Embryos produced in vitro from bulls carrying 16;20 and 1;29 Robertsonian translocations: efficiency and kinetics of oocyte fertilization and embryo development

The present experiments were designed to study the effects of Robertsonian translocations on the efficiency and kinetics of in vitro fertilization and early and advanced embryo development. Spermatozoa from bulls with rob(16;20), rob(1;29) and normal karyotype (A, B and C, respectively) were used. Oocytes were matured, fertilized and cultured by the standard protocol described previously. Twenty-four hours after fertilization, adequate numbers of oocytes were fixed, stained and examined. The development of embryos was evaluated on days 2 (D2), 7 (D7) and 8 (D8) after fertilization. The rate of normally fertilized oocytes was significantly lower (p < or = 0.01) for bull A than for bulls B and C. However, no significant differences in the kinetics of fertilization were found between bulls A, B and C. The D2 cleavage rate of embryos was significantly lower (p < or = 0.01) for bull A than for bulls B and C. Both D7 and D8 blastocyst rates for bull A or bull B were significantly lower (p < or = 0.01 or p < or = 0.05) than those for bull C. The percentages of both D7 advanced blastocysts and D8 expanded blastocysts were significantly lower (p < or = 0.01) for bulls A and B than for bull C. In conclusion, for rob(16;20), the efficiency of fertilization was strongly reduced; it resulted in low early and advanced embryo development. On the other hand, for the rob(1;29), neither fertilization nor early embryo development were affected and only advanced embryo development was decreased. But for both translocations, blastocyst formation was significantly delayed.     

Fecal androgens of bison bulls during the rut

The influence of sex hormones is a key proximate factor underlying male reproductive behavior in mammals. Effective conservation policies for the remaining purebred plains bison (Bison bison bison) herds require knowledge of the physiology underlying bison reproductive biology. We used fecal steroid analysis to characterize androgen levels in adult bison bulls before, during, and after the rut, and to examine androgen levels of bulls differing in reproductive status, age, and mating success. Fieldwork was carried out at the Fort Niobrara National Wildlife Refuge in north-central Nebraska. All adult bison in the herd were individually known by unique brands. Fecal samples were collected during 2003 from bulls during pre-rut (June), rut (July-August), and post-rut (September), and behavioral observations focused on reproductive status and mating success during the rut. Matched sample data indicated that androgen levels (ng/g feces) of bulls peaked during the rut, doubling from pre-rut to rut and then declining by 75% during post-rut. Dominant bulls that tended (guarded) cows maintained higher androgen levels than bulls that were not tending. There was a positive correlation between bull age (associated with mating success) and androgens, with higher androgen levels in prime-aged bulls compared with younger bulls. Nonetheless, there was no correlation between mating success (measured by number of copulations observed) and androgen level. This suggests that while androgens may provide the proximate motivation to compete for matings, other factors determine the mating success of bison bulls.     

Cortisol and prolactin concentrations during three different seasons in relocated Brahman and Hereford bulls

The objective of this study was to evaluate seasonal changes of cortisol and prolactin (PRL) concentrations in Brahman and Hereford bulls moved to locations that differ in geographical and environmental conditions. Postpubertal Hereford bulls from Montana (n = 15) and Nebraska (n = 15) and Brahman bulls from Texas (n = 18) were located in or relocated to Montana, Nebraska or Texas so that each location had 5 Montana Herefords, 5 Nebraska Herefords and 6 Texas Brahman bulls. Blood samples were collected at 20-minute intervals for 8 hours in November (Fall 1), April (Spring) and November (Fall 2) of the next year. These dates corresponded to 6, 12 and 18 months, respectively, after relocation in May of the first year. Cortisol concentrations were higher (P<0.05) in Fall 1 than in Fall 2 and were higher (P<0.05) for bulls in Montana than for bulls in Texas. The decrease in cortisol concentrations from Fall 1 to Fall 2 was negatively related (P<0.05) to age and weight. There was a three-way interaction (P<0.05) of breed-type origin, location and season for PRL concentrations. Seasonal patterns of PRL concentrations differed between relocated Texas Brahman and Hereford bulls, and patterns for relocated bulls differed from those of the nonrelocated bulls. Seasonal patterns of PRL were influenced to a greater extent by relocation in Texas Brahman bulls than in Hereford bulls.     

In vitro assessment of sperm from bulls of high and low field fertility

The aim of this study was to investigate the reasons for differences in field fertility of bulls following insemination with frozen-thawed semen. The study was carried out in two separate parts over two years and comparisons were made between 5 high and 4 low fertility Holstein Friesian bulls as determined by their either 90 day non-return rate (Year 1) or calving rate (Year 2). Two high fertility Limousin bulls were included in Year 1 for comparative purposes. The ability of sperm from each bull to penetrate artificial mucus was assessed (Year 1 = 7 replicates; Year 2 = 5 replicates). Glass capillary tubes (2 per bull per replicate) were filled with artificial mucus and incubated with sperm stained in 1% Hoechst 33342 for 30 min at 37 °C. The number of sperm were subsequently counted at 10 mm intervals along the tube between 40 and 80 mm markers. Sperm mitochondrial activity of each bull was assessed by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay (4 replicates in each year). Sperm were incubated with MTT for 1 h at 37 °C following which the absorbance of formazan was read using a spectrophotometer. Sperm viability after thawing was assessed for each bull using a live/dead sperm viability kit (Year 1 = 3 replicates; Year 2 = 4 replicates). A minimum of 250 cells were assessed per bull in each replicate and classified as either live or dead. Finally, the ability of sperm to fertilize oocytes in vitro and their ability to develop to blastocyst stage embryos were assessed (5 replicates in each year involving 220 to 306 oocytes per bull). Data transformation to normalize residuals was required for mucus sperm penetration (square root) and IVF (cleavage and blastocyst rate) results (arcsin). The mean number of sperm counted at each 10 mm mark between 40 and 80 mm was higher in the high fertility (56.0; 95% CI 39.5 to 75.3) compared to the low fertility (42.9; 95% CI 29.3 to 59.1) Holstein Friesian bulls but the difference did not reach formal significance (P = 0.09). Fertility status had no effect on the ability of sperm to reduce MTT to formazan (mean absorbance 0.34 ± 0.051 and 0.30 ± 0.044) or on the percentage of live sperm per straw (mean 47.3 ± 5.47 and 32.4 ± 4.66) for high and low fertility Holstein Friesian bulls respectively. Oocyte cleavage rate following insemination with sperm from high fertility Holstein Friesian bulls was significantly higher than with sperm from low fertility Holstein Friesian bulls [76.7% (95% CI 60.9 to 89.4) and 55.3 (95% CI 40.4 to 69.7) respectively, P = 0.04]. There was no significant effect of bull fertility on blastocyst rate [34.7% (95% CI 21.1 to 49.6) and 24.2 % (95% CI 14.1 to 36.0) for the high and low fertility Holstein Friesian bulls, respectively; P = 0.2]. In conclusion, sperm from high fertility bulls tended to be more effective in penetrating artificial mucus and to have an increased ability to fertilize oocytes in vitro; however, once fertilization occurred subsequent embryo development was not significantly affected by fertility status.     

Epidemiology of Tritrichomonas foetus in beef bull populations in Florida

The objectives of this study were to estimate the prevalence of herd and individual bull infection with Tritrichomonas foetus in a survey of beef bulls in the state of Florida and to perform an epidemiological investigation of risk factors for the disease. Bulls were tested for T. foetus colonization by a single preputial scraping and culture. Bull infection prevalence within herds was calculated and relationships with bull, herd factors, and production measurements were determined. The survey included 1984 beef bulls in 59 herds throughout Florida; nine bulls in three small herds (<100 cows) were later excluded from the models. An overall prevalence for T. foetus-infected bulls was 6.0% (within-herd prevalence ranged from 0 to 27%). The herd prevalence was 30.4% (i.e. at least one infected bull); infected bulls were found in 11.1 and 39.5% of herds sampled in North and South Florida, respectively. The likelihood of disease was greatest in larger herds in more extensive management settings (> or = 500 cows, 53.9% prevalence; medium-sized herds of 100-499 cows, 10.0% prevalence). Tritrichomonas foetus infection was associated with several bull factors, including age, breed, herd, and herd management practices (bull-to-cow ratio, bulls per breeding group). Tritrichomonas foetus infection continues to be prevalent in beef herds in Florida that use natural service.     

Embryos produced in vitro from bulls carrying 16;20 and 1;29 Robertsonian translocations: detection of translocations in embryos by fluorescence in situ hybridization

Robertsonian translocation rob(16;20) in the heterozygous state was discovered in a subfertile bull of the Czech Siemmental breed. A chromosomal analysis of its family has shown that this dicentric fusion is formed de novo. The present experiments were designed to detect rob(16;20) and determine its incidence for in vitro produced embryos, using fluorescence in situ hybridization (FISH) and rob(1;29) as a detection control. To characterize semen of both bulls with the rob translocations, their sperm was examined for DNA integrity by the sperm chromatin structure assay (SCSA). For in vitro fertilization of oocytes, spermatozoa from a rob(16;20) bull carrier (Czech Siemmental breed) and those from a rob(1;29) bull carrier (Charolais breed) were used. Embryos at the 6- to 8-cell stage were cultured in a vinblastine-supplemented medium for 17 h, and embryos at the blastocyst stage were cultured in a colcemide-supplemented medium for 4 h. The embryos were fixed in methanol and acetic acid with Tween-20. Painting probes for chromosomes 16 (Spectrum Green) and 20 (Spectrum Orange) and chromosomes 1 (Spectrum Orange) and 29 (Spectrum Green) were simultaneously hybridized. In the embryos derived from the rob(16;20) bull, the presence of this translocation was not detected. On the other hand, 52.5% of the embryos derived from the rob(1;29) bull were translocation carriers. There was no significant difference in the frequency of this translocation between early and advanced embryos.     

Effect of reducing sperm concentration during IVF on the ability to distinguish between bulls of high and low field fertility: work in progress

The objectives of this study were to evaluate the effect of sperm dose and sire on the fertilization rate, cleavage rate and blastocyst yield following insemination in vitro, to examine the relationship between these parameters and field fertility in cattle, and to examine the relationship between blastocyst quality and sire used in IVF. Frozen semen from four bulls with 150-day nonreturn rates ranging from 57 to 78% was used. In Experiment 1, oocytes were inseminated with sperm from one of the four bulls at concentrations ranging from 0.016 to 0.5 x 10(6)sperm/ml. A proportion of presumptive zygotes were fixed at 17 h post-insemination (hpi), while the remainder was transferred to in vitro culture (IVC) in droplets of synthetic oviduct fluid (SOF). Cleavage at 48 hpi and the percentage of oocytes reaching the blastocyst stage by Day 8 were recorded. In Experiment 2, to assess blastocyst quality, after insemination with semen from one of the four bulls, presumptive zygotes were cultured in SOF until Day 7. Blastocysts for each bull were removed and vitrified/warmed and survival was recorded at 24, 48 and 72 h after warming. Regardless of bull used, a concentration of 0.125 x 10(6)sperm/ml or above resulted in higher blastocyst yields than any lower concentration used. Fertilization and cleavage rates were also higher at higher sperm concentrations. The best predictor of field fertility was fertilization rate at a concentration of 0.5 x 10(6)sperm/ml (r=0.94, P<0.0001). There was also a significant correlation between cleavage rate at a concentration of 0.5 x 10(6)sperm/ml and nonreturn rate (r=0.90, P<0.0001). In Experiment 2, blastocysts derived from one bull, HTA, were of superior quality as measured by survival 24h after thawing, although these differences were less significant at the subsequent time points measured. In conclusion, these data show that differences between the field fertility of bulls can be determined at sperm concentrations routinely used in IVF. Lowering the sperm concentration does not increase the likelihood of optimizing the differences in fertility or cleavage rate between bulls of different field fertility. We have also demonstrated that the bull can have a significant effect on the quality of blastocysts produced using IVF techniques.     

The use of in vitro fertilization techniques to investigate the fertilizing ability of bovine sperm with proximal cytoplasmic droplets

The objective of this experiment was to determine the effect of proximal droplets on sperm-oocyte binding, zona penetration, fertilization, and the developmental competence of oocytes fertilized by sperm with proximal droplets (PD) in an in vitro fertilization (IVF) and culture system. Frozen semen from three bulls (PD1, PD2 and PD3) with varying proportions of normal appearing sperm with proximal droplets and semen from a normal control bull (C) were used in this experiment. The mean number of sperm bound to the zona pellucida (26.8 +/- 2.0, n=100; 15.2 +/- 1.1, n=100; 16.2 +/- 1.0, n=100) for bulls PD1, PD2, and PD3, respectively, were significantly lower (P<0.05) than that of the control bull C (47.4+/- 1.9; n=114). No spermatozoa with PD were found bound to the zona pellucida and this finding was consistent among the three bulls. The percentage penetration of zonae for the bulls PD1, PD2 and PD3 (74%, 74/100; 71%, 71/100 and 69%, 69/100, respectively) were not different than that of bull C (72%, 179/245). Similarly, the mean number of sperm penetrating the zona pellucida (1.43+/- 1.2, 1.24 +/- 1.1 and 1.20 +/- 1.1, for bulls PD1, PD2 and PD3, respectively) were not different than that of bull C (1.45 +/- 1.1). However, fertilization rates (8.8%, 8/90; 16.8%, 16/95; and 10.6%, 11/103, for bulls PD1, PD2 and PD3, respectively) were lower (P<0.001) than that of bull C (68.7%; 77/112). Similarly, cleavage rates (5%, 10/200; 8%, 8/100 and 14%, 15/111) for the bulls PD1, PD2 and PD3, respectively, were lower than that of the control bull, C (60.7%; 79/130). Cleaved zygotes resulting from the fertilization of oocytes by apparently normal sperm from bulls with PD did not develop beyond cleavage, whereas, 43.8% (57/130) morulae and 20% (26/130) blastocysts were produced by oocytes fertilized by sperm from bull C. In summary, normal appearing sperm with PD did not bind to the zona pellucida. Apparently normal sperm with out proximal droplets co-existing in the semen along with sperm containing PD were also functionally deficient, resulting in reduced zonae binding and zygotes resulting from insemination with semen with a high percentage of PD did not develop beyond cleavage.     

Weekly bull exchange shortens postpartum anestrus in suckled beef cows

The duration of anestrus in cattle was usually shortened when cows were exposed to bulls. The objective of the present experiment was to determine if weekly bull exchange accelerated the resumption of cyclicity in postpartum suckled beef cows. We tested the hypothesis that exposure of postpartum, anestrous, suckled beef cows (extensively managed) to weekly exchange of bulls, accelerates the resumption of cyclicity, compared to continuous exposure to the same bulls. Ninety-one multiparous suckled Hereford and Hereford x Angus cows, <60d postpartum, were assigned to two homogeneous groups. Beginning on December 1st (late spring), the control group (C, n=45) remained with one pair of bulls throughout the breeding period (7 weeks), whereas in the "exchanged" (E, n=46) group two pairs of bulls were exchanged weekly. Based on weekly ultrasonographic examinations of all cows, none had a CL at the start of the experiment and for 2 weeks after the start of bull exposure. However, the accumulated frequency of cows with a CL was greater in group E than in group C cows on week 4 (P=0.024), as well as on weeks 5-7 (P<0.001) after the start of bull exposure. Furthermore, in group E versus group C, there was a higher pregnancy rate 30d after the end of bull exposure (26 of 46, 56.2% vs. 16 of 45, 35.6%; P=0.045). In conclusion, weekly exchange of two pairs of bulls shortened postpartum anestrus in suckled multiparous cows, compared to continuous exposure to a single pair of bulls.     

Sperm nuclei analysis of 1/29 Robertsonian translocation carrier bulls using fluorescence in situ hybridization

In 1964, Gustavsson and Rockborn first described the 1/29 Robertsonian translocation in cattle. Since then, several studies have demonstrated the negative effect of this particular chromosomal rearrangement on the fertility of carrier animals. During the last decade, meiotic segregation patterns have been studied on human males carrying balanced translocations using FISH on decondensed sperm nuclei. In this work, we have applied the 'Sperm-FISH' technique to determine the chromosomal content of spermatozoa from two bulls heterozygous for the 1/29 translocation and one normal bull (control). 5425 and 2702 sperm nuclei were scored, respectively, for the two heterozygous bulls, using whole chromosome painting probes of chromosomes 1 and 29. Very similar proportions of normal (or balanced) spermatozoa resulting from alternate segregation were observed (97.42% and 96.78%). For both heterozygous bulls, the proportions of nullisomic and disomic spermatozoa did not follow the theoretical 1:1 ratio. Indeed, proportions of nullisomic spermatozoa were higher than those of disomic sperma tozoa (1.40% vs 0.09% (bull 1) and 1.29% vs 0.15% (bull 2) for BTA1, and 0.65% vs 0.40% (bull 1) and 1.11% vs 0.63% (bull 2) for BTA29). The average frequencies of disomic and diploid spermatozoa in the normal bull were 0.11% and 0.05%, respectively.     

Paternity Tests in Multisired Beef Herds by Blood Grouping

The number of offspring sired by individual bulls in multisired beef herds was determined by blood grouping. Six series of tests, each having from 3 to 18 bulls as possible sires,, yielded from 35 to 114 dam-offspring pairs. The paternity was established for 45 to 90 % of the calves tested in each series. The rest were cases with either more than 1 bull not being excluded as the possible sire, or all bulls excluded from being the sire. The causes for the non-established paternities are discussed. Highly significant differences were found in the number of offspring sired by each bull. In each series, bulls ranking in the top third of the siring order sired 65–100 % of the calves with established paternities. Since a minority of the bulls sired the majority of the calf-crop, the generally recomended number of bulls for herds on the range can perhaps be reduced.     

Effects of bull exposure on the cyclic activity of beef cows

In Experiment I, 38 crossbred suckled beef cows grazing fescue pastures and 34 crossbred beef cows grazing bluestem pastures were randomly allocated at the time of calving into a group with 4 teaser bulls or no bulls. Two blood samples were collected 7 d apart from the cows to determine cyclic activity 67 and 76 d after calving in the fescue and bluestem pastures, respectively. Progesterone greater than 1.0 ng/ml in one or both samples indicated cyclic activity. There was no difference in the percentage of cows cyclic among the different groups. The number of cyclic cows in the fescue pasture with bulls was 16/19 (84%); in the fescue pasture with no bulls, 14/19 (74%); in the bluestem pasture with bulls, 17/17 (100%); and in the bluestem pasture with no bulls, 16/17 (94%). Overall cyclic activity among all cows for teaser bull-exposed and no bull was similar, 33/36 (91%) and 30/36 (83%). Overall cyclic activity was greater (P < 0.05) in cows grazing bluestem (33/34), 97% than fescue pastures (30/38), 80%. Measurements of cyclic activity were initiated too late in the postcalving period to quantify differences in estrous activity between the bull and no bull treatment groups. Another trial was planned for the following year with a modified protocol. In Experiment II, blood samples were collected for progesterone concentrations soon after calving and were repeated at intervals to characterize both the occurrence and duration of estrous cycles. In this experiment, 29 crossbred suckled beef cows grazing fescue pastures were randomly allocated 12 d after calving (Day 0) into 1 of 2 groups with teaser bulls or without bulls. Nineteen crossbred beef cows grazing bluestem pastures were allocated similarly 10 d after calving (Day 0). Bulls were added to the groups with bulls in fescue and bluestem pastures on day 6 after the initial allocations. Blood samples were collected from all cows on Day 0 and every 3 d until Day 46. Means (+/- SEM) of the cumulative progesterone concentrations (ng/ml) per cow for the 16 samples from cows grazing fescue were 12.5 +/- 3.5 for cows exposed to bulls, 2.5 +/- 0.16 for cows not exposed to bulls, 27.6 +/- 4.42 for cows grazing bluestem pastures and exposed to bulls, and 16.0 +/- 2.75 for cows without exposure to bulls. Progesterone concentrations were higher in cows exposed to bulls (P < 0.01). The percentages of both short and normal cycles increased (P < 0.01) in groups exposed to bulls (88%, 21/24 and 63%, 15/24) when compared with the no bull groups (29%, 7/24 and 21%, 5/24), respectively. Cows exposed to bulls also showed increased cyclic activity.     

The prevalence of chlamydiae of bulls from six bull studs in Germany

Although there are indications for venereal transmission of chlamydiae in cattle, epidemiological data on the presence of these bacteria in bulls and bull semen in particular is still incomplete. We investigated semen (n=120), preputial washing samples (n=121) and faeces (n=122) of bulls from six bull studs located within five Federal States of Germany for the presence of chlamydiae using omp1-PCR and partial omp1 sequencing. Blood serum was examined for chlamydial antibodies using an indirect ELISA (n=122). Chlamydiae were found in 11 (9.2%), 13 (10.7%) and 22 (18.0%) of the semen, preputial washing and faecal samples, respectively. Among individual chlamydial species identified, Chlamydophila (Cp.) psittaci predominated in semen and preputial washing samples, and Cp. pecorum in faeces. Cp. abortus was the third frequently observed species. Chlamydial antibodies were detected in a total of 62 (50.8%) bulls. Bull studs differed in regard to the number of bulls found chlamydia-positive in faeces and serologically positive. No correlation was observed between serological data and PCR of semen, preputial washing samples or faeces. Standard ejaculate parameters did not differ between bulls that were chlamydia-positive and -negative in semen. In conclusion, detection of chlamydiae in semen of bulls suggests a potential for venereal transmission. Chlamydiae appear to be widespread within the bull population in Germany. Serological testing failed to identify bulls shedding chlamydiae in their semen.     

Binding of the anti-human sperm monoclonal antibody HS-11 to bull spermatozoa is correlated with fertility in vitro

The present study aimed to determine if there is bull to bull variation in the binding of the anti-human sperm monoclonal antibody (MAb) HS-11 to bull spermatozoa, and to investigate if there is any correlation between HS-11 binding to spermatozoa and in vitro fertility of the bulls tested. Semen samples of a single collection (split frozen in 0.5-ml straws) from 8 dairy bulls were used. Swim-up separated motile spermatozoa were incubated in 90-microl drops of capacitation medium (TALP+10 microg/ml heparin) at 39 degrees C, 5% CO2, 95% air. At 0, 2, 4 and 6 h of incubation HS-11 was added (1:1000 final concentration), and the MAb binding was assessed by indirect immunofluorescence assay (IIFA). The HS-11 binding was indicated by a bright green fluorescence of the sperm acrosome region. In vitro-matured, good quality bovine oocytes were randomly allocated to spermatozoa of each bull for in vitro fertilization. Sperm samples of 2 to 3 bulls were used in each trial until 4 replicates per bull were attained for IVF (n approximately 100 oocytes/bull) and IIFA experiments. Sperm capacitation status was assessed simultaneously using an egg yolk lysophosphatidylcholine- (LC) induced acrosome reaction assay. The binding of HS-11 to spermatozoa was maximum at 4 h of incubation in most (6/8) of the bull semen samples. Significant (P < 0.01) differences were observed between bulls in the binding of HS-11 to their spermatozoa (range 22 +/- 8 to 52 +/- 5%) at 4 h, but not within replicates. Similarly, variations (P < 0.05) in the cleavage rate were also seen (range 22 +/- 9 to 58 +/- 7%) between bulls. The HS-11 binding and cleavage were significantly correlated (r = 0.43; n = 32; P < 0.05). The highest percentage of spermatozoa underwent acrosome reaction in response to LC treatment at the 4-h incubation period. This and the linear relationship between HS-11 binding and the cleavage rate observed in the present study together strengthen our earlier suggestion that the binding of the monoclonal antibody HS-11 to bull spermatozoa on a time-dependent manner, may indicate capacitation changes. We conclude that 1) between-bull differences exist in HS-11 binding to spermatozoa, and in the cleavage rate, and 2) HS-11 binding to spermatozoa is correlated with fertility, as determined by the cleavage of bovine oocytes matured and fertilized in vitro.     

Kinetics of fertilization and development, and sex ratio of bovine embryos produced using the semen of different bulls

The between bulls variation in in vitro fertility and the shift of sex ratio towards male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the kinetics of fertilization, embryo development and the sex ratio of the resulting embryos using the frozen/thawed semen of four different bulls. In a first experiment, the kinetics of pronucleus (PN) formation was evaluated at 8, 12 and 18 h post-insemination (hpi). Based upon the pronuclei sizes and the distance between the two pronuclei, inseminated oocytes were classified in three PN stages. Differences between bulls were observed at each time point, but were more important at 12 hpi. At 8 and 12 hpi bull III showed a significantly faster PN evolution by comparison with the three other bulls (P<0.05), while at 18 hpi, the proportion of the three PN stages was similar to those of bulls I and IV, bull II being delayed. In a second experiment, the kinetics of in vitro embryo development was compared using time-lapse cinematography. The analysis of embryos reaching the blastocyst stage revealed significant differences in the mean time of first cleavage (range of 22.7-25.6h, P<0.05), while the lengths of the subsequent three cell cycles did not differ between bulls. The early mean time of first cleavage with bull III was associated with an early blastulation and a high blastocyst rate at Day 7, in opposition to what was observed with bull II showing a later timing of first cleavage (first cleavage 22.1 hpi versus 25.5 hpi; blastulation 140.4 hpi versus 152.5 hpi; D7 blastocyst rates: 31.3% versus 21.9%; P<0.05). In a third experiment, 65-76 Day 8 blastocysts per bull were sexed by PCR. Only blastocysts obtained with bull III showed a shift in sex ratio towards male embryos (76% male embryos; P<0.05). Such shift was already observed at the 2-cell and morula stages. In conclusion, the bull influences the kinetics of PN formation, of embryo development and the sex ratio of the embryos. Moreover, those parameters might be related.     

Prediction of fertility of bovine semen: Preliminary studies with the hamster egg penetration test

The ability of liposome-treated fresh and frozen spermatozoa from two bulls to interact with zona-free hamster oocytes was examined to show whether the in vitro test results would correspond with in vivo fertility as indicated by the 60 to 90 d nonreturn to service rates which, using frozen semen, were 77 and 59%, respectively. The motility of spermatozoa in washed suspensions was also rated. Hamster test results were obtained using three ejaculates from each bull both as fresh and frozen semen. The results with frozen semen corresponded with fertility. The averages of three hamster tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte comparing spermatozoa from the bull with the higher fertility with spermatozoa from the bull with the lower fertility were 91% and 2.7 versus 56% and 1.4, respectively. Spermatozoa washed from frozen semen from the bull with the higher fertility interacted with hamster oocytes at the higher rate even when sperm motility was rated the same for both bulls. By contrast, fresh spermatozoa from the lower fertility bull interacted with hamster oocytes at a higher rate than spermatozoa from the higher fertility bull in six tests, comparing six ejaculates of fresh semen from both bulls. Comparing the higher fertility bull with the lower fertility bull, the average of six tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte were 60% and 1.6 versus 89% and 3.0, respectively. This suggests that this hamster test cannot be used with fresh semen to predict relative levels of fertility of frozen semen. Also, the subjective rating of sperm motility did not correspond with the in vitro oocyte penetrating ability of the spermatozoa.     

Repeatability of reproductive performance of beef bulls as yearlings and two-year-olds at pasture

Bull fertility, measured as pregnancy rate, percentages of calves born during the first 4 and 6 weeks of calving and means and medians of calving date distributions, was studied in 62 single-sire breeding herds of three breed groups over a 10-year period. Each bull was used as a yearling and then again as a 2-year-old. Average bull-to-cow ratio was approximately 1:20, and the breeding season started on 1 July and extended for approximately 60 days. The average pregnancy rate of cows mated to yearling bulls was similar to that for 2-year-old bulls (83.9% vs. 82.5%, respectively). There was a tendency towards earlier breeding among 2-year-old compared to yearling bulls as shown by 6.8% more (P = 0.09) calves born during the first 4 weeks of calving and 2.5 days advancement (P = 0.10) in the median of calving date distribution. Year and breed group of bull had no significant effect on the measures of bull fertility. The repeatability estimates of the measures of bull fertility were low, indicating that the reproductive performance of a 2-year-old bull cannot be accurately predicted from his performance as a yearling.     

Dilauroylphosphatidylcholine liposome effects on the acrosome reaction and in vitro penetration of zona-free hamster eggs by bull sperm: I. A fertility assay for fresh semen

Fresh sperm from five bulls having nonreturn rates ranging from 48% to 77% were treated with 15.7, 21.0, 26.2, 31.5, 36.7, and 42.0 μM dilauroylphosphatidylcholine (PC12) to induce the sperm acrosome reaction (AR). Treated sperm were incubated 3 hr with zona-free hamster eggs at 39°C prior to fixation. The eggs were then stained and examined for sperm penetration. Differences in the percentages of motile sperm and of sperm exhibiting an AR among bulls were small when compared on a within-liposome-concentration basis. Increasing the PC12 concentration from 15.7 μM to 42.0 μM increased the percentage of sperm exhibiting an AR for all bulls. At the lowest lipid concentration (15.7 μM), the percentage of eggs penetrated by sperm from the five bulls was 6% to 36%, with 0% in controls. When sperm were incubated with increasing lipid concentrations, the egg penetration rate increased to over 80%, and the total number of sperm increased to over 100 per 36 eggs in each treatment for every bull. These penetration rates decreased at the highest lipid concentration. A correlation between the PC12 concentration maximizing egg penetration and the nonreturn rate of ?.63 was found. The correlation between the PC12 concentration maximizing the total number of penetrated sperm per treatment and the bull nonreturn rate was ?.96. It was concluded that PC 12 liposomes induce the AR in bull spermatozoa, which enables them to penetrate zona-free hamster eggs. High fertility bulls required less lipid to induce the AR than did lower fertility bulls. Consequently, this assay of fresh semen could provide a laboratory method to estimate the fertility of a bull.     

Evaluation of bull semen fertility by homologous in vitro fertilization tests

In vitro fertilization assays were performed to investigate their validity in evaluating artificial insemination (AI) bull fertility. A total of 1,532 oocytes, collected from ovaries at the abattoir, were subsequently used in a 4 x 6 x 2 factorial design: 4 doses of heparin added into the capacitation and fertilization medium (0; 0.05; 0.1 and 0.2 micrograms/ml), 6 different bulls with known on-field non-return (NR) rates (range: 64.6-75.3%) and 2 different ejaculates for each bull, collected within a approximately 1-month interval. Oocytes were considered fertilized when 2 pronuclei (or more) were seen in the ooplasm. Both the heparin dose and bull exerted a highly significant effect on the in vitro fertilization (IVF) rates which ranged, per oocyte group, from 30-80%; bull x dose of heparin interaction was significant (P less than 0.001). The 0.05 micrograms/ml dose of heparin was optimal for discriminating individual bulls. At that dose, the correlation coefficients between the bulls, NR rates and the IVF rates from each ejaculate (within-bull or the mean of two ejaculates), were highly significant (r = 0.83). The rates of polyspermy were also significantly influenced by bull and heparin dose, but there was no interaction. In conclusion, capacitation and fertilization in a modified Tyrode medium containing 0.05 micrograms/ml of heparin may be a valuable tool for evaluating AI bull fertility.     

Phylogenetic analysis of Buggy Creek virus: evidence for multiple clades in the Western Great Plains, United States of America

We present the first detailed phylogenetic analysis of Buggy Creek virus (BCRV), a poorly known alphavirus with transmission cycles involving a cimicid swallow bug (Oeciacus vicarius) vector and cliff swallows (Petrochelidon pyrrhonota) and house sparrows (Passer domesticus) as the principal avian hosts. Nucleotide sequences of a 2,075-bp viral envelope glycoprotein-coding region, covering the entire PE2 gene, were determined for 33 BCRV isolates taken from swallow bugs at cliff swallow colonies in Nebraska and Colorado in the summer of 2001 and were compared with the corresponding region of BCRV isolates collected from Oklahoma in the 1980s. We also analyzed isolates of the closely related Fort Morgan virus (FMV) collected from Colorado in the 1970s. Phylogenetic analysis indicated that BCRV falls into the western equine encephalomyelitis complex of alphaviruses, in agreement with antigenic results and a previous alphavirus phylogeny based on the E1 coding region. We found four distinct BCRV/FMV clades, one each unique to Nebraska, Colorado, and Oklahoma and one containing isolates from both Nebraska and Colorado. BCRV isolates within the two clades from Nebraska showed 5.7 to 6.2% nucleotide divergence and 0.7 to 1.9% amino acid divergence, and within these clades, we found multiple subclades. Nebraska subclades tended to be confined to one or a few cliff swallow colonies that were close to each other in space, although in some cases, near-identical isolates were detected at sites up to 123 km apart. Viral gene flow occurs when cliff swallows move (bugs) between colony sites, and the genetic structure of BCRV may reflect the limited dispersal abilities of its insect vector.