Suppression of ovarian progesterone production in dairy cows using an implant of GnRH-agonist (deslorelin) for the purpose of evaluating progesterone metabolism

OBJECTIVE: To evaluate the potential of an implant of a GnRH-agonist (deslorelin) to create a progesterone free animal suitable for studying progesterone (P4) metabolism in intact cows by measuring blood P4 and faecal P4 metabolites. METHODS: Experiment 1: Eighteen non-lactating cycling Holstein-Friesian cows, 4 to 7 years old, were allocated to one of three groups to study plasma P4 concentrations preceding an intravaginal insert. These groups comprised: i) a deslorelin group (GnRH-agonist implanted); ii) a PGF group receiving two injections of prostaglandin (PGF2alpha) 12 days apart; and, iii) an ovariectomised (OVX) group. An intravaginal device (CIDR) was inserted into the vagina of each animal and left in place for 11 days. Plasma P4 concentrations were measured during the study period. Experiment 2: Twelve non-lactating cycling Holstein-Friesian cows, 4 to 7 years old, were allocated to two groups: i) a deslorelin group (GnRH-agonist implanted); and ii) an ovariectomised group. Plasma P4 and faecal P4 metabolites (20-oxo-pregnanes, 20alpha-OH and 20beta-OH) were monitored for a period of 5 weeks. RESULTS: Experiment 1: Average plasma P4 concentration did not differ between the three groups (1.28, 1.43 and 1.55 ng/mL for deslorelin, OVX and PGF cows, respectively, P = 0.8) during the period of supplementation. Experiment 2: There was no difference in plasma P4 (mean plasma P4 < 0.02 ng/mL, P = 0.9) and faecal P4 metabolites between deslorelin and OVX cows 2 weeks after the implantation (P = 0.7). CONCLUSIONS: These data showed that a GnRH-agonist (deslorelin) implant may be used as an alternative to ovariectomy to create a progesterone free animal suitable for studying the metabolism of administered P4.     

Survey of internal parasites in Western Australian pig herds. 1. Prevalence

Between September 1982 and March 1984, 101 Western Australian piggeries with 15 or more sows were surveyed to determine the prevalence of internal parasites and examine the relationship between parasitism and management practices. Faecal samples were collected from 20 pigs in 4 age groups in randomly selected piggeries, and examined for the presence of eggs of helminth parasites and protozoan cysts. Evidence of nematode parasites was found in 79% of piggeries. Sows were more commonly affected than other classes of pigs with worm eggs being found in 68% of herds. Oesophagostomum spp was the most prevalent worm species, being found in pigs from 65% of piggeries and in sows in 60% of herds. Ascaris suum was the most common species of worm found in growing pigs. There was no evidence of infection with either Metastrongylus spp or Strongyloides spp in any of the herds sampled. Oocysts of coccidia were found in pigs from 56% of piggeries and Balantidium coli cysts were detected in pigs from 42% of piggeries sampled.     

Interactions between different media and follicle‐stimulating hormone supplementation on in vitro culture of preantral follicles enclosed in ovarian tissue derived from collared peccaries (Pecari tajacu Linneaus, 1758)

The aim was to verify the effect of follicle‐stimulating hormone (FSH) supplementation to α‐MEM+ or TCM199+ media on the in vitro development of ovarian preantral follicles (PFs) derived from collared peccaries. Ovaries (n = 5 pairs) were collected and divided into fragments destined to control group (non‐cultured) or treatments that were cultured for 7 days. The PFs morphology, growth and activation were evaluated by classical histology. The immunohistochemistry markers Ag‐NOR and PCNA were used for nuclear proliferation analysis, and the picrosirius red labelling was used for ovarian extracellular matrix (ECM) evaluation. After 7‐day culture, only the TCM199+ treatment maintained the proportion of intact PFs similar to day 1 (63.2%), but no differences were found among treatments (p > .05). In addition, a significant increase in the growing follicles proportion was verified for all the treatments, indicating follicular activation (p > .05). By the Ag‐NOR analysis, only the TCM199+/FSH maintained the nuclear proliferation similar to the first day (p > .05). The picrosirius red staining revealed that the ECM remained intact in all the treatments (p > .05). We suggest the use of TCM199+ medium supplemented of FSH for the in vitro development of peccaries PFs under 7‐day culturing conditions.     

Rumen microbial sequestration of [2-(13)C]acetate in cattle

To investigate the impact of rumen microbial sequestration of VFA carbon on estimates of acetate availability based on intraruminal infusion of [2-(13)C] acetate, three nonlactating or low-yielding dairy cows were continuously intraruminally infused with [2-(13)C]acetate for 26 h. The 13C content of ruminal VFA, duodenal carbon, and fatty acids (FA) and AA isolated from liquid-associated ruminal microbes and duodenal DM was measured by an isotope ratio mass spectrometer interfaced to an elemental analyzer or a gas-liquid chromatograph. The ruminal gross production of acetate was 38 +/- 4 mol/d and could account for about 38% of the DE intake. Of the intraruminally infused 13C in [2-(13)C]acetate, 7.6 +/- 0.9% was recovered at the duodenum. The 13C content of ruminal propionate, butyrate, and valerate increased (P < 0.05) with intraruminal infusion of [2-(13)C]acetate. It was estimated that about 28% of the 13C intraruminally infused in [2-(13)C]acetate could be accounted for by duodenal 13C flow and absorption of non-acetate VFA. A number of FA isolated from liquid-associated ruminal microbes (C6, C12, C14, anteiso C15, and iso C15) were enriched with 13C (P < 0.05) at a level comparable to the enrichment of ruminal butyrate. Any absorption of these FA from the rumen would further contribute to non-acetate 13C uptake. A maximum of 72% of the ruminal gross production of acetate represented acetate absorption from the rumen in the present study. Consequently, previously used models using intraruminal isotope dilution techniques seem not to be appropriate for measuring acetate availability in ruminants. The number of metabolites exchanging carbon with acetate was found to be so high that assessments of the entire range of inter conversions seem to be practically impossible. Portal absorption studies are discussed as an alternative method of estimating VFA availability to the metabolism in ruminants.     

Effects of Clinical Mastitis on Ovarian Function in Post-partum Dairy Cows

Mastitis-induced ovarian abnormalities were studied in a field trial. At 1-3 day after calving, > or = 2 parity cows not affected with chronic recurrent mastitis and yielding < 400,000/ml somatic cell count (SCC) individual milk in the previous lactation, were enrolled in the study. Thereafter milk samples were collected three times weekly for 95-100 day for progesterone (P4) assay. Individual P4 profiles were used to monitor ovarian cyclicity. When mastitis was diagnosed in the first 80 day post-partum (pp), clinical signs were recorded and scored, and aseptic milk samples were taken to identify the mastitis pathogens. Depending on the isolated pathogens the cows were blocked into one of the three sub-groups affected by either Gram-positive (GP), or Gram-negative (GN) bacteria, or of those with no detected pathogens (NDP). Cows suffering from any type of mastitis between days 15 and 28 (n = 27) showed a delay in the onset of ovarian cyclicity, and estrus was postponed compared to cows affected during the first 14 day pp (n = 59) and controls (n = 175) (38.6 +/- 2.3 vs 33.4 +/- 2.1 and 32.0 +/- 1.0 day, respectively, for onset of ovarian cyclicity and 90.7 +/- 2.5 vs 80.2 +/- 2.8 and 83.9 +/- 2.1 day, respectively, for estrus; both p < 0.05). The percentage of cows ovulating by day 28 was lower in those affected by mastitis between days 14 and 28 compared to cows between days 1 and 14 and controls (22.2% vs 47.5 and 50.3%, respectively; p < 0.05). A significantly higher rate of premature luteolysis was observed in GN + NDP compared to GP mastitis and healthy cows (46.7% vs 8.3 and 2.0%, respectively; p < 0.001). If the mastitis outbreak occurred during the follicular phase, the duration of this cycle segment was lengthened in GN + NDP mastitis compared to GP mastitis and healthy cows (10.8 +/- 0.9 vs 7.9 +/- 0.1 and 7.2 +/- 0.1, respectively; p < 0.001). The results indicate that mastitis can affect the resumption of ovarian activity in pp dairy cows. Mastitis may also impair reproduction also in cyclic cows: this effect can be the consequence of premature luteolysis or a prolonged follicular phase.     

CONTRAST-ENHANCED ULTRASONOGRAPHY IN NORMAL CANINE LIVER. EVALUATION OF IMAGING AND SAFETY PARAMETERS

Contrast-enhanced ultrasonography, a new imaging modality in veterinary medicine, can provide data on tissue perfusion. The objective of this study was to use the ultrasonographic contrast agent SonoVue to evaluate various transit time indices in the normal canine liver, to examine the effect of anesthesia on these parameters, and to evaluate the safety of this agent in dogs. The liver of 11 healthy dogs was studied by ultrasound during an intravenous bolus injection of SonoVue. Each dog underwent the examination twice, first with and later without the use of anesthesia. A time-intensity curve was generated from a selected region of interest within the liver from each scanning session. Ratios derived from peak enhancement, time to peak enhancement, up-slope and full-width half-maximum (FWHM) of the curve were calculated from the time-intensity curves, and are reported. There were no statistically significant differences (P > 0.05) in peak enhancement, up-slope and FWHM between dogs that were anesthetized and dogs that were not. Time to peak enhancement, however, was significantly shorter when the dogs were anesthetized than when they were nonanesthetized (P < 0.05). There were no biologically significant changes in clinical laboratory findings. This study indicates that contrast-enhanced ultrasound using SonoVue gives reproducible liver perfusion data, and appears to be a safe and well-tolerated agent for use in dogs. When considering normal values, the use of anesthetic drugs has to be considered.     

Decreased platelet function in Cavalier King Charles Spaniels with mitral valve regurgitation

With aggregometry, increased platelet activity has been reported in Cavalier King Charles Spaniels (CKCS) without mitral regurgitation (MR). In contrast, dogs with MR have been found to have decreased platelet activity. The purpose of this study was to test an easy bedside test of platelet function (the Platelet Function Analyzer [PFA-100]) to see if it could detect an increase in platelet activity in CKCS without MR and a decrease in platelet activity in CKCS with MR. This study included 101 clinically healthy dogs > 1 year of age: 15 control dogs of different breeds and 86 CKCS. None of the dogs received medication or had a history of bleeding. The PFA-100 evaluates platelet function in anticoagulated whole blood under high shear stress. Results are given as closure times (CT): the time it takes before a platelet plug occludes a hole in a membrane coated by agonists. The CT with collagen and adenosine-diphosphate as agonists was similar in control dogs (median 62 seconds; interquartile interval 55-66 seconds) and CKCS with no or minimal MR (55; 52-64 seconds). The CT was higher in CKCS with mild MR (regurgitant jet occupying 15-50% of the left atrial area) (75; 60-84 seconds; P = .0007) and in CKCS with moderate to severe MR (jet > 50%) (87: 66-102 seconds; P < .0001). CKCS with mild, moderate, and severe, clinically inapparent MR have decreased platelet function. The previous finding of increased platelet reactivity in nonthrombocytopenic CKCS without MR could not be reproduced with the PFA-100 device.     

Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class I) were cloned and sequenced for two haplotypes (H4 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs. The extracellular domain of SLA-I was connected to porcine beta2 microglobulin by glycine-rich linkers. The engineered single-chain proteins, consisting of fused SLA-I and beta2 microglobulin, were overexpressed as inclusion bodies in Escherichia coli. Also, variants were made of the single-chain proteins, by linking them through glycine-rich linkers to peptides representing T-cell epitopes from classical swine fever virus (CSFV) and foot-and-mouth disease virus (FMDV). An in vitro refold assay was developed, using a monoclonal anti-SLA antibody (PT85A) to gauge refolding. The single best-defined, SLA-I restricted porcine CD8(+) T-cell epitope currently known is a 9-residue peptide from the polyprotein of CSFV (J. Gen. Virol. 76 (1995) 3039). Based on results with the CSFV epitope and two porcine haplotypes (H4 and H7), the in vitro refold assay appeared able to discriminate between peptide-free and peptide-occupied forms of SLA-I. It remains to be seen whether the rapid and technically very simple in vitro refold assay described here will prove generally applicable for the screening of virus-derived peptides for SLA-I binding.