computer assisted semen analyzer (CASA) and flow cytometry, for the intervals 5 wk before (Pre-exposure period), 9 wk during (Exposure period) and 9 wk after (Post-exposure period) treatment. Weekly plasma samples were analyzed for prolactin by radioimmunoassay. Plasma prolactin concentrations decreased markedly (mean ± SEM, 16.74 ± 3.70 in Exposure and 33.42 ± 3.08 ng/mL in Post-Exposure periods; P < 0.01) compared to Control (67.54 ± 21.47 and 42.59 ± 15.06 ng/mL). Treatment did not affect (P ≥ 0.17) body weight gain, sperm concentration, sperm count/ejaculate, motility or percent live sperm. Averaged over the exposure and post-exposure durations, the scrotal circumference was smaller (P = 0.02) by 2.7% in the Ergot group. Progressive motility remained unchanged from 59.92 ± 2.31% in Exposure to 59.61 ± 2.59% in Post-Exposure periods, compared to marked increase in Control (61.42 ± 1.60% to 67.52 ± 1.47%; P = 0.02). Straight-line sperm velocity decreased (−3.15 ± 1.53 μm/s) from exposure to post-exposure periods in Ergot group (P = 0.04) versus an increase (2.96 ± 2.17 μm/s) in Control. Midpiece defects decreased from Exposure to Post-exposure periods in Control group but remained unchanged in Ergot group (trt∗age, P < 0.01). Ergot feeding resulted in a smaller proportion of sperm with medium mitochondrial potential (Ergot: 22.65 ± 0.98%, Control: 24.35 ± 1.05%, P = 0.04). In conclusion, feeding ergot at Canadian permissible limit for 9-wk resulted in a 4-fold decrease in plasma prolactin concentrations. Semen end points were not significantly affected, although there were subtle effects on progressive motility, midpiece defects and mitochondrial membrane potential. Clinical relevance of observed changes requires further evaluation. Results supported our hypothesis that prolonged low-level ergot will adversely affect plasma prolactin. However, semen parameters were partially affected, supporting similar work on fescue toxicosis. • Ergot alkaloid feeding at 3.4 mg/kg of DMI/day for 9 weeks of yearling bulls transiently reduced plasma prolactin by 4-folds. • Body weight, scrotal circumference, rectal temperature, or sperm concentrations were not remarkably affected. • Subtle effects on progressive motility, mid-piece defects, and mitochondrial membrane potential were recorded. • Current Canadian ergot standards (3 mg/kg of feed) are adequate for bull reproduction; prolactin decrease is concerning. [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]