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Bovine viral diarrhea virus (BVDV) replicates in embryo co-culture systems and remains associated with developing IVF bovine embryos, despite washing and trypsin treatment. Previous research demonstrated that 2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan (DB606) inhibits replication of BVDV in cultured cells. The objective of this study was to evaluate the capability of IVF embryos to develop into normal, weaned calves after exposure to antiviral concentrations of DB606 during IVC. Oocytes were obtained from cows via transvaginal, ultrasound-guided follicular aspiration. Presumptive zygotes ( n = 849) that resulted from fertilization of these oocytes were cultured for 7 d in medium supplemented with 0.4 μM DB606 or medium lacking antiviral agent. All blastocysts ( n = 110) were transferred individually into the uterus of a synchronized recipient. The pregnancy status of recipients was determined using transrectal ultrasonography at 21–23 d after embryo transfer. Additional pregnancies as controls ( n = 21) were initiated by natural breeding. Developing fetuses and resulting calves were evaluated every 27–34 d. Blastocyst development, pregnancies per transferred embryo, pregnancies maintained per pregnancies established, gestation length, gender ratio, birth weights, viability of neonates, complete blood counts, and serum chemistry profiles at 3 mo of age and adjusted 205 d weaning weights were compared for research treatments. Development to weaning after exposure to DB606 did not differ significantly from controls. In conclusion, bovine embryo cultures can be safely supplemented with antiviral concentrations of DB606; addition of DB606 agent might prevent viral transmission if BVDV were inadvertently introduced into the embryo culture system.

Gametes, somatic cells and materials of animal origin in media are potential sources for introducing bovine viral diarrhea virus (BVDV) into systems for production of IVF bovine embryos. Further, the efficacy of washing and trypsin treatment for removal of BVDV from IVF embryos is questionable. Washing and trypsin treatments recommended by the International Embryo Transfer Society for in vivo-derived embryos were applied to in vitro-derived, virus-exposed, bovine embryos in this side-by-side comparison of treatments. Embryos for the study were produced in a virus-free system in which follicular oocytes were matured and fertilized in vitro and presumptive zygotes were co-cultured with bovine uterine tubal cells for 7 d. A total of 18 trials was performed, 9 using a noncytopathic BVDV and 9 using a cytopathic BVDV. In each trial, 4 equal groups of 10 or less, zona pellucida-intact embryos/ova were assembled, including 2 groups of morulae and blastocysts (M/B) and 2 groups of nonfertile or degenerated ova (NFD). Each group was prewashed and exposed to 104 to 106 TCID50/mL of either noncytopathic (SD-1) or cytopathic (NADL) BVDV for 2 h. Following in vitro viral exposure, one group of M/B and one group of NFD were washed. The other groups of M/B and NFD were trypsin-treated. Both treatments were consistent with IETS guidelines. After in vitro exposure to noncytopathic BVDV and washing, viral assays of 100 % ( 9 9 ) and 78 % ( 7 9 ) of the groups of M/B and NFD ova, respectively, were positive. After in vitro exposure to cytopathic BVDV and washing, viral assay of 33 % ( 3 9 ) of the groups of both M/B and NFD ova were positive. After in vitro exposure to noncytopathic BVDV and trypsin treatment, viral assay of 44 % ( 4 9 ) of groups of M/B and 67 % ( 6 9 ) of groups of NFD ova were positive. Finally, after in vitro exposure to cytopathic BVDV and trypsin treatment, viral assay of 22 % ( 2 9 ) of the groups of M/B and 44 % ( 4 9 ) of the groups of NFD ova were positive. Contingency table analysis, in which data was stratified by embryo type and virus biotype, was used to compare results. While a difference existed between results of the 2 treatments of groups of M/B within the noncytopathic biotype (P = 0.01, Mantel Haenszel Chi-square), no difference was observed between comparison of treatment between all groups in both biotypes (P > 0.05).

Techniques for in vitro production of bovine embryos have evolved to the extent that applications for the commercial production of calves have been proposed. However, little is known about the epidemiological implications of the procedures. One concern is the introduction of noncytopathic bovine viral diarrhea virus (BVDV). In this study, follicular oocytes (n=247) collected from 10 cows were matured and fertilized in vitro and presumptive zygotes were cultured for 7 d. Primary cultures of bovine oviductal epithelial cells for use during in vitro fertilization and culture were divided into 2 groups. Treated oviductal cells were infected with BVDV while control cells were not exposed to the virus. Two approximately equal groups of mature oocytes from each cow were inseminated, and the presumptive zygotes were cultured with infected or noninfected oviductal cells. After 7 d in culture, zona pellucida-intact morulae/blastocysts and degenerated ova were washed, sonicated and assayed for the presence of virus. The rates of cleavage and development were also compared by Chi-square analysis. After washing, virus was not isolated from morulae and blastocysts but was isolated from some groups of degenerated ova. Infections of oviductal cells were inapparent and did not significantly (P>0.05) affect rates of cleavage or development.

Bovine zonae pellucidae (ZP) from follicular oocytes and from embryos and degenerated ova collected on Day 7 from superovulated cows were examined by scanning electron microscopy, by dimensional measurement, and by total protein determination. The number of plaque-forming units (PFU) of infectious bovine rhinotracheitis virus (IBRV) that were associated with ZP-intact embryos/ova from each of the 3 sources after in vitro exposure was also determined. Scanning electron microscopy revealed that the surfaces of Day-7 embryos and degenerated ova were smoother than those of follicular oocytes. Mean dimensional measurements of the diameter/thickness of the ZP from follicular oocytes, Day-7 embryos, and degenerated Day-7 ova were 156.7 μm/12.3μm, 161.3μm/12.6μm, and 158.9μm/12.8μm, respectively. The mean total protein per ZP of follicular oocytes, embryos, and degenerated ova was 0.331 μg, 0.349 μg, and 0.254 μg, respectively. Considerable variability existed within groups, but significantly greater quantities of IBRV were associated with follicular oocytes (mean PFU/oocyte = 68.1) than with Day-7 embryos (mean PFU/embryo = 43.0; P The reliability of using an assay for IBRV associated with nontransferable ova/embryos as an indicator of the presence or absence of the virus in transferable embryos from the same collection (Day 7) was supported. Although structural differences between the ZPs of follicular oocytes and Day-7 embryos were observed in this study, further investigation is needed to determine if there are differences in the protective function of the respective ZPs.

Thirty superovulatory treatments were administered to 19 mixed-breed, nonlactating cows. In 10 superovulatory treatments, the cows were primed with follicle stimulating hormone (FSH) on the second and third day of the estrous cycle, and in another 10 superovulatory treatments, the cows received no priming dosage of FSH. Initiation of the superovulatory treatments in both groups was determined by ultrasonically monitoring for regression of the dominant anovulatory follicle. Still another 10 superovulatory treatments were begun on Day 10 without regard for regression of the dominant anovulatory follicle and without a priming dosage of FSH. The mean days for starting the superovulatory treatment in the FSH-primed cows, in the nonprimed cows and in the controls were 10.5, 11.9 and 10 days, respectively. All cows were treated with eight injections of FSH at 12-hour intervals in a declining dosage (36 mg total). Cows were bred naturally and embryos collected nonsurgically seven days later. There was no significant difference (P>0.05) between the total number of embryos or transferable embryos in the three treatment groups. In this study neither priming on Days 2 or 3 nor initiating the superovulatory treatment, based on the morphologic regression of the dominant anovulatory follicle, was an effective means for improving the superovulatory response in cattle.

Zona pellucida-intact ova collected from ewes seronegative to Brucella ovis were exposed in vitro to B ovis and washed 10 times in medium that contained no antibiotics. After exposure and washing, nontransferable ova were cultured for isolation of Brucella , and the viable ova were transferred into seven B ovis seronegative ewes. No pregnancies resulted, thus recipient ewes were bred during the next breeding season, and blood samples were collected for bacteriological and serological examination until one month after lambing. Brucella ovis was isolated from all of the nontransferable ova, indicating that the transferred ova had viable organisms adhered to them. Although no recipient was found to be pregnant at Day 45, all seven ewes responded to the transferred ova by producing anti- Brucella antibodies. With the exception of a ewe that was euthanized early in the project due to a traumatic injury, all recipients lambed normally during the following breeding season. Brucella was not found in any sample collected from ewes or lambs. However, ELISA titers for B ovis remained in the suspicious range and a ewe was positive on the CF test within 2 wk of lambing.

Oviductal-stage embryos were surgically collected from 27 superovulated adult cows of various breeds, ages, and parity. A total of 88 surgeries was performed via a caudal flank grid approach, with the animals in lateral recumbency and the reproductive tract irrigated with sterile glycerol solution prior to surgical closure. Eight cows were operated on twice and five cows were operated on three or more times. The maximum number of surgeries for a single cow was five. Successful ova collection was accomplished in each surgical attempt, and all cows submitted to this procedure subsequently became pregnant following return to the breeding herd. This technique provided greater exposure of the ovary, uterine tube, and uterine horn, with less adhesion formation than traditional ventral midline techniques.

This study quantified the relationship between calibrated caliper and ultrasonographic derived measurements of bovine testicles in vivo with actual testicular length, width, volume and weight. The prolate spheroid formula was tested to accurately predict testicular volume and a modification to predict weight. Ten bulls were employed to derive caliper and ultrasound testicle (n = 20) length and width measurements in vivo. Caliper length measurements were more reliable than ultrasound derived lengths, with correlations of r2= 0.8023; P < 0.05 and r2= 0.5111; P < .05, respectively. Width for both the calipers and ultrasound measurements when compared to actual width measurements were r2= 0.7313; P < 0.05 and r2= 0.8310; P < 0.05, respectively. The prolate spheroid formula is reliable in determining testicle (n = 116) volume (r2= 0.8928; P < 0.05). Testicular volume and weight are highly correlated (r2= 0.9776; P < 0.05); therefore, a modification of the prolate spheroid formula was used to predict weight (r2= 0.9084; P < 0.05) against the actual weight. Caliper-derived length and width measurements used in the prediction of volume and weight had correlation coefficients against actual volume and weight of r2= 0.5497; P < 0.05 and r2= 0.6340; P < 0.05, respectively. Ultrasound in vivo measurements for prediction of testicular volume and testicular weight had a correlation of r2= 0.3276; P < 0.05 and r2= 0.6249; P

In vitro embryo production has been used extensively in research and is now offered as a commercial service, yet the hazards of introducing specific infectious agents into in vitro embryo production systems have not been completely defined. The introduction of noncytopathic bovine viral diarrhea virus (BVDV) is a special concern. One objective of this study was to determine if noncytopathic BVDV-infected uterine tubal cells in IVF and IVC systems affected the rate of cleavage and development. An additional objective was to determine if either degenerated ova or embryos produced in the presence of the infected cells had virus associated with them after washing. Follicular oocytes (n = 645) collected from slaughterhouse ovaries were matured and fertilized in vitro, and presumptive zygotes were cultured for 7 d. Primary cultures of uterine tubal cells for use during IVF and IVC were divided into 2 groups. One-half of the cultures was infected with noncytopathic BVDV while the other half was not exposed to the virus. Approximately equal groups of mature oocytes were inseminated, and the presumptive zygotes were cultured with infected or noninfected uterine tubal cells. After 7 d in IVC, zona pellucida-intact (ZP-I) morulae and blastocysts and degenerated ova were washed and assayed for the presence of infectious virus. Infections of uterine tubal cells were not apparent and did not reduce rates of cleavage and development (P > 0.05; Chi-square test for heterogeneity). After washing, BVDV was isolated at a significantly higher rate from groups of virus-exposed degenerated ova (79%) than from individual virus-exposed morulae and blastocysts (37%; P = 0.0002; Mantel-Haenszel summary, Chi-square).

This study was conducted to determine the relationship between testicular shape, scrotal circumference (SC) and sperm production. Twenty-seven mature Holstein bulls were evaluated subjectively and objectively for testicular shape as indicated by testicular length and width, then placed in 1 of 3 groups. Group 1 contained 17 bulls with a normal ovoid testicular shape and a length to width ratio of 1.61:1 +/- 0.01 (SEM). Group 2 was composed of 4 bulls with a long, slender testicular shape and a length to width ratio of 1.95:1 +/- 0.06 (SEM). Group 3 was comprised of 6 bulls with spheroid-shaped testicles and a length to width ratio of 1.3:1 +/- 0.03 (SEM). All the groups were statistically different for length to width ratios (P0.05). Length measurements from cranial to caudal pole of the testis proper were also different between groups (P0.05). Width or testicular diameter was different between Group 2 and Group 3 at P0.05; however, there was no difference between Group 1 and Group 2 or between Group 1 and Group 3. Predicted volumes and weights of testicles were not significantly different between groups. Scrotal circumference measurements were significantly different between groups (P0.05). Group 1 had an average SC of 43.07 +/- 0.36 cm (SEM), Group 2 of 39.33 +/- 1.18 cm (SEM) and Group 3 of 46.22 +/- 0.69 cm (SEM). Sperm production for a twice daily, 2-day-per-week collection schedule revealed a statistically significant difference for sperm output. A total of 2742 ejaculates was evaluated. A total of 1818 ejaculates was evaluated in Group 1, 440 ejaculates in Group 2 and 484 ejaculates in Group 3. The mean spermatozoal harvest per day for Group 1 bulls was 13.62 +/- 0.09 x 10(9) (SEM). Group 2 bulls with the longer-shaped testicles produced 14.82 +/- 0.18 x 10(9) (SEM) spermatozoa per day, and Group 3 bulls, with the more rounded testicle shape and the significantly larger SC produced 11.72 +/- 0.64 x 10(9)(SEM) sperm cells per day. All 3 groups were statistically different at the P = 0.05 level. The results suggest that prediction of sperm production may be dependent on factors other than SC, testicular volume, or weight. Testicular shape may influence sperm output in mature Holstein bulls.

The main purpose of the study was to assess the feasibility of a combined system for in vitro maturation of oocytes, in vitro fertilization, and in vitro culture of embryos for production of calves from cows that have to be removed prematurely from production units. Eighteen cows that were to be culled from experimental dairy production units were ovariectomized. An average of 45.7 oocytes per cow was collected from the ovaries. After in vitro maturation and fertilization of the oocytes, an average of 40.8 presumptive zygotes was placed into in vitro culture, with an average of 16.1 cleaving by day 2 and an average of 5.7 developing to morulae/blastocysts by day 6 or 7. A greater mean quantity of oocytes was collected from cows that were ovariectomized between day 5 and day 13 of the estrous cycle than from those that were ovariectomized between day 0 and day 3 of the estrous cycle. Correspondingly larger mean numbers of cleaved zygotes and morulae/blastocysts were produced from the cows that were ovariectomized between day 5 and day 13 of the cycle. Transferable embryos were produced from 17 of the 18 cows. Eighteen embryos from six oocyte donor cows were transferred to recipients. Six of the eighteen recipients were confirmed to be pregnant after 40 days. Three of the pregnant recipients delivered live calves at term. Two others remain pregnant but have not reached term. The sixth recipient aborted at approximately 120 days of gestation. Results from the preliminary study indicate that this system can be used for production of calves from cull cows. Although transferable embryos were produced from all except one cow, there was a high degree of variability among cows in total number of oocytes recovered and embryos produced. More donors need to be evaluated to determine the effects of age, breed, reason for culling, and source of semen.

Semen was collected from 5 mature beef bulls by electroejaculation before, during, and after 20 days of scrotal insulation. Thermally-induced testicular degeneration was irreversible in 3 of the bulls. Analysis of sperm and seminal plasma polypeptides revealed that 15 to 30 sperm polypeptides and 25 to 30 seminal plasma polypeptides were indistinguishable between bulls prior to the insulation treatment. Changes in the sperm polypeptides pattern appeared as early as 2 days after the insulation treatment and persisted for at least 11 months in 2 of the bulls. In the spermatozoa, there was a detectable loss of 31, 34, 49 and 58 kDa polypeptides and an appearance of 6 to 8 new major polypeptides, ranging from 32 to 83 kDa. The 83 kDa polypeptide was most prominent in the 2 bulls that regained normal sperm motility and morphology following the insulation period. The post-insulation appearance of a seminal plasma polypeptide (circa 60 kDa) was also identified in these 2 bulls. Seminal plasma polypeptides remained qualitatively unaltered by the insulation treatment in the 3 bulls with irreversible testicular degeneration.

An early study demonstrated that washing embryos was effective for removal of virus after artificial exposure of in vivo-derived embryos to a cytopathic isolate of BVDV (Singh et al., 1982 Theriogenology 17,437–444). However, a recent study using representative noncytopathic isolates of BVDV demonstrated that washing was less beneficial for removing some isolates of BVDV than for others (Waldrop et al., 2000 Theriogenology 57, 575). Thus, the objective of this study was to determine if the quantity of a high affinity isolate of BVDV that remains associated with single-washed or trypsin-treated embryos is sufficient to cause infection in vivo. Twenty zona pellucida-intact Day 7 morulae and blastocysts (MB) were collected from superovulated cows. After collection, all MB were washed according to International Embryo Transfer Society (IETS) standards, and all but 4 (negative controls) were exposed for 2h to approximately 106 cell culture infective doses (50% endpoint) per mL (CCID50/mL) of viral strain SD-1. Following exposure, one-half of the MB were washed and one-half were trypsin-treated according to IETS standards. All MB were then individually sonicated, and sonicate fluids were injected intravenously into seronegative calves. Blood was collected from each calf on Days 0, 3, 6, 9, 12, 15, 20, 25, and 30, and sera were assayed for BVDV and anti-BVDV antibodies. All cattle used in the study were determined to be virus- and antibody-negative 30d prior to and the day of intravenous inoculation of sonicate fluids into calves. Viremia was not detected in any calf following injection, possibly due to intermittent sampling and/or small amount of embryo-associated virus present. However, seroconversion of 38 and 13% of the calves occurred following injection with sonicate fluids from washed and trypsin-treated embryos, respectively. Findings demonstrated that the quantity of a high affinity isolate of BVDV associated with single-washed or trypsin-treated embryos is sufficient to be infective in vivo as evidenced by seroconversion. These results emphasize the need for studies to determine if the virus associated with exposed individual embryos constitutes an infective dose when placed into the uterus.

The main purpose of the study was to assess the feasibility of a combined system for in vitro maturation of oocytes, in vitro fertilization, and in vitro culture of embryos for production of calves from cows that have to be removed prematurely from production units.

Semen collection was attempted from 18 stallions with an artificial vagina (AV) and without use of a mare or dummy mount. After the stallions were sexually excited by the presence of another horse (mares, and in some instances, geldings) an AV was placed over the erect penis. Thirteen of the 18 stallions manifested thrusting and ejaculation in at least one attempt at semen collection. Semen was collected from 12 of the stallions on the first attempt with all subsequent attempts at semen collection from 11 of these 12 stallions being successful. The ejaculate from one of the stallions was judged incomplete based upon spermatozoal concentration compared with the concentration of a subsequent ejaculate obtained with a mount.

Twelve attempts were made to isolate caprine arthritis-encephalitis virus (CAEV) from the uterine flushings of serologically positive superovulated does mated to serologically positive bucks. Embryos were transferred to eight serologically negative estrus-synchronized recipient does and the recipients were monitored serologically following embryo transfer. Virus isolation was attempted from colostrum and placental tissues from does that kidded following embryo transfers and the surviving kid was monitored serologically until four months of age. The CAEV was not isolated from any of the uterine flushings, colostrum or placental tissues. All recipients and the kid remained seronegative throughout the trial.

Fifty-three zona pellucida-intact ova were collected surgically from superovulated, Brucella -free mixed-breed ewes. Groups containing two to seven ova were incubated in medium containing Brucella abortus . All groups of ova were then washed 10 times, and ova and sequential washes were cultured for the isolation of B. abortus . Brucella were not found beyond the fifth wash for any group of ova, but were isolated from one of 12 groups of ova. Results indicate that mechanical washing in the absence of antibiotics is advantageous, but alone, is not totally reliable for removing B. abortus from exposed, zona pelucida-intact ovine ova.

Seventy-three zona pellucida-intact ova were collected surgically from 15 superovulated, Brucella -free mixed-breed ewes. Twenty-one groups containing one to five ova were incubated in medium containing Brucella ovis . Subsequently, seven and five groups were incubated for 24 and 4 h, respectively, at 37°C in medium containing penicillin and streptomycin, while nine groups were not treated with antibiotics. All groups of ova were washed 10 times, and ova and sequential washes were cultured for the presence of B. ovis . Brucella were isolated from seven of the nine groups of nontreated ova and from the 10th wash for six of these groups. While Brucella were detected in fewer washes after antibiotic treatment, the organism was still isolated from 11 of the 12 treated groups. Results indicate that standard washing techniques are not reliable for removing B. ovis from exposed, zona pellucida-intact, ovine ova.