Comparison of reproductive performance of primiparous dairy cattle following revaccination with either modified-live or killed multivalent viral vaccines in early lactation.

The objective of this randomized clinical trial was to compare the effect of revaccination in primiparous dairy cows with modified live viral (MLV) or killed viral (KV) vaccines containing bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BoHV-1) on (1) pregnancy rate following estrus synchronization-timed artificial insemination (TAI), (2) serum progesterone concentrations, and (3) serum neutralizing antibody titers at revaccination and at TAI. Primiparous dairy cows (n=692) that had been previously vaccinated with 4 doses of MLV vaccine as calves or heifers were randomized to receive either an MLV or a KV vaccine between 21 and 28 d in milk and 17 d before initiation of a double-Ovsynch-TAI protocol. Serum was collected within the double-Ovsynch protocol for determination of progesterone concentrations, and at vaccination and TAI for serum neutralizing antibody titers. Ultrasound pregnancy determinations were made at 30 and 60 d after TAI. No differences in pregnancy rates were observed between cows receiving MLV vaccine (44%; n=326) or KV vaccine (43%; n=336). No differences were observed in serum progesterone concentrations during a double-Ovsynch-TAI protocol between cows receiving MLV and KV vaccines. No differences were observed in BVDV 1 or BVDV 2 antibody titers at vaccination and TAI between cows receiving MLV or KV vaccine; however, BoHV-1 antibody titers were greater at TAI in cows receiving KV vaccine. Overall response to vaccination-defined as the percent of all individual cows that had any detectable increase in antibody titer from vaccination to TAI-was 39% for BVDV 1, 45% for BVDV 2, and 61% for BoHV-1. In this research, use of an MLV vaccine did not impede reproduction when revaccination was performed between 21 and 28 DIM and just before enrollment in an estrus synchronization-TAI program in primiparous dairy cows; however, response to vaccination as defined by increases in virus-specific antibody titers could be considered less than ideal for this population of cattle.

Bovine viral diarrhea virus is inactivated when whole milk from persistently infected cows is heated to prepare semen extender.

Bovine viral diarrhea virus (BVDV) can be present in cryopreserved bovine semen and be transmitted through artificial insemination. Because BVDV can be shed in milk, the virus might also be introduced as a contaminant of milk-based semen extenders. Thus, the purpose of this study was to evaluate the epidemiologic risk of using heated, BVDV-contaminated milk to prepare semen extender. Milk was obtained from cows free of and persistently infected (PI) with BVDV. Six replicates of milk samples were processed by heating (85-92.2 degrees C, 10min). Samples of milk collected before and after heating were assayed for BVDV. Additionally, milk was injected intravenously into eight BVDV seronegative calves to monitor for seroconversion and viral infection. Virus was not detected in any milk samples from negative animals. Virus was consistently isolated from unheated milk samples from PI cows by passage of somatic cells, ultracentrifugation, and animal inoculation. Virus was usually detected in these samples by RT-nPCR (reverse transcription nested polymerase chain reaction). In heated milk samples from PI cows, no infectious BVDV was detected using any technique, but viral RNA was detected using RT-nPCR in four of six replicates. Bovine viral diarrhea virus in milk from PI cows was inactivated by heating. Therefore, properly heated milk used in semen extenders will not result in transmission of infectious BVDV. Although RT-nPCR detected the presence of viral RNA in milk samples after heating, the virus was not infectious as demonstrated by lack of replication despite using multiple sensitive techniques.

Bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced preimplantation bovine embryos following artificial exposure.

The objective was to determine the average amount of bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced bovine embryos following recommended processing procedures for embryos. In vivo-derived and in vitro-produced bovine embryos at 7d post-fertilization were exposed (for 2h) to 2 x 10(5-7) cell culture infective dose (CCID(50))/mL of SD-1 (a noncytopathic, Type 1a strain of BVDV), and then washed according to International Embryo Transfer Society (IETS) guidelines prior to testing. Of the 87 in vivo-derived embryos tested, 27% were positive for virus by quantitative polymerase chain reaction (qPCR). The range in amount of virus associated with 99% of the contaminated embryos was

Safety and efficacy of vaccination of seronegative bulls with modified-live, cytopathic bovine viral diarrhea viruses.

The objectives were to vaccinate peri-pubertal bulls with a modified-live vaccine consisting of cytopathic BVDV strains Singer and 296 and evaluate the resulting: (a) transient shed of modified-live, cytopathic BVDV in semen; (b) risk of prolonged testicular infection; and (c) protection against subsequent testicular infection due to viral challenge. Seronegative, peri-pubertal bulls were vaccinated subcutaneously with a standard dose of vaccine (n=11) or were maintained as unvaccinated controls (n=11). Forty-nine days after vaccination, all bulls were intranasally inoculated with a noncytopathic field strain of BVDV. Semen and testicular biopsies collected after vaccination and challenge were assayed for BVDV using virus isolation, reverse transcription-nested PCR, or immunohistochemistry, and the identity of viral strains was determined by nucleotide sequencing of PCR products. Vaccination of peri-pubertal bulls with this vaccine caused a short-term, transient shed of only the type 1a strain of modified-live, cytopathic BVDV in semen for up to 10d after vaccination. The vaccine did not cause prolonged testicular infection. Vaccination with this product prevented development of prolonged testicular infections after subsequent exposure to a field strain of BVDV.

Amplification of bovine viral diarrhoea virus introduced into an in vitro embryo production system via oocytes from persistently infected cattle.

The purpose of this study was to determine whether or not embryos derived from in vitro fertilization of oocytes from persistently infected (PI) cattle would contain infectious virus.Three in vitro embryo production treatment groups were assessed: 1) oocytes and uterine tubal cells (UTC) free of bovine viral diarrhoea virus (BVDV) (negative control), 2)oocytes free of BVDV fertilized and cultured in media containing UTC obtained from PI heifers, and 3) oocytes from PI heifers fertilized and cultured in media containing UTC free of BVDV. The developmental media, UTC and embryos (individual or groups of five) were assayed for virus.Virus was not isolated from any samples in treatment group 1.As shown in previous studies, a proportion of embryo samples were positive for BVDV in treatment group 2. In treatment group 3, the virus associated with the oocytes contaminated the developmental media and infected susceptible co-culture cells used during fertilization and culture. In addition, 65% (11/17) of the degenerated ova from treatment group 3 had infectious virus associated with them. While none of the ova developed into transferable embryos, the study did confirm that use of oocytes from PI cows could lead to amplification of BVDV and cross contamination during in vitro embryo production.

Efficacy of a recombinant trypsin product against bovine herpesvirus 1 associated with in vivo- and in vitro-derived bovine embryos.

Although porcine-origin trypsin will effectively remove bovine herpesvirus 1 (BHV-1) associated with in vivo-derived embryos, TrypLE, a recombinant trypsin-like protease, has not been evaluated. In Experiment 1, 17 groups of 10 in vivo-derived embryos were exposed to BHV-1, treated with TrypLE Express or TrypLE Select (10x concentration) for varying intervals, and assayed as 2 groups of 5 embryos. TrypLE Select treatment for 5 and 10 min (two and seven groups of five embryos, respectively) effectively inactivated BHV-1. In Experiment 2, 22 groups of 10 IVF embryos were treated and assayed. Treatment with TrypLE Select for 7 and 10 min (six groups of five embryos each) and with TrypLE Select diluted 1:2 for 10 min (seven groups of five embryos) was also effective. In Experiment 3, 17 groups of 10 IVF embryos were further evaluated with TrypLE Select undiluted and diluted 1:2 for 10 min. Treatment with the diluted product was effective (18 groups of five embryos), whereas the undiluted product was not completely effective (virus isolated from 2 of 16 groups). In Experiment 4, IVF embryos were treated as described in Experiment 3 and then cultured individually or as groups of five on uterine tubal cells (UTCs) for 48 h; 60% of UTC samples associated with groups of embryos and 35% of UTC associated with individual embryo samples were positive for BHV-1. Therefore, although TrypLE Select appeared to have promise for the treatment of in vivo-derived embryos, it cannot be recommended for treatment of in vitro-derived embryos.

Detection of bovine viral diarrhea virus (BVDV) in single or small groups of preimplantation bovine embryos.

The objectives of this study were to develop techniques to detect BVDV associated with single or small groups of bovine embryos contained in small aliquots of medium using either virus isolation (VI) or real time quantitative polymerase chain reaction (RT-QPCR) assays. In vivo-derived and in vitro-produced bovine embryos at 7 d post-fertilization were exposed to SD-1, a high affinity strain of BVDV, for 2 h and then processed according to the International Embryo Transfer Society (IETS) guidelines prior to testing. Groups of five or two in vivo-derived embryos, and single in vivo-derived embryos, were VI positive for BVDV 100, 50, and 33% of the time, and were RT-QPCR positive 100, 75, and 42% of the time, respectively. The virus was detected by the VI technique in all of the groups of five or two in vitro-produced embryos and in all of the single in vitro-produced embryos, and it was detected in 100, 80, and 50%, using RT-QPCR. Techniques for RT-QPCR were sufficiently sensitive to detect 10 copies of viral RNA in a sample and to detect BVDV associated with single embryos. Application of this new technology, RT-QPCR, will facilitate additional studies to further assess the risk of transmission of BVDV through embryo transfer.

Normal calves produced after transfer of in vitro fertilized embryos cultured with an antiviral compound.

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 microM 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.

Effects of aromatic cationic molecules on bovine viral diarrhea virus and embryonic development.

Bovine viral diarrhea virus (BVDV) has been shown to replicate in embryo culture systems and remain associated with bovine embryos developing in vitro. In this study, novel antiviral agents were evaluated for capability to inhibit replication of BVDV without affecting embryonic development. Serial concentrations of 2-[5(6)-{2-imidazolinyl}-2-benzimidazolyl]-5-(4-aminophenyl)furan (DB456) or 2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan (DB606) were prepared in IVC medium. Then, bovine uterine tubal epithelial cells (UTC) were placed in IVC media with varying concentrations of DB456 or DB606. Within 1h, a genotype I or II strain of BVDV was added to the cultures. Cultures were maintained for 7 days. Infectious virus was quantitated in IVC media collected on days 3 and 7 and in UTC lysates harvested on day 7. The effective antiviral concentrations of DB606 were much lower than effective antiviral concentrations of DB456. In subsequent experiments, IVF presumptive zygotes were cultured in IVC medium with or without DB456 or DB606 at multiple concentrations for 7 days to evaluate effect of the compound on conceptus development. On day 7, stage of embryonic development was observed, and blastocysts were harvested and stained using Hoechst 33342 to enumerate embryonic cells. While DB456 inhibited blastocyst development, DB606 at 20 times the effective antiviral concentration did not hinder blastocyst development or reduce the mean number of cells per blastocyst. These preliminary results indicated that bovine embryo cultures might be safely supplemented with effective concentrations of an antiviral agent.

Uterine tubal cells remain uninfected after culture with in vitro-produced embryos exposed to bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) has been isolated from washed and sonicated, in vitro-produced embryos, but the infectivity of BVDV associated with intact, developing, embryos has not been demonstrated. The objective of this study was to determine if a dose of BVDV infective for co-culture cells was associated with individual, developing embryos, following artificial exposure to the virus and washing. In 5 replicates, zona pellucida-intact, in vitro-produced embryos were assigned to a negative control embryo group, or were incubated in 10(5)-10(6) cell culture infective doses (50%, CCID50) per milliliter of a type I, noncytopathic (strain SD-1) BVDV for 2 h. Unexposed negative control embryos and exposed positive control embryos were washed, sonicated and assayed for BVDV using virus isolation with immunoperoxidase monolayer assay. Immediately or following cryopreservation, remaining virally-exposed, washed embryos were co-cultured individually with BVDV-negative cultures of bovine uterine tubal cells in a medium free of BVDV-neutralizing activity. After two days in culture, uterine tubal cells and embryos (including the zona pellucida) were separated and washed. The culture medium, uterine tubal cells and embryos were then assayed for BVDV. Bovine viral diarrhea virus was not isolated from any negative control embryo group, but was isolated from all positive control embryo groups. Although all uterine tubal cell populations were confirmed to be susceptible to BVDV, virus was never isolated from uterine tubal cells or embryos from post-exposure culture. In conclusion, although BVDV remains associated with washed in vitro-produced embryos, the virus associated with unsonicated embryos was not infective for uterine tubal cells in vitro.

Interaction of Mycoplasma bovis and Mycoplasma bovigenitalium with preimplantation bovine embryos.

In vitro exposures of bovine embryos to Mycoplasma bovis and Mycoplasma bovigenitalium were conducted to determine if these organisms adhered to the zona pellucida-intact (ZP-I) bovine embryo, and standard procedures for washing and treating embryos were evaluated to determine their effectiveness for removing or killing mycoplasmas. Mycoplasma bovis and M. bovigenitalium were isolated from 19 of 19 and 24 of 24 ZP-I embryos, respectively, after in vitro exposure and subsequent washing, thus demonstrating adherence of the two species of Mycoplasma to the ZP. Additionally, M. bovis was isolated from 20 of 20 and 23 of 23 embryos, while M. bovigenitalium was isolated from 25 of 25 and 22 of 22 embryos after antibiotic and trypsin treatment, respectively. It was concluded that neither of the standard procedures currently used for cleansing embryos should be relied upon for insuring freedom from mycoplasmas.

Replication and persistence of different strains of bovine viral diarrhea virus in an in vitro embryo production system.

Recent studies have shown that exposed, in vitro-derived embryos remain contaminated with bovine viral diarrhea virus (BVDV) after washing. However, introduction of a Genotype II versus Genotype I strain of BVDV into an IVF system was reported to provide greater potential for transmission of disease. The primary objective of this study was to compare the potentials for different strains of noncytopathic BVDV to replicate in an IVF system, associate with IVF embryos and infect co-cultured cells via association with washed embryos. The secondary objective was to compare the effect of different strains of BVDV on embryonic development. Two Genotype I (SD-1 and NY-1) and 2 Genotype II (CD-87 and PA-131) strains of BVDV were evaluated. After IVM and IVF of oocytes, presumptive zygotes were washed and transferred into in vitro cultures containing uterine tubal cells (UTC) and medium that was free of BVDV-neutralizing activity. Immediately before addition of zygotes, the cultures were inoculated with 10(5) cell culture infective doses (50%, CCID50) of a strain of BVDV or maintained as a negative control. Cultures of zygotes were then incubated for 7 d. Embryonic development was observed on Days 3 and 7, and attempts were made to isolate BVDV from UTC and medium on Day 7. Also on Day 7, groups of intact, washed blastocysts were either transferred into virus-free secondary cultures containing UTC or sonicated with sonicate fluid assayed by both virus isolation and single-closed-tube reverse transcription nested polymerase chain reaction (RT-nPCR). After 3 d in secondary culture, hatched embryos were enumerated, and medium from the cultures, washed UTC and embryos were tested for BVDV by virus isolation. In addition, washed UTC and embryos were tested for BVDV using RT-nPCR. All strains of BVDV persisted and replicated in the embryo culture environment, but cleavage beyond the 4-cell stage, blastocyst development and hatching varied among cultures contaminated with different strains of virus. Further, the quantity of BVDV associated with washed embryos from both initial and secondary cultures varied among strains, but the variation was unrelated to difference in genotype (SD-1 and PA-131 greater than NY-1 and CD-87). Although all strains of BVDV replicated in UTC in the initial in vitro cultures and remained associated with washed blastocysts, susceptible UTC in the secondary in vitro cultures were seldom infected by any strain of virus.

Quantity and infectivity of embryo-associated bovine viral diarrhea virus and antiviral influence of a blastocyst impede in vitro infection of uterine tubal cells.

In previous studies, bovine viral diarrhea virus (BVDV) remained associated with IVF embryos after viral exposure and washing. However, uterine tubal cells (UTC) were not infected when exposed embryos were washed and individually co-cultured with them. The objective of this study was to evaluate quantity and infectivity of embryo-associated virus and antiviral influence of a blastocyst as possible explanations for failure to infect the UTC in vitro. Morulae and blastocysts were produced in vitro and washed. A portion of the embryos were incubated for 2 h in medium containing 10(6) to 10(8) cell culture infective doses (50%, CCID50) of a genotype I, noncytopathic BVDV per milliliter and then washed again. Virus isolation was attempted on sonicated negative (virus unexposed) and positive (virus exposed) control embryo groups after washing. The influence of quantity and infectivity of embryo-associated virus was evaluated by transferring exposed, washed embryo groups (2, 5, and 10 embryos/group) or sonicate fluid of exposed, washed, sonicated embryo groups (2, 5, and 10 embryos/group) to cultures containing bovine UTC in IVC medium that was free of BVDV neutralizing activity. The antiviral influence of an embryo was evaluated by adding 1 to 10(5) CCID50 of BVDV to UTC in the presence or absence of a single unexposed blastocyst in IVC medium. After 2 d in co-culture, the UTC, IVC medium and washed embryos (when present) were tested separately for the presence of BVDV using virus isolation. Virus was isolated from sonicate fluids of all positive but no negative controls. Virus was not isolated from any UTC following 2 d of culture with virally exposed groups of intact embryos. However, virus was isolated from UTC cultured with sonicate fluids from some groups of 5 (60%) and 10 (40%) embryos. Infective virus also remained associated with some groups of 2 (20%), 5 (40%) and 10 (60%) intact embryos after 48 h of post-exposure culture. Finally, primary cultures of UTC were more susceptible to infection with BVDV in the absence of a blastocyst (P = 0.01). Results indicate that insufficient quantity and reduced infectivity of embryo-associated virus as well as an antiviral influence of intact IVF blastocysts may all contribute to failure of embryo-associated virus to infect UTC in vitro.

Validation of a reverse transcription nested polymerase chain reaction (RT-nPCR) to detect bovine viral diarrhea virus (BVDV) associated with in vitro-derived bovine embryos and co-cultured cells.

Sensitive RT-nPCR assays can be used for the rapid detection of viruses. The objective of this research was to validate an RT-nPCR assay for detection of BVDV associated with various samples collected from an IVF system. In 12 research replicates, we maintained matured COCs as negative controls or exposed them to 1 of 4 noncytopathic strains (SD-1, NY-1, CD-87, or PA-131) of BVDV for 1 h immediately before IVF. After 4 d of IVC, we harvested groups of 5 nonfertile ova or degenerated embryos (NFD) and some associated cumulus cells and transferred developing embryos and the remaining cumulus cells into secondary IVC drops. On the seventh d of IVC, cumulus cells, groups of 5 washed NFD and groups of 5 developed, washed embryos were harvested. We also collected single developed embryos after washing, washing with trypsin, washing and cryopreservation in ethylene glycol, or washing with trypsin and cryopreservation in ethylene glycol. All washes were performed according to International Embryo Transfer Society standards. Developed embryos and NFD were sonicated prior to assay. All samples were assayed for BVDV using virus isolation and RT-nPCR. The virus isolation and RT-nPCR assays determined that all negative control samples were BVDV-free. Virus was detected in association with all exposed cumulus cells and groups of developed embryos using both virus isolation and RT-nPCR. Results from viral assays of other exposed samples indicate enhanced sensitivity of the RT-nPCR assay. The RT-nPCR assay used in this research exhibited acceptable sensitivity, specificity, predictive value and repeatability for rapid detection of BVDV associated with the various samples obtained from an IVF system.

Quality controls for bovine viral diarrhea virus-free IVF embryos.

Introduction of bovine viral diarrhea virus (BVDV) with cumulus-oocyte-complexes (COCs) from the abattoir is a concern in the production of bovine embryos in vitro. Further, International Embryo Transfer Society (IETS) guidelines for washing and trypsin treatment of in-vivo-derived bovine embryos ensure freedom from a variety of pathogens, but these procedures appear to be less effective when applied to IVF embryos. In this study, COCs were exposed to virus prior to IVM, IVF and IVC. Then, virus isolations from cumulus cells and washed or trypsin-treated nonfertile and degenerated ova were evaluated as quality controls for IVF embryo production. The effect of BVDV on rates of cleavage and development was also examined. All media were analyzed prior to the study for anti-BVDV antibody. Two approximately equal groups of COCs from abattoir-origin ovaries were washed and incubated for 1 h in minimum essential medium (MEM) with 10% equine serum. One group was incubated in 10(7) cell culture infective doses (50% endpoint) of BVDV for 1 h, while the other was incubated without virus. Subsequently, the groups were processed separately with cumulus cells, which were present throughout IVM, IVF and IVC. Cleavage was evaluated at 4 d and development to morulae and blastocysts at 7 d of IVC. After IVC, groups of nonfertile and degenerated ova or morulae and blastocysts were washed or trypsin-treated, sonicated and assayed for virus. Cumulus cells collected at 4 and 7 d were also assayed for virus. Anti-BVDV antibody was found in serum used in IVM and IVC but not in other media. A total of 1,656 unexposed COCs was used to produce 1,284 cleaved embryos (78%), 960 embryos > or = 5 cells (58%), and 194 morulae and blastocysts (12%). A total of 1,820 virus-exposed COCs was used to produce 1,350 cleaved embryos (74%), 987 embryos > or = 5 cells (54%), and 161 morulae and blastocysts (9%). Rates of cleavage (P = 0.021), cleavage to > or = 5 cells (P = 0.026) and development to morula and blastocyst (P = 0.005) were lower in the virus-exposed group (Chi-square test for heterogeneity). No virus was isolated from any samples from the unexposed group. For the exposed group, virus was always isolated from 4- and 7-d cumulus cells, from all washed nonfertile and degenerated ova (n = 40) and morulae and blastocysts (n = 57) and from all trypsin-treated nonfertile and degenerated ova (n = 80) and morulae and blastocysts (n = 91). Thus, virus persisted in the system despite the presence of neutralizing antibody in IVM and IVC media, and both washing and trypsin treatment were ineffective for removal of the virus. Presence of virus in 4- and 7-d cumulus cells as well as in nonfertile and degenerated ova were good indicators of virus being associated with morulae and blastocysts.

The effects of FSH-priming and dominant follicular regression on the superovulatory response of cattle.

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.

In vitro fertilization and in vitro culture of bovine embryos in the presence of noncytopathic bovine viral diarrhea virus.

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).

Healing of segmental grafts of digital pad skin in dogs.

Pad grafts would be indicated in instances of severe paw trauma when there has been loss of the major weight-bearing pads (ie, metatarsal and metacarpal pads) as well as loss of the digital pads. A practical technique for replacing pad tissue on the remaining paw tissue could avert limb amputation for lack of weight-bearing tissue in the area. Small segmental digital pad grafts were placed in granulation tissue beds in dogs. Although the grafts were from thick pad skin, they healed well. However, intervening wound areas did not become covered with the heavier keratinized epithelium of the pads. The thinner, more rapidly growing, less keratinized epithelium from the wound edges covered most of the wound.

Evaluation of the dermal effects of cast padding in coaptation casts on dogs.

Three configurations of cast padding and no cast padding were evaluated for their effects on skin in dogs. Padding was placed over bony prominences, between bony prominences, and over both areas for full-length padding under short-limb walking casts applied to 1 pelvic limb of Greyhounds. Evaluations were performed by pressure measurement over the calcaneal tuberosity, measurement of skin thromboxane B2 (TxB2) concentrations in skin over bony prominences, and measurement of plasma TxB2 concentrations. Pressure studies were performed to evaluate cutaneous pressures related to no cast padding and various configurations of cast padding. Concentrations of TxB2 in the skin were determined to evaluate the skin inflammatory effects of no padding and the padding configurations, and TxB2 concentrations in the plasma were analyzed to ascertain whether they could be used to predict impending dermal pressure lesions. Flexion of casted limbs revealed the greatest pressure over the calcaneal tuberosity with full-length cast padding. This was followed in decreasing order by no cast padding, padding over the prominences, and padding between the prominences. Compared with all other bony prominences and padding configurations, TxB2 skin concentrations were significantly higher over the calcaneal tuberosity when no padding was used and over the lateral base of metatarsal V when padding was placed between the prominences. Over the calcaneal tuberosity, this was attributed to the sharpness of the prominence and its potential for movement. This high TxB2 concentration corresponded to the high pressure found in the pressure studies. Over the lateral base of metatarsal V, the increase in TxB2 concentration was related to the mass of the prominence and the tendency for localized padding to settle around the area.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of surgical scrub and antiseptic solutions for surgical preparation of canine paws.

The purpose of the prospective study reported here was to evaluate surgical preparation of canine paws. Three combinations of surgical scrub solutions and antiseptic solutions were used: (1) 7.5% povidone-iodine scrub/10% povidone-iodine solution; (2) 2% chlorhexidine acetate scrub/2% chlorhexidine diacetate solution; and (3) tincture of green soap/70% isopropyl alcohol. The control was warm (38 to 42 C) tap water. Four microbial colony counts were used to evaluate surgical preparation of 4 paws of 8 dogs. Specimens were obtained from the paws for a baseline microbial flora count. After surgical scrub was performed, additional specimens were obtained for bacteriologic culturing. Antiseptic was applied followed by collection of another specimen for bacteriologic culturing. A final specimen was obtained following a 24-hour period under a sterile occlusive bandage. The 3 scrub solutions and the tap water control resulted in lower colony counts following scrubbing of the paws; however, only the 3 antiseptic solutions resulted in further colony count reduction after their application. Evaluation of residual colony counts isolated from specimens taken after a 24-hour period under a sterile occlusive bandage revealed chlorhexidine and povidone-iodine scrub/antiseptic combinations to be similar in antibacterial activity, with significantly (P less than or equal to 0.05) lower colony counts than those from specimens of paws treated with either the tincture of green soap/isopropyl alcohol combination or the tap water control. The lack of a significant difference between the bacterial counts immediately after surgical preparation with povidone-iodine and chlorhexidine and their respective 24-hour residual counts, indicated no particular advantage to surgical preparation and occlusive bandaging 24 hours prior to surgery.(ABSTRACT TRUNCATED AT 250 WORDS)