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1.  In vitro exposure of bovine morulae to Ureaplasma diversum. 
Ureaplasma diversum has been associated with infertility in the cow experimentally and in naturally occurring cases. However, the pathogenic mechanism is undetermined. The purpose of this study was to determine whether ureaplasmas are pathogenic for bovine morulae in vitro. Twenty-one morulae were recovered from three superovulated, mature, Holstein cows six or seven days postestrus. The embryos were divided into three groups (A,B,C) and incubated for 16 hours at 37 degrees C in humidified air with 10% CO2. Group A was incubated in embryo culture medium alone, Group B was incubated in culture medium with sterile ureaplasma broth added and Group C was incubated in culture medium containing 1.7 X 10(6) colony forming units Ureaplasma diversum strain 2312. After incubation, the morulae were examined using an electron microscope. Structures morphologically identical to U. diversum were present on the outer surface of the zonae pellucidae of all the morulae exposed to the organism and none were present on the unexposed control embryos. No other morphological differences were observed in either the ureaplasma-exposed embryos or the two groups of control embryos. Ureaplasma diversum was isolated from three of the five embryos incubated in culture medium with sterile ureaplasma broth added. These three embryos were recovered from one donor cow which cultured positive for U. diversum from the vulva and flush fluid. This finding suggests that the contaminating organisms entered the embryo culture wells either in the embryo collection medium or attached to the embryos.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1255303  PMID: 3607652
2.  Reinfection with Chlamydophila abortus by Uterine and Indirect Cohort Routes Reduces Fertility in Cattle Preexposed to Chlamydophila  
Infection and Immunity  2004;72(5):2538-2545.
This study investigated the effects of controlled reinfection on fertility of cattle naturally preexposed to Chlamydophila abortus. All animals had high prechallenge levels of immunoglobulin M (IgM), IgG, IgG1, and IgG2 serum antibodies against ruminant C. abortus in a chemiluminescent enzyme-linked immunosorbent assay. Twenty virgin heifers were estrus synchronized with prostaglandin F2, artificially inseminated 2 to 3 days later, and challenged immediately by intrauterine administration of 0, 104, 105, 106, or 108 inclusion-forming units (IFU) of C. abortus. Ten heifers were estrus synchronized, inseminated, and uterine challenged 2 weeks later. These animals were also indirectly exposed to C. abortus infection (cohort challenged) by contact with their previously challenged cohorts. Pregnancy was determined by rectal palpation 42 days after insemination. All anti-C. abortus antibody isotypes increased in heifers following uterine challenge with 108 IFU. A total of 11, 83, 50, 66, and 0% of heifers were pregnant after uterine challenge with 0, 104, 105, 106, and 108 IFU of C. abortus, respectively. A total of 50 and 65% of heifers were pregnant with and without cohort challenge, respectively. Uterine inoculum dose and cohort challenge (or, alternatively, a negative pregnancy outcome [infertility]) correlated highly significantly with a rise in postchallenge anti-C. abortus IgM levels over prechallenge levels. Logistic regression modeled fertility, with uterine challenge dose and cohort challenge or prechallenge IgM as predictors (P < 0.05). The models predict that the uterine C. abortus inoculum causing infertility is 8.5-fold higher for heifers without cohort exposure and 17-fold higher for heifers with high IgM levels than for heifers with cohort exposure or with low IgM levels.
PMCID: PMC387841  PMID: 15102761
3.  Isolation of Ureaplasma diversum and mycoplasmas from genital tracts of beef and dairy cattle in Saskatchewan 
We report herein a survey in which cultures of bovine reproductive tracts for Ureaplasma diversum and mycoplasmas were carried out in order to better understand the role of these organisms in granular vulvitis (GV). Samples cultured were vulvar swabs from clinically normal cows or ones with GV, preputial swabs or raw semen from bulls, and abomasal contents of aborted fetuses.
Ureaplasma diversum was isolated from 104 (43.3%) of 240 dairy cows, 32 (27.1%) of 118 beef cows, 43 (47.2%) of 91 beef heifers, 23 (67.6%) of 34 beef bulls, and three (60%) of five dairy bulls. Mycoplasmas were isolated from 18 (7.5%) dairy cows, two (1.6%) beef cows, three (8.8%) beef bulls, and one dairy bull. No isolation was made from 97 aborted fetuses. For 65 dairy cows and 30 beef heifers with vulvar lesions, the isolation rates for ureaplasmas of 62.5% and 69.7%, respectively, were significantly higher (X2) than those for normal animals (37.5% and 30.3%). On immunofluorescent serotyping of 137 of the 205 isolates, there were 66 in serogroup C (strain T44), 18 in serogroup B (strain D48), eight in serogroup A (strain A417 or strain 2312), 14 cross-reacting, and 31 that were not identified. It was concluded that U. diversum is commonly present in the lower reproductive tract of beef/dairy cattle in Saskatchewan and is associated with granular vulvitis.
PMCID: PMC1481172  PMID: 17423929
4.  Immune response of heifers to vaginal submucosal or subcutaneous vaccination and intravaginal challenge with Ureaplasma diversum. 
Twenty beef heifers were randomly assigned to five equal groups and vaccinated: Group 1--in vaginal submucosa (VM) with Ureaplasma diversum ultrasonicated whole cells (WC) in complete Freund's adjuvant (CFA); Group 2--in VM with U. diversum cell membranes (CM) in CFA; Group 3--subcutaneously (SC) with CM in CFA; Group 4--in VM with CM alone; and Group 5--in VM with phosphate buffered saline (PBS) in CFA. A second vaccination with the same antigens in incomplete Freund's adjuvant was given after four weeks, and three weeks later, all heifers were challenged intravaginally with 3.6 x 10(7) colony-forming units (CFU) of U. diversum strain 2312. Immunoglobulins that reacted with U. diversum were measured in serum and cervicovaginal mucus (CVM) by an enzyme-linked-immunosorbent assay. In groups 1 and 2, vaccination by the VM route with WC or CM antigens, stimulated high levels of U. diversum-reactive IgG1 and IgG2 antibodies in serum as well as CVM, but a low IgA response only in CVM. In group 4, VM vaccination with CM (no adjuvant) elicited a minimal IgG1 and IgG2 response in serum and CVM. In group 3, SC vaccination with CM antigen stimulated high IgG1 and IgG2 reactivity in both serum and CVM, but no IgA reactivity. Very little IgM reactivity was detected in the four vaccinated groups. Intravaginal challenge resulted in characteristic granular vulvitis in all vaccinated and control heifers, with all animals remaining culture-positive for the 35 day observation period. The infection stimulated a marked increase in the specific IgA response in CVM of the three groups vaccinated with either, adjuvanted antigen.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1263675  PMID: 8004535
5.  The effects of two Ureaplasma diversum strains on early pregnancy in heifers. 
Two field isolates of Ureaplasma diversum spp. were used to infect heifers at the time of insemination in a preliminary study to observe the effect of infection on early pregnancy. M84-14c-1 was a field isolate from a bull's prepuce typed by immunofluorescence to be similar to U. diversum strain T-44 (Group C). M84-477c-4 was a field isolate from bovine semen typed by immunofluorescence to be similar to U. diversum strain T-288 (Group A). All three heifers infected with M84-477c-4 had a mild granular vulvitis at some time during the trial. None was pregnant when slaughtered 27 days after infection. The result of infection with M84-14c-1, a preputial isolate, was not consistent. One heifer had no infection and a normal pregnancy, one heifer was infected with an abnormal pregnancy, and one heifer was open but ureaplasmas were not detected until day 17 of the trial.
PMCID: PMC1255382  PMID: 3453276
6.  Effects of Fertility on Gene Expression and Function of the Bovine Endometrium 
PLoS ONE  2013;8(8):e69444.
Infertility and subfertility are important and pervasive reproductive problems in both domestic animals and humans. The majority of embryonic loss occurs during the first three weeks of pregnancy in cattle and women due, in part, to inadequate endometrial receptivity for support of embryo implantation. To identify heifers of contrasting fertility, serial rounds of artificial insemination (AI) were conducted in 201 synchronized crossbred beef heifers. The heifers were then fertility classified based on number of pregnancies detected on day 35 in four AI opportunities. Heifers, classified as having high fertility, subfertility or infertility, were selected for further study. The fertility-classified heifers were superovulated and flushed, and the recovered embryos were graded and then transferred to synchronized recipients. Quantity of embryos recovered per flush, embryo quality, and subsequent recipient pregnancy rates did not differ by fertility classification. Two in vivo-produced bovine embryos (stage 4 or 5, grade 1 or 2) were then transferred into each heifer on day 7 post-estrus. Pregnancy rates were greater in high fertility than lower fertility heifers when heifers were used as embryo recipients. The reproductive tracts of the classified heifers were obtained on day 14 of the estrous cycle. No obvious morphological differences in reproductive tract structures and histology of the uterus were observed in the heifers. Microarray analysis revealed differences in the endometrial transcriptome based on fertility classification. A genome-wide association study, based on SNP genotyping, detected 7 moderate associations with fertility across 6 different chromosomes. Collectively, these studies support the idea that innate differences in uterine function underlie fertility and early pregnancy loss in ruminants. Cattle with defined early pregnancy success or loss is useful to elucidate the complex biological and genetic mechanisms governing endometrial receptivity and uterine competency for pregnancy.
PMCID: PMC3734181  PMID: 23940519
7.  Invasion of Ureaplasma diversum in bovine spermatozoids 
BMC Research Notes  2011;4:455.
Ureaplasma diversum has been associated with infertility in cows. In bulls, this mollicute colonizes the prepuce and distal portion of the urethra and may infect sperm cells. The aim of this study is to analyze in vitro interaction of U. diversum isolates and ATCC strains with bovine spermatozoids. The interactions were observed by confocal microscopy and the gentamycin internalization assay.
U. diversum were able to adhere to and invade spermatozoids after 30 min of infection. The gentamicin resistance assay confirmed the intracellularity and survival of U. diversum in bovine spermatozoids.
The intracellular nature of bovine ureaplasma identifies a new difficulty to control the reproductive of these animals.
PMCID: PMC3219583  PMID: 22032232
Ureaplasma diversum; bovine spermatozoid; invasion; confocal microscopy
8.  Ureaplasma diversum infection in vitro alters prostaglandin E2 and prostaglandin F2a production by bovine endometrial cells without affecting cell viability. 
Infection and Immunity  1994;62(5):1528-1533.
Bovine epithelial and stromal cells of the endometrium were inoculated with Ureaplasma diversum, pathogenic strain 2312, at 10(6) or 10(3) color-changing units (ccu)/ml in the presence of 1% fetal bovine serum (depleted of steroids by dextran-charcoal treatment) to assess the effect of infection on prostaglandin biosynthesis. When the inoculum of U. diversum was 10(6) ccu/ml, the concentration of U. diversum in the culture medium decreased with time. U. diversum was found on the epithelial and stromal cell monolayers, increasing in titer 100-fold, indicating that attachment and eventually growth occurred. When the inoculum was 10(3) ccu/ml, the titer of U. diversum remained the same or increased in the supernatant and increased on epithelial and stromal cells. The effect of infection was evaluated by measurement of the primary prostaglandin produced by each cell type, prostaglandin F2a for epithelial cells and prostaglandin E2 for stromal cells. Infection with U. diversum significantly decreased prostaglandin F2a accumulation, by 44.7% +/- 6.0% at 10(6) ccu/ml (P < or = 0.005) and 15.8% +/- 5.3% at 10(3) ccu/ml (P < or = 0.05) in epithelial cells. Prostaglandin E2 accumulation by stromal cells was decreased by 34.0% +/- 4.0% at 10(6) ccu/ml (P < or = 0.001) and by 13.5% +/- 2.7% at 10(3) ccu/ml (P < or = 0.005). Infection with 10(6) ccu/ml did not alter endometrial cell viability, as shown by protein measurement, trypan blue dye exclusion, and cell plating efficiency tests. Thus, alterations in prostaglandin production were not due to cell deterioration. These observations suggest that U. diversum can alter prostaglandin E2 and prostaglandin F2a patterns in primary cultures of bovine endometrial cells without affecting cell viability.
PMCID: PMC186347  PMID: 8168914
9.  Protein A gold identification of ureaplasmas on the bovine zona pellucida. 
The object of this study was to develop a prefixation protein A gold labelling technique for Ureaplasma diversum and to apply this to bovine embryos. Sixteen hour cultures of Ureaplasma diversum strain 2312 were incubated with either specific antiserum or nonimmune serum, followed by exposure to protein A gold and negative staining. The ureaplasmas which were incubated with specific antiserum were labelled with gold particles while those ureaplasmas which were incubated with nonimmune serum were not labelled. Twenty-three unhatched, day 7 bovine embryos were then incubated in either embryo culture medium (ECM) alone, ECM with sterile ureaplasma broth added or ECM with 1.7 X 10(6) colony forming units of Ureaplasma diversum strain 2312 per embryo. After 16 hours, the embryos were washed twice and incubated with either specific antiserum or nonimmune serum. The embryos were then incubated with medium containing protein A gold and examined by electron microscopy. No ureaplasmas were identified on the zona pellucida of the control embryos. Ureaplasmas were identified on the outer surface of the zona pellucida of 13 of the 17 embryos which had been exposed to the organism. Of these, the embryos which were incubated with specific antiserum had labelled ureaplasmas while the embryos which were incubated with nonimmune serum had unlabelled ureaplasmas on the zona pellucida. It was concluded that the protein A gold method was a suitable technique for the identification of ureaplasmas in EM preparations. The presence of ureaplasmas on the outer surface of the bovine zona pellucida following in vitro exposure to the organism was confirmed.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1255543  PMID: 2469532
10.  Effects of Ureaplasma diversum on bovine oviductal explants: quantitative measurement using a calmodulin assay. 
Calmodulin (CAM) acts as an intracellular regulator of calcium, an important mediator of many cell processes. We used the CAM assay and electron microscopy to investigate the effects of Ureaplasma diversum on bovine oviductal explants obtained aseptically from slaughtered cows. A stock suspension of U. diversum (treated specimens) and sterile broth (controls) was added to replicates of cultured explants and incubated at 38 degrees C in an atmosphere of 5.5% CO2 for 48 hours. Explants were examined for ciliary activity, extracellular CAM loss, and for histological and ultrastructural changes. Explants and their culture media were examined for changes in CAM concentration. All experiments were replicated three times. In addition, U. diversum, medium and broth were assayed for CAM content. The concentrations of CAM in explants and media changed significantly (p < 0.05) in samples which were inoculated with U. diversum when compared to controls. The controls and infected specimens did not differ histologically or ultrastructurally, but U. diversum was seen to be closely associated with infected explant tissue. In view of this close affinity it is assumed the loss of CAM from the oviductal cells was causally related, but this was not proven. The failure to show cell membrane injury on light and electron microscopic examination was probably related to the short duration of the experiment and may only point out the sensitivity of the CAM assay in detecting early cell membrane injury. Compromise in characteristics of the medium to support both, the viability of oviductal cells and U. diversum limited the experimental time to 48 hours.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1263676  PMID: 8004536
11.  Humoral and secretory antibodies to Ureaplasma diversum in heifers following subcutaneous vaccination and vaginal infection. 
We measured antibody levels in serum and cervicovaginal mucus (CVM) of four heifers vaccinated with two inoculations of killed Ureaplasma diversum strain 2312 in incomplete Freund's adjuvant (IFA) two weeks apart, and six heifers given a placebo. Two weeks later, the vaccinates and four placebo heifers, were challenged by intravaginal inoculation with 6.4 x 10(8) colony-forming units of the homologous U. diversum strain. The remaining two placebo heifers served as unvaccinated, unchallenged controls. Antibody levels in serum and CVM of all heifers were determined by an enzyme-linked immunosorbent assay (ELISA). Vaccination stimulated specific IgG1 and IgG2 responses in serum and CVM but only a slight IgM and no IgA response. In both vaccinate and placebo heifers, subsequent intravaginal challenge resulted in a granular vulvitis (GV) with a predominant IgA response in the CVM. The GV gradually subsided during the 35 day observation period but ureaplasmas were consistently demonstrated by culture. We concluded that subcutaneous vaccination stimulated a specific, albeit nonprotective, IgG response in serum and CVM. In contrast, vaginal infection primarily induced a mucosal IgA response.
PMCID: PMC1263674  PMID: 8004534
12.  Activation of murine macrophages and lymphocytes by Ureaplasma diversum. 
Ureaplasma diversum is a pathogen in the bovine reproductive tract. The objective of the research was to study interactions with macrophages and lymphocytes which might elucidate aspects of pathogenetic mechanisms of this organism. We studied the activation of murine macrophages of C3H/HeN (LPS-responder) and C3H/HeJ (LPS-low-responder) genotype for TNF-alpha, IL-6, IL-1 and nitric oxide production and blastogenic response of C3H/HeJ splenocytes after Ureaplasma diversum stimulation. Live and heat-killed U. diversum induced TNF-alpha, IL-6 and IL-1 in peritoneal macrophage cultures of both C3H/HeN and C3H/HeJ mice in a dose dependent manner. Interferon-gamma modulated the cytokine production, by increasing the production of TNF-alpha, IL-6 and nitric oxide, but IL-1 secretion was only enhanced in C3H/HeJ macrophages stimulated by live ureaplasmas. Supernatant of U. diversum sonicate was mitogenic for murine spleen lymphocytes. The blastogenic response was dose dependent, and stimulation with both U. diversum and Concanavalin A seemed to have an additive effect. These results suggest that U. diversum, similar to other mycoplasmas, activates murine macrophages and lymphoid cells. The studies should be repeated with bovine cells in order to elucidate pathogenetic aspects of inflammation in cattle caused by U. diversum.
PMCID: PMC1263712  PMID: 7889459
13.  Gamete Therapeutics: Recombinant Protein Adsorption by Sperm for Increasing Fertility via Artificial Insemination 
PLoS ONE  2013;8(6):e65083.
A decrease in fertility can have a negative economic impact, both locally and over a broader geographical scope, and this is especially the case with regard to the cattle industry. Therefore, much interest exists in evaluating proteins that might be able to increase the fertility of sperm. Heparin binding proteins (HBPs), specifically the fertility associated antigen (FAA) and the Type-2 tissue inhibitor of metalloproteinase (TIMP-2), act to favor the capacitation and acrosome reaction and perhaps even modulate the immune system’s response toward the sperm. The objective of this research was to determine the effect on fertility of adding recombinant FAA (rFAA) and recombinant TIMP-2 (rTIMP-2) to bovine semen before cryopreservation for use in an artificial insemination (AI) program in a tropical environment. For this experiment, 100 crossbred (Bos taurus x Bos indicus) heifers were selected based on their estrus cycle, body condition score (BCS), of 4 to 6 on a scale of 1 to 9, and adequate anatomical conformation evaluated by pelvic and genital (normal) measurements. Heifers were synchronized using estradiol benzoate (EB), Celosil® (PGF2α) (Shering-Plough) and a controlled internal drug release (CIDR) device was inserted that contained progesterone. Inseminations were performed in two groups at random, 50 animals per group. The control group was inseminated with conventional semen. The treatment group was inseminated with semen containing rFAA (25 µg/mL) and rTIMP-2 (25 µg/mL). In the control group a 16% pregnancy rate was obtained versus a 40% pregnancy rate for the HBP treatment group, resulting in a significant difference (P = 0.0037). Given the results herein, one may conclude that the HBPs can increase fertility and could be an option for cattle in tropical conditions; however, one needs to consider the environment, nutrition, and the genetic interaction affecting the final result in whatever reproductive program that is implemented.
PMCID: PMC3677874  PMID: 23762288
14.  Mycoplasmas and bovine respiratory disease: studies related to pathogenicity and the immune response--a selective review. 
Three species of mycoplasma have been established as being of importance as causes of pneumonia in housed calves, based on pathogenicity studies and frequency of association with the disease. These three species are Mycoplasma bovis, M. dispar, and Ureaplasma diversum. M. bovis is the most pathogenic of these species but the disease outbreaks with which it is associated are sporadic. M. dispar is regularly isolated from pneumonic calves but is also found causing mild superficial and asymptomatic infections of the respiratory mucosa. The bovine ureaplasmas are serologically complex. They are distinct from ureaplasmas isolated from other non-ruminants by PAGE analysis, G + C content of DNA, and serology. A second species within the genus ureaplasma has been proposed to accommodate the bovine ureaplasmas, U. diversum. Control of mycoplasma respiratory infections of cattle based on immunization might be possible. Calves have been immunized against M. bovis and immunity has been related to antibody in the lung. M. dispar appears less immunogenic in calves than M. bovis and this may contribute to its pathogenicity.
PMCID: PMC2590553  PMID: 6382831
15.  Susceptibilities of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma diversum strains to antimicrobial agents in vitro. 
The purpose of this study was to determine the susceptibility of various strains of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma diversum, which are prevalent causes of pneumonia in calves, to 16 antimicrobial agents in vitro. The MICs of the antimicrobial agents were determined by a serial broth dilution method for 16 field strains and the type strain of M. bovis, for 19 field strains and the type strain of M. dispar, and for 17 field strains of U. diversum. Final MICs for M. bovis and M. dispar were read after 7 days and final MICs for U. diversum after 1 to 2 days. All strains tested were susceptible to tylosin, kitasamycin, and tiamulin but were resistant to nifuroquine and streptomycin. Most strains of U. diversum were intermediately susceptible to oxytetracycline but fully susceptible to chlortetracycline; most strains of M. bovis and M. dispar, however, were resistant to both agents. Strains of M. dispar and U. diversum were susceptible to doxycycline and minocycline, but strains of M. bovis were only intermediately susceptible. Susceptibility or resistance to chloramphenicol, spiramycin, spectinomycin, lincomycin, or enrofloxacin depended on the species but was not equal for the three species. The type strains of M. bovis and M. dispar were more susceptible to various antimicrobial agents, including tetracyclines, than the field strains. This finding might indicate that M. bovis and M. dispar strains are becoming resistant to these agents. Antimicrobial agents that are effective in vitro against all three mycoplasma species can be considered for treating mycoplasma infections in pneumonic calves. Therefore, tylosin, kitasamycin, and tiamulin may be preferred over oxytetracycline and chlortetracycline.
PMCID: PMC187660  PMID: 8452363
16.  Effect of narrow sperm head shape on fertility in cattle 
Seven experiments were done in feedlot heifers to determine the importance of various degrees of narrowness of the sperm head on fertility in feedlot heifers. Frozen semen used in these experiments was selected to be normal in all respects except for very high numbers of a single specific type of sperm head aberration. Semen with the sperm aberration in question and control semen were selected to be as similar as possible in dose and postthaw viability so that differences in fertility would be attributable to the morphological variant under study. Fertilization rates were determined by collecting embryos from the reproductive tracts of superovulated heifers which had been slaughtered seven days after insemination. Pregnancy rates and rates of embryonic loss were studied in estrus-synchronized heifers by repeated transrectal ultrasound examinations from day 22 to day 55 after insemination. Reproductive tracts were collected and examined after slaughter at 60 days postinsemination.
The combined results of these experiments show that a moderate degree of sperm head narrowness, in the absence of other seminal signs of a disturbance of spermatogenesis, is not detrimental to fertility. However, extreme narrowness of the postacrosomal region of the sperm head of most spermatozoa, as was found in two bulls without other seminal signs of a disturbance of spermatogenesis, resulted in significantly reduced fertility. The data suggest that, although a decision between normal and abnormal sperm morphology may contain a degree of subjectivity, of the defects studied only sperm with extreme narrowness of the post-acrosomal region are likely to reduce fertility.
PMCID: PMC1481173  PMID: 17423927
17.  Estrus synchronization and pregnancy rates in beef cattle given CIDR-B, prostaglandin and estradiol, or GnRH. 
The Canadian Veterinary Journal  2000;41(10):786-790.
Two experiments were conducted to determine estrous response and pregnancy rate in beef cattle given a controlled internal drug release (CIDR-B) device plus prostaglandin F2 alpha (PGF) at CIDR-B removal, and estradiol or gonadotropin releasing hormone (GnRH). In Experiment I, crossbred beef heifers received a CIDR-B device and 1 mg estradiol benzoate (EB), plus 100 mg progesterone (E + P group; n = 41), 100 micrograms gonadotropin releasing hormone (GnRH group; n = 42), or no further treatment (Control group; n = 42), on Day 0. On Day 7, CIDR-B devices were removed and heifers were treated with PGF. Heifers in the E + P group were given 1 mg EB, 24 h after PGF, and then inseminated 30 h later. Heifers in the GnRH group were given 100 micrograms GnRH, 54 h after PGF, and concurrently inseminated. Control heifers were inseminated 12 h after onset of estrus. The estrous rate was lower (P < 0.01) in the GnRH group (55%) than in either the E + P (100%) or Control (83%) groups. The mean interval from CIDR-B removal to estrus was shorter (P < 0.01) and less variable (P < 0.01) in the E + P group than in the GnRH or Control groups. Pregnancy rate in the E + P group (76%) was higher (P < 0.01) than in the GnRH (48%) or Control (38%) groups. In Experiment II, 84 cows were treated similarly to the E + P group in Experiment I. Cows received 100 mg progesterone and either 1 mg EB or 5 mg estradiol-17 beta (E-17 beta) on Day 0 and either 1 mg of EB or 1 mg of E-17 beta on Day 8 (24 h after CIDR-B removal), in a 2 x 2 factorial design, and were inseminated 30 h later. There were no differences among groups for estrous rates or conception rates. The mean interval from CIDR-B removal to estrus was 44.2 h, s = 11.2. Conception rates were 67%, 62%, 52%, and 71% in Groups E-17 beta/E-17 beta, E-17 beta/EB, EB/E-17 beta, and EB/EB, respectively. In cattle given a CIDR-B device and estradiol plus progesterone, treatment with either EB or E-17 beta effectively synchronized estrus and resulted in acceptable conception rates to fixed-time artificial insemination.
PMCID: PMC1476379  PMID: 11062836
18.  The Management of Drug-induced Manipulation of the Estrous Cycle in Normal Cows and Heifers 
The Canadian Veterinary Journal  1987;28(6):366-373.
Part I Prostaglandin-induced Synchronization of Estrus in Beef Cattle
Prostaglandin-induced regression of the mature cyclic corpus luteum in cows and heifers triggers a sequence of physiological events that results in a return to estrus in two to five days. There are several breeding management programs based on this premise. These programs range from attempts to synchronize estrus in entire herds with two injections of prostaglandin eleven days apart and breeding artificially, to simply shortening diestrus in responsive cattle with a single injection and bullbreeding.
In this paper, several programs are discussed. No single program will be successful in all situations. Programs must be modified to fit each herd and its management. The factors that most commonly lead to program failure include inadequate nutrition, short postpartum interval, and mismanagement of heifers and first-calf heifers.
Part II The Synchronization of Estrus in Embryo Transfer Recipients Using Various Synchronization Compounds
Approximately 1800 recipient cows were synchronized for embryo transplants in three separate trials.
The response rates, distribution of estrus, cull rates, and pregnancy rates of the synchronization products were compared. The pregnancy rates in prostaglandin-induced estrus in embryo transfer recipients were found to be no different from those in recipients that were used after natural noninduced estrus.
The specific prostaglandin analog fenprostalene was tested for efficacy using various combinations of route of administration and antibiotic addition. There were no adverse reactions and neither the addition of oxytetracycline nor the route of administration had any effect on estrus rate, distribution, or pregnancy rates, which were not different from those achieved with the control prostaglandin analog cloprostenol.
Part III Timed Breeding in Prostaglandin-synchronized Dairy Heifers
Five groups of 20 to 40 Holstein heifers were treated with two doses of cloprostenol eleven days apart. The control groups were bred 12 to 16 hours after first seen in standing estrus. Treatment groups were bred at either 64 or 72 hours postinjection (with no detection) or at more than 24 hours postdetection of estrus.
Optimum results were achieved when heifers were bred within 16 hours of first observed estrus or 72 hours after the second synchronizing injection with no detection of estrus.
PMCID: PMC1680664  PMID: 17422808
19.  Survey of immunoglobulin A protease activity among selected species of Ureaplasma and Mycoplasma: specificity for host immunoglobulin A. 
Infection and Immunity  1985;47(3):704-709.
Because immunoglobulin A (IgA) is the predominant immunoglobulin at mucosal surfaces, IgA proteases produced by pathogenic bacteria are considered potential virulence factors for organisms that cause disease or gain entry at mucous membranes. To determine the role of IgA protease in the pathogenicity of mycoplasmal disease, a variety of human and animal mycoplasma and ureaplasma species were examined for IgA protease activity with human, murine, porcine, and canine IgA. None of the mycoplasma species examined showed detectable IgA protease activity with any of the IgAs tested. Twenty-eight strains of Ureaplasma urealyticum isolated from human urogenital tissues cleaved human IgA1, but no cleavage of human IgA2 or murine, porcine, or canine IgA was observed. Ureaplasmas isolated from nonhuman hosts (feline, canine, avian, and bovine [Ureaplasma diversum]) did not cleave human IgA1. Two strains of canine ureaplasmas were able to cleave canine IgA, but not murine IgA. Thus, ureaplasmas from other species can produce IgA protease, but the specificity of the enzyme was restricted to the IgA of the appropriate host. This finding suggests that IgA proteases could play a role in the selective host specificity of mucosal pathogens.
PMCID: PMC261363  PMID: 3882564
20.  Effect of Heifer Frame Score on Growth, Fertility, and Economics 
A non-traditional forage-based protocol was employed to evaluate replacement heifer growth, fertility, and economics between small frame (SF, 3.50; n = 50) and large frame (LF, 5.56; n = 50) heifers using three increasing gain growth phases. Preceding an 85 d growing-breeding period (Phase 3; P3) the heifers were managed as a common group for Phases 1 and 2 (P1 and P2). During P1, heifers grazed common fields of unharvested corn and corn residue (total digestible nutrients [TDN] 56%) with supplemental hay. For P2, heifers grazed early spring crested wheatgrass pasture (CWG; TDN 62%) that was followed by the final P3 drylot growing and breeding period (TDN 68%). Small frame heifers were lighter at the end of P1 in May and at the start of P3 breeding in August (p = 0.0002). Percent of mature body weight (BW) at the end of P1 (209 d) was 48.7% and 46.8%, respectively, for the SF and LF heifers and the percent pubertal was lower for SF than for LF heifers (18.0% vs 40.0%; p = 0.02). At breeding initiation (P3), the percentage of mature BW was 57.8 and 57.2 and the percentage pubertal was 90.0 and 96.0 (p = 0.07) for the SF and LF heifers, respectively; a 5-fold increase for SF heifers. Breeding cycle pregnancy on days 21, 42, and 63, and total percent pregnant did not differ (p>0.10). In drylot, SF heifer dry matter intake (DMI) was 20.1% less (p = 0.001) and feed cost/d was 20.3% lower (p = 0.001), but feed cost/kg of gain did not differ between SF and LF heifers (p = 0.41). Economically important live animal measurements for muscling were measured in May and at the end of the study in October. SF heifers had greater L. dorsi muscle area per unit of BW than LF heifers (p = 0.03). Small frame heifer value was lower at weaning (p = 0.005) and the non-pregnant ending heifer value was lower for SF heifers than for the LF heifers (p = 0.005). However, the total development cost was lower for SF heifers (p = 0.001) and the net cost per pregnant heifer, after accounting for the sale of non-pregnant heifers, was lower for SF heifers (p = 0.004). These data suggest that high breeding efficiency can be attained among March-April born SF and LF virgin heifers when transitioned to a more favorable May-June calving period through the strategic use of grazed and harvested forages resulting in a lower net cost per pregnant SF heifer.
PMCID: PMC4283190  PMID: 25557677
Beef Heifer; Heifer Production Economics; Fertility; Frame Score; Increasing Energy Management; Percent Mature Body Weight
21.  Natural Besnoitia besnoiti infections in cattle: chronology of disease progression 
Bovine besnoitiosis is an emerging protozoan disease in cattle. Neither vaccines nor chemotherapeutic drugs are currently available for prevention and treatment of Besnoitia besnoiti infections. Therefore the implementation of appropriate disease management strategies is of utmost importance. The aim of this longitudinal study was to complement current knowledge on the chronology of disease progression. This was realized by correlating clinical findings in early stages of naturally acquired bovine besnoitiosis with results of real-time PCR of skin biopsies and of two western immunoblots and an immunofluorescent antibody test (IFAT).
Animals for this study were obtained by i) closely monitoring a cow-calf operation with a high prevalence of bovine besnoitiosis for cases of acute disease, and by ii) conducting a 12-week cohabitation experiment on pasture with five healthy heifers, a healthy bull and five B. besnoiti infected cows. A control group of six healthy heifers was kept at a minimal distance of 20 m. Further, the spectrum of potential insect vectors was determined.
Infected cattle were followed up to a maximum of 221 days after first detection of B. besnoiti antibodies. Two severely affected cows developed visible and palpable alterations of skin, a decrease in body condition despite good feed intake, and chronic bovine besnoitiosis-associated laminitis leading to non-healing sole ulcers. The cows also had high reciprocal IFAT titers and high loads of parasite DNA in skin samples. Two heifers developed a mild clinical course characterized by few parasitic cysts visible in the scleral conjunctivae and vestibula vaginae. Both heifers became infected during the time of high insect activity of the species Musca domestica, Musca autumnalis, Haematobia irritans, and Stomoxys calcitrans. When a third heifer became subclinically infected, low insect activity was recorded. None of the six control heifers contracted a B. besnoiti infection.
In chronic besnoitiosis, the severe clinical course apparently corresponded with high reciprocal IFAT titers and high loads of parasite DNA in skin, whereas mild and subclinical cases displayed lower values. Bovine besnoitiosis-associated laminitis represents an important complication in severe chronic disease which severely impairs animal welfare.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-015-0344-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4357170  PMID: 25886463
Besnoitia besnoiti; Bovine besnoitiosis; Cattle; Natural infection; Acute disease; Chronic disease; Laminitis
22.  Experimental Bovine Genital Ureaplasmosis II. Granular Vulvitis, Endometritis and Salpingitis Following Uterine Inoculation 
Twenty-three virgin Holstein heifers received uterine inoculations with ureaplasma and were necropsied one to thirteen days later. Three heifers inoculated intracervically were necropsied on days 3, 5 and 11.
Granular vulvitis was produced on average by 3.6 days in fourteen of sixteen uterine inoculated heifers monitored for four or more days. Two cervically inoculated heifers monitored for over four days also developed granular vulvitis by the fourth day.
At necropsy, ureaplasma was recovered from 94% of uterine horn cultures for the first four days postinoculation and 50% during days 5 to 7. Thereafter all uterine cultures were negative. The percentage of positive ureaplasma recoveries from uterine tube flushings was lower than for uterine horns but remained positive for a longer period. By day 7, three of four uterine tube flushings were still positive. No bacterial pathogens were isolated from the uterine horns or uterine tube flushings.
On histopathology 50% of uterine inoculated heifers had endometritis up to six days postinoculation and a slightly higher percentage (58%) had salpingitis. Endometritis was not found in any heifers after day 6. Residual salpingitis was present in one heifer on day 7. Endometritis was present in cervically inoculated heifers necropsied on days 3 and 5 but not on day 11. Salpingitis was not found in any of the three cervically inoculated animals.
The study concluded that some strains of ureaplasma are pathogenic for the upper reproductive tract of the cow and should be considered significant when isolated from cases of granular vulvitis, endometritis or salpingitis.
PMCID: PMC1320071  PMID: 7427773
23.  Ureaplasma infection of cell cultures. 
Infection and Immunity  1986;52(2):437-444.
Studies were performed to characterize the effects of ureaplasmas in HeLa, 3T6, and CV-1 cell cultures. The ureaplasmas studied were human Ureaplasma urealyticum T960 (serotype VIII), bovine U. diversum T95, simian strain T167-2, ovine strain 1202, canine strain D1M-C, and feline strains 382 and FT2-B. FT2-B was the only ureaplasma to grow in the cell free culture medium, Dulbecco modified Eagle-Earle medium containing 10% fetal bovine serum. The growth pattern of the ureaplasmas varied in the different cell cultures, but each strain grew in at least two of the cell cultures, suggesting a requirement for a product of the cell culture and for low concentrations of urea. When growth occurred, organisms grew to concentrations that approached, but did not equal, those observed in 10B broth. Most, but not all, ureaplasmas grew quickly, reaching peak titers 2 days after infection. Canine strain D1M-C did not grow in 3T6, but showed rapid growth in HeLa and CV-1 cells, killing both cultures, In some systems, e.g., U. urealyticum T960 and simian strain T167-2, the infection persisted, and ureaplasmas could be recovered from cell cultures four passages after infection, when studies were terminated. The cell culture ureaplasmas grew on T agar, but not on mycoplasma agar medium.
PMCID: PMC261018  PMID: 3699891
24.  Pregnancy Rates in Repeat-breeder Heifers Following Multiple Artificial Inseminations during Spontaneous Oestrus 
Acta Veterinaria Scandinavica  2005;46(1):1-12.
Hormonal asynchronies during oestrus, related to the presence of suprabasal plasma-progesterone (P4) concentrations and a delayed ovulation, interfere with the fertility of repeat-breeder heifers (RBH). Since tubal dysfunction can occur in connection with hormonal asynchronies and constrained availability of fertile spermatozoa at the time of ovulation, the present study tested the hypothesis that frequent sperm deposition from onset of oestrus to ovulation may improve pregnancy rates in RBH. Five RBH and five virgin heifers (VH; controls) were repeatedly artificially inseminated (AI) at 6 h intervals from onset of oestrus to spontaneous ovulation. Hormone analyses revealed suprabasal P4 concentrations and a delay in the occurrence of the luteinising hormone (LH) surge, but a normal cortisol profile in RBH. Compared with controls, RBH presented longer interval from onset of oestrus to ovulation, and therefore, received more AIs. Pregnancy rates in RBH reached control levels (60%; NS), indicating that the hypothesis might be correct. Pregnancy rates in VH were below the expected range, presumably attributed to a deleterious influence of the frequent handling. The study suggests that pregnancy rates can be improved in RBH by frequent AI in relation to spontaneous ovulation. However, this practice of repeated manipulations, while seeming not to show adverse effects, lacks practicality for routine use.
PMCID: PMC2202785  PMID: 16108207
Pregnancy rates; Repeat-breeder; Heifer; AI; Oestrus
25.  Transmission of border disease virus from a persistently infected calf to seronegative heifers in early pregnancy 
This study describes the transmission of border disease virus (BDV) from a persistently infected calf to seronegative heifers in early pregnancy, resulting in persistently infected fetuses. On day 50 of pregnancy (= day 0 of the infection phase), six heifers were co-housed in a free stall with a bull calf persistently infected with BDV (pi BVD) for 60 days. The heifers underwent daily clinical examination, and blood samples were collected regularly for detection of pestiviral RNA and anti-pestivirus antibodies. After day 60 (= day 110 of pregnancy), the heifers were slaughtered, and the fetuses and placentae underwent post-mortem and immunohistochemical examination and RT-PCR for viral RNA detection.
Three heifers had mild viraemia from day 8 to day 14, and by day 40 all heifers had pestivirus antibodies identified as anti-BDV antibodies in the serum neutralisation test. The placenta of the three viraemic heifers had histological evidence of inflammation, and fetal organs from these heifers were positive for pestivirus antigen by immunohistochemical examination and for BD viral RNA by RT-PCR and sequencing. Thus, co-housing of heifers in early pregnancy with a pi-BDV calf led to seroconversion in all heifers and persistent fetal infection in three.
Considering that pi-BDV cattle can infect other cattle and lead to persistent infection of the fetus in pregnant cows, BDV should not be ignored in the context of the mandatory BVDV eradication and monitoring program. This strongly suggests that BDV should be taken into account in BVD eradication and control programs.
PMCID: PMC4336514  PMID: 25889936
Cattle; Border disease; Early pregnancy; Persistent infection; BVDV; Pestivirus

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