Participation of phosphofructokinase,malate dehydrogenase and isocitrate dehydrogenase in capacitation and acrosome reaction of boar spermatozoa

The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well‐known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential–interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/10¹⁰ spermatozoa, the activity of NAD‐ and NADP‐dependent IDH was 0.111 ± 0.005 U/10¹⁰ and 2.22 ± 0.14 U/10¹⁰ spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/10¹⁰ spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.     

Sites of Persistence of Lumpy Skin Disease Virus in the Genital Tract of Experimentally Infected Bulls

The objectives of this work were to determine the site of persistence of lumpy skin disease virus (LSDV) in bulls shedding the virus in semen for a period longer than 28 days, to determine if the virus is present in all fractions of semen and to study lesions that developed in the genital tract. Six serologically negative postpubertal bulls were experimentally infected with a virulent field isolate of LSDV. The polymerase chain reaction (PCR) was performed on sheath washes, vesicular fluid, supernatant and cell‐rich fractions of semen from day 10 to day 26 postinfection (p.i.). Bulls that were positive by PCR on the whole semen sample collected on day 28 p.i. were slaughtered and tissue samples from their genital tracts submitted for histopathological evaluation, immunoperoxidase staining, virus isolation and PCR. Two of the bulls developed severe lumpy skin disease (LSD) and were found to be shedding viral DNA in their semen on day 28 p.i. Viral DNA was identified in all semen fractions from all bulls, but mostly from the cell‐rich fraction and from the severely affected bulls. The PCR assay was positive on postmortem samples of testes and epididymides from the two severely affected bulls. Virus could be recovered from the testes of these two bulls and from the epididymis of one of them. Immunoperoxidase staining was positive for LSDV staining in sections of testes and epididymides exhibiting necrosis. This study suggests that the testis and epididymis are sites of persistence of LSDV in bulls shedding virus in semen for prolonged periods and revealed that viral DNA is present in all fractions of the ejaculate.     

Vascular leakage induced by histamine, bradykinin, serotonin and prostaglandin E2 in Greyhounds

SUMMARY Vascular leakage induced by intradermal injection of histamine, bradykinin and serotonin alone and co-injected with prostaglandin E₂ was measured in Greyhounds using ¹²⁵Iodine-labelled human serum albumin (¹²⁵I-HSA) as a marker in the blood. Histamine and bradykinin produced dose-dependent vascular leakage. At equimolar concentrations, histamine was more than twice as potent as bradykinin. Serotonin did not induce vascular leakage and was irritant. Prostaglandin E₂ did not induce significant vascular leakage (maximum 5μL) when injected alone, but when co-injected with histamine and bradykinin, the vascular leakage of both histamine and bradykinin was increased. This effect was more pronounced if lower concentrations of histamine and bradykinin were injected. The induced vascular leakage was greatest during the first five minutes of lesion development for histamine, during the second five minutes of lesion development for bradykinin, and the synergistic effect of prostaglandin E₂ was maximal during the third five minute period of lesion development.     

Effects of inflammation-associated acute-phase response on hepatic and renal indices in the horse

Objectives To determine the effect of an acute soft tissue inflammatory response on biochemical and haematological indices of hepatic and renal function in the Thoroughbred horse.
Procedure Soft tissue inflammation was induced in four Thoroughbred horses by intramuscular injections of Freunds complete adjuvant. The horses were clinically examined and blood and urine samples were collected before and after the adjuvant injections. Biochemical and haematological indices were measured in samples collected and used to determine the onset of the acute-phase response and to assess hepatic and renal function at this time.
Results After adjuvant injection, significant increases (P< 0.01) in total white (13.1 ± 1.4 times 10⁹/L) and neutrophil (10.2 ± 1.2 times109/L) cell counts, rectal temperature (39.7 ± 0.5A°C) and various plasma protein concentrations, including fibrinogen (6.6 ± 1.2 g/L), haptoglobin (1.3 ± 0.1 g/L) and total protein (88.1 ± 2.7 g/L), indicated the induction of an acute-phase response. This corresponded with significant reductions (P< 0.01) in the plasma elimination half-lives (t½β) sodium bromo-sulphthalein (3.13 ± 0.05 to 2.82 ± 0.07 min) and sodium sulphanilate (38.29 ± 4.04 to 19.60 ± 5.68 min) and reductions in the plasma activities of aspartate aminotransferase, gluta-mate dehydrogenase, creatine kinase, alkaline phosphatase, gamma glutamyl transferase; the urinary creatinine clearance ratios of sodium, chloride and potassium; and the urinary gamma glutamyl transferase-to-creatinine clearance ratios. (All values mean ± SD.)
Conclusions The effects of the acute-phase response on indices of hepatic and renal function in the horse suggest that the disposition of pharmacological agents administered at this time may be altered and that indices of acute inflammation should be interpreted cautiously.     

The Origin of Membrane Vesicles in Ram Seminal Plasma

The hypothesis tested in this study was that the membrane vesicles present in ram seminal plasma are of testicular origin, rather than being secreted by the accessory sex glands as has been previously reported for a number of species. Membrane vesicles were present in cellular extracts from reproductive organs and accessory sex glands of six rams, and in the seminal plasma of a further eight rams. When four of the latter rams were subjected to vasectomy, to isolate ejaculate contents to only the secretions of the accessory sex glands, the vesicles were largely eliminated from their ejaculates, while vesicles were still present in the ejaculates of the four control rams. The constituents of the cytoplasmic droplets and membrane vesicles derived from the seminal plasma were compared by transmission electron microscopy (TEM). Vesicles present in the cytoplasmic droplets were similar in morphology but smaller on average than those in the seminal plasma. It was concluded that the membrane vesicles in ram seminal plasma originate from either the cytoplasmic droplets, or a combination of vesicles from the droplets and the epididymis.     

Autologous Whole Ram Seminal Plasma and its Vesicle-free Fraction Improve Motility Characteristics and Membrane Status but not In Vivo Fertility of Frozen–Thawed Ram Spermatozoa

Motility characteristics (assessed subjectively and with computer-assisted semen analysis) and membrane status (after staining with chlortetracycline) of washed and non-washed frozen-thawed ram spermatozoa were evaluated after incubation in buffer and buffer containing autologous whole seminal plasma or one of its two fractions: the pellet of membrane vesicles obtained by ultracentrifugation (and used at three times normal protein concentration) or the vesicle-free supernatant fraction. Whole seminal plasma and supernatant, but not membrane vesicles, improved the motility characteristics of spermatozoa after 3 and 6 h of post-thaw incubation compared with the control buffer. Resuspension and incubation with whole seminal plasma, supernatant or membrane vesicles lowered the proportion of acrosome-reacted frozen-thawed spermatozoa compared with the control buffer. Unwashed frozen-thawed semen from three rams, incubated with autologous whole seminal plasma or its fractions and inseminated using cervical or intrauterine artificial insemination, had no effect on pregnancy rates of ewes in synchronized oestrus. However, fertility was higher after laparoscopic than cervical insemination (44.9 vs 12.3%, p < 0.001). In conclusion, resuspension and incubation of frozen-thawed ram spermatozoa in autologous whole seminal plasma or its vesicle-free supernatant fraction improved their motility characteristics and, with membrane vesicles, membrane status, but these benefits were not reflected in improved fertility after cervical or intrauterine insemination.     

The minor role of pigs in outbreaks of foot-and-mouth disease of northern Thailand

SUMMARY: A study was undertaken in northern Thailand to examine the involvement of pigs in outbreaks of foot-and-mouth disease (FMD). Data were collected by surveying selected villages, by serological monitoring of pigs and by investigating outbreaks. Fifty-three of 58 villages (91%) surveyed reported that pigs did not develop FMD during the most recent outbreak. The source of 49/60 (82%) outbreaks was attributed to either recent purchases of infected cattle and buffalo or commingling of cattle and buffalo with stock from an infected neighbouring village. One of 60 villages (1.7%) reported that the source was introduced infected pigs. There was no association between the various hypothesised risk factors relating to the management of pigs and the frequency of FMD outbreaks in the survey. The percentage of seropositive pigs during 3 rounds of serological monitoring conducted at 6-monthly intervals in selected villages was 3.5%, 2.6% and 0%, respectively. No clinically affected pigs were observed in 11 outbreak investigations. It was concluded that pigs did not commonly become infected when there were outbreaks of FMD in village cattle and buffalo in northern Thailand. This was probably due to the pig feeding and housing practices employed by villagers that protected pigs from exposure to virus from infected cattle or buffalo, or their products.     

Surface-active phospholipid (surfactant) in equine tendon and tendon sheath fluid

AIM: To investigate the presence of surface-active phospholipid (SAPL, or surfactant) in equine tendon and tendon sheath fluid.

METHODS: The left front flexor tendon and sheath were removed from five Thoroughbred horses. Phospholipid was extracted from tendon sheath fluid using Folch reagent and quantifi ed using spectroscopy. Transmission electron microscopy (TEM) was used to observe the tendon surfaces.

RESULTS: The presence of phospholipid (90.6 (SD 4.3) μg/ml) in tendon sheath fluid, plus the appearance of oligolamellar layers and lamellar bodies on the tendon surface were indicative of SAPL.

CONCLUSIONS: Evidence of SAPL was found in equine tendon, and may have a similar lubricating function as reported for synovial joints.

CLINICAL RELEVANCE: These findings may have important implications for normal tendon function and possible therapeutic adjuncts for tendon and tendon sheath injuries.