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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]

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]

Abstract: The objective of this study was to determine the effect of in utero and lactational exposure to nicotine on the male reproductive tract. Dams were randomly assigned to receive saline or nicotine bitartrate (1mg/kg-d s.c.) daily for two weeks prior to mating until weaning (postnatal day 21). Male offspring were sacrificed at 7 (peri-pubertal) and 26 (adult) weeks of age. Nicotine-exposure resulted in retention of spermatids after stage VIII, tubular vacuolation, degeneration of pachytene and round spermatids at stage VII in the testes; and lymphocyte infiltration, germ cell exfoliation, and hypospermia in epididymides, at 7 weeks of age. Nicotine-exposure had no effect on testis or epididymal morphology, daily sperm production, epididymal sperm reserve, sperm viability at 26 weeks of age, and circulating testosterone levels at either age examined. We conclude that maternal nicotine-exposure during pregnancy and lactation can induce transient structural changes in the testis and epididymis of male offspring. [Copyright &y& Elsevier]

Background: A suitable culture system is important for follicle growth in adult bovine ovarian tissue. This study aimed to assess the avian chorioallantoic membrane (CAM) for short-term culture of adult bovine ovarian tissues compared with a traditional in vitro culture system. Methods: Ovarian cortical tissues (1–2 mm3), collected from slaughtered adult cows, were randomly assigned to control, CAM or in vitro culture groups. In the control group, ovarian tissues were fixed with paraformaldehyde without culture. In CAM and in vitro culture groups, the ovarian tissues were cultured for up to 5 days and then fixed. Ovarian tissues were examined on culture days 0, 1, 3 and 5 for angiogenesis, follicle morphology and growth. In all groups, primordial and growing (healthy and atretic) follicle densities were determined. Results: In the CAM culture, the avian blood vessel density increased (p < 0.01) over time with a decline (p < 0.001) in the bovine blood vessel density. Healthy primordial, atretic primordial and healthy growing follicle densities were higher (p < 0.05) in CAM-cultured ovarian tissues than in vitro-cultured tissues. Regardless of the culture system, the density of healthy primordial follicles decreased (p < 0.001) over time with an increase in healthy growing follicles on day 3 (p < 0.01) and an increase in atretic (primordial and growing) follicles during the 5-day culture period (p < 0.001). The proportions of healthy primordial and atretic growing follicles were also affected by culture day (p < 0.001). Conclusions: The CAM culture in chick embryos supported the bovine ovarian tissue grafts for 3 days demonstrating that CAM can be used as a satisfactory short-term culture system to assess ovarian tissue health, and to study follicle activation and development. [ABSTRACT FROM AUTHOR]

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]

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]

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 (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]

Experiments were done to compare the in vivo and in vitro maturational characteristics of cumulus-oocyte complexes (COC) collected from live wood bison. In Experiment 1 (anovulatory season), follicular ablation was done to synchronize follicle wave emergence among bison on Day −1, and FSH was given on Days 0 and 2. Bison were then assigned to 5 groups (n = 5/group) in which COC were collected by transvaginal follicle aspiration on Day 4 and either fixed immediately with no maturation (control), matured in vitro for 24 or 30 h, or collected on Day 5 after in vivo maturation for 24 or 30 h ( i.e ., after hCG treatment). In Experiment 2 (ovulatory season), bison were treated as described for Experiment 1, but PGF2α (cloprostenol) was given to control the luteal phase on Days −9 and 3. In both experiments, cumulus cell expansion was more extensive following in vivo than in vitro maturation, and the percentage of fully expanded COC was highest in the in vivo 30 h groups. Nuclear maturation occurred more rapidly in vitro ; 60–70% of oocytes were at the MII stage 24 h after in vitro maturation while only 25–27% of oocytes had reached the MII stage after 24 h of in vivo maturation. In conclusion, nuclear maturation occurred more rapidly during in vitro vs . in vivo maturation, but was associated with less cumulus expansion than in vivo maturation. In vivo oocyte maturation was more complete at 30 vs . 24 h after hCG treatment. Season had no effect on the maturational capacity of wood bison oocytes. [ABSTRACT FROM AUTHOR]

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. [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]

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. • Pregnancy rate up to 43% was achieved using protein-free frozen bison semen. • Bison calves were born following fixed-time AI with protein-free frozen semen. • Protein-free extender yielded acceptable post-thaw bison semen quality. • Cholesterol-cyclodextrin complex replaced egg yolk from semen extender. [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]

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]

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. • Antioxidants minimize the detrimental effect of cryo-injuries in bull sperm. • Semen with poor post-thaw motility had a better response to the antioxidants. • Motility and flow cytometric parameters were improved in low-cryotolerance semen. [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]

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 < 0.001) in the Vitrified group than in the Control, VS1 and VS1 + VS2 groups (40.9 and 1.6% vs 92.2 and 34.4%, 79.4 and 25.2%, and 80.2 and 20.8%, respectively in Experiment 1, and 25.0 and 1.7% vs 75.3 and 27.2%, 67.9 and 19.5%, and 62.7 and 22.5%, respectively in Experiment 2). 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. [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]

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 mm3) 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. [ABSTRACT FROM AUTHOR]