Pharmacokinetics of ketamine in plasma and milk of mature Holstein cows.

The purpose of this study was to evaluate the pharmacokinetics of ketamine in mature Holstein cows following administration of a single intravenous (i.v.) dose. Plasma and milk concentrations were determined using a high-performance liquid chromatography assay. Pharmacokinetic parameters were estimated using a noncompartmental method. Following i.v. administration, plasma T(max) was 0.083 h and plasma C(max) was 18,135 ± 22,720 ng/mL. Plasma AUC was 4484 ± 1,398 ng·h/mL. Plasma t(½ß) was 1.80 ± 0.50 h and mean residence time was 0.794 ± 0.318 h with total body clearance of 1.29 ± 0.70 L/h/kg. The mean plasma steady-state volume of distribution was calculated as 0.990 ± 0.530 L/kg and volume of distribution based on area was calculated as 3.23 ± 1.51 L/kg. The last measurable time for ketamine detection in plasma was 8.0 h with a mean concentration of 24.9 ± 11.8 ng/mL. Milk T(max) was detected at 0.67 ± 0.26 h with C(max) of 2495 ± 904 ng/mL. Milk AUC till the last time was 6593 ± 2617 ng·h/mL with mean AUC milk to AUC plasma ratio of 1.99 ± 2.15. The last measurable time that ketamine was detected in milk was 44 ± 10.0 h with a mean concentration of 16.0 ± 9.0 ng/mL.

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.

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.

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

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.

Pharmacokinetics of lidocaine in serum and milk of mature Holstein cows.

The purpose of this study was to evaluate the pharmacokinetics of lidocaine in mature Holstein cows following an inverted L and caudal epidural nerve block. Plasma and milk concentrations were determined using high-performance liquid chromatography assay. Pharmacokinetic parameters were estimated using a noncompartmental method. Following administration via inverted L nerve block, serum T(max) was 0.521 +/- 0.226 h and serum C(max) was 572 +/- 207 ng/mL. Serum AUC was 1348 +/- 335 ng.h/mL. Apparent serum t((1/2)beta) was 4.19 +/- 1.69 h and MRT was 5.13 +/- 2.33 h with clearance uncorrected for the extent of absorption of 2.75 +/- 0.68 L/kg/h. The last measurable time of lidocaine detection in serum was 8.5 +/- 1.4 h with a mean concentration of 51 +/- 30 ng/mL. Milk T(max) was detected at 1.75 +/- 0.46 h with C(max) of 300 +/- 139 ng/mL. Milk AUC till the last time was 1869 +/- 450 ng.h/mL with the mean AUC milk to AUC serum ratio of 1.439 +/- 0.374. The last measurable time of lidocaine detection in milk was 32.5 +/- 16.2 h with a mean concentration of 46 +/- 30 ng/mL. There was no detectable lidocaine concentration in any samples following caudal epidural administration.

Approaches to biosecurity in bovine embryo transfer programs.

Although transfer of bovine embryos is much less likely to result in transmission of pathogens than transport of postnatal cattle, the epidemiologic risk associated with bovine embryo transfer merits examination. Much research has validated the efficacy of internationally approved processing protocols to render bovine in vivo-derived embryos free of specified pathogens. The purpose of this review is to summarize current sanitary recommendations for bovine embryo transfer, while emphasizing recent research to develop and validate novel approaches to biosecurity. Continued research will enable the development and validation of novel embryo treatments and culture reagents to minimize requirements for testing of embryo or oocyte donors, and testing of embryo recipients.

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.

Review: Risks of disease transmission through semen in cattle.

The purpose of this paper is to review scientific evidence concerning pathogens that could potentially be transmitted via bovine semen. As a result of a careful analysis of the characteristics of infections that may cause transmission of disease through semen, effective control procedures can be identified that provide minimal constraint to the introduction of new bulls into herds for natural breeding and importation of valuable novel genetics through artificial insemination. The potential for transmission through bovine semen and corresponding effective control procedures are described for bovine herpesvirus 1, bovine viral diarrhea virus, bovine leukemia virus, lumpy skin disease virus, bluetongue virus, foot-and-mouth disease virus, and Schmallenberg virus. Brief consideration is also provided regarding the potential for transmission via semen of Tritrichomonas foetus, Campylobacter fetus venerealis, Brucella abortus, Leptospira spp., Histophilus somni, Ureaplasma diversum, Mycobacterium avium subsp. paratuberculosis, Chlamydiaceae, Mycobacterium bovis, Coxiella burnetii, Mycoplasma mycoides ssp. mycoides and Neospora caninum. Thoughtful and systematic control procedures can ensure the safety of introducing new bulls and cryopreserved semen into cattle production systems.

Bovine viral diarrhea virus (BVDV): epidemiologic concerns relative to semen and embryos.

Artificial insemination and embryo transfer are used commonly in cattle production and exchange of germplasm between populations of cattle. If properly monitored, assisted reproductive techniques can be used to prevent the spread of infectious agents. However, these techniques potentially represent unnatural routes for transmission of diseases. Bovine viral diarrhea virus (BVDV) is broadly distributed among the world's populations of cattle. Fluids, gametes and somatic cells from infected animals are likely contaminated with the virus. Thus, use of semen or embryos from infected animals could result in spread of BVDV. This paper provides an overview of the risks of transmitting this virus by AI or production and transfer of embryos and summarizes the precautions needed to prevent such transmissions of disease from occurring.

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.

Analytical sensitivity of assays used for detection of bovine viral diarrhea virus in semen samples from the Southeastern United States.

Bovine viral diarrhea virus (BVDV) is a significant pathogen that can be shed in the semen of infected bulls. Thus, screening for BVDV in semen of bulls is recommended prior to their entry into an artificial insemination center. No previous research has compared the analytical sensitivity of reverse transcription-nested polymerase chain reaction (RT-nPCR) and virus isolation assays for detection of BVDV in semen from an infected bull. Therefore, the goals of this research were to compare the analytical sensitivity of RT-nPCR and virus isolation assays for BVDV in semen and to apply these assays to determine the prevalence in the Southeastern United States of bulls that lack viremia yet shed BVDV in semen. Semen collected from a bull that was persistently infected with BVDV was serially diluted (1/10) in semen from uninfected bulls and frozen in liquid nitrogen as raw, partially extended or fully extended semen. Subsequently, samples of semen were assayed by virus isolation and RT-nPCR. Viral detection was more sensitive in extended semen samples than in raw semen samples and more sensitive by RT-nPCR than virus isolation. After this evaluation of analytical sensitivity, serum and semen were collected from 558 post-pubertal bulls in our region. These samples were tested for BVDV by virus isolation. Partially extended semen was also assayed for BVDV by RT-nPCR. All samples were negative by all assays for BVDV. The application of analytically sensitive assays reveals a very low prevalence (

Epidemiologic concerns relative to in vivo and in vitro production of livestock embryos.

Evidence indicates low potential for transmission of pathogens with in vivo-derived embryos of cattle when appropriate precautions are taken. In apparent contrast, results of research with in vivo-derived embryos of small ruminants and swine and with in vitro-derived embryos of cattle suggest a greater tendency for their association with pathogens after artificial exposure. However, regardless of donor species, investigations involving collection of embryos from artificially or naturally infected animals and assessment of health of recipients and offspring after transfer of these embryos have indicated low potential for transmitting disease. In this paper, results of embryo-pathogen research are summarized, emphasizing potential for spread of pathogens under natural circumstances. Also, safe embryo handling practices and their application to multiple species are discussed.

Infectious agents in bovine embryo production: hazards and solutions.

Infectious agents in systems for producing bovine embryos might reduce the number and quality of embryos generated, result in transmission of disease to recipients and offspring, or confound findings of research. Embryo-associated pathogens might also jeopardize human health when the goal of embryo production is creating transgenic animals intended to be a source of pharmaceuticals or organs. This paper addresses risks and resulting hazards of pathogen and microbial contaminant introduction into in vivo or in vitro embryo production systems. Additionally, methods for prevention and quality control are discussed.

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.