American Bison (Bison bison) reproductive endocrinology: serum Pregnancy Associated Glycoproteins (PAG), Progesterone, Estrone and Estrone-Sulfate in non pregnant animals and during gestation.

This study describes concentrations of Pregnancy Associated Glycoproteins (PAG), progesterone (P4), estrone (E1) and estrone-sulfate (E1S) in American Bison sera. In 2 ranches, mature American Bison were sampled once a year for 2 yr. Subsequent American Bison cows calving days were reported. PAG concentration was determined by Radio-Immuno Assay, whereas P4, E1 and E1S were assayed using Liquid Chromatography and Mass Spectrometry. Concentrations were compared between American Bison bulls (B, n = 7), Nonpregnant cows (NP, n = 32), first (1TP, n = 3), second (2TP, n = 26) and third (3TP, n = 15) trimester of pregnancy. Seven American Bison bulls and 92 cows were sampled, 51 calved during these 2 yr. Calving occurred mostly in spring (74.5%), but also in summer (13.7%) and fall (11.8%). PAG and P4 were higher in 2TP and 3TP than B and NP (P< 0.0001). P4 was non-basal in B and NP. E1 and E1S were correlated (P< 0.0001; r = 0.76) and increased in 2TP and 3TP when compared with B and NP (P< 0.01). Moreover, E1S was higher in 3TP than in 2TP (P< 0.0001) and correlated to pregnancy day (P< 0.0001; r = 0.60). Breeding American Bison in Belgium induces a calving seasonality loss. P4 slowly increases in 1TP and remains steady and high in 2 and 3TP. P4 non-basal and variable concentrations in B or NP disable its use as gestation marker. American Bison produce PAG in the 2 and 3TP, but Estrone-sulfate assay seems to be the best pregnancy marker during the 2 last trimesters as it could help to estimate the gestation period.

Concentration, activity and biochemical characterization of myeloperoxidase in fresh and post-thaw equine semen and their implication on freezability.

Myeloperoxidase (MPO) is a pro-oxidant enzyme associated with decreased motility in thawed equine semen. This study aimed to describe MPO concentration, activity and subunits in raw and thawed semen and to correlate these data with motilities in raw and thawed semen. Semen samples from five stallions were collected four times. Motilities were assessed in raw and thawed semen. MPO assays were performed in raw seminal plasma, raw sperm-rich pellet and thawed semen. Total and active MPO concentrations were, respectively, assayed by enzyme-linked immunosorbent assay and specific immunological extraction followed by enzymatic detection. MPO subunits present in semen were characterized by Western blot. Purified active MPO was added in saline solution and freezing extender to control its activity during freezing procedure. Differences between medians were determined using Kruskal-Wallis test, and correlations were determined using Spearman's test for nonparametric data. Active MPO concentration was low in seminal plasma and thawed semen, but high in pellet (p = 0.0058), as the opposite relation was observed for total MPO concentration (p < 0.0001). In seminal plasma and post-thaw semen, inactive 86-kDa MPO precursor was mainly observed. Purified MPO activity was decreased in the extender (p = 0.0286). MPO activity in pellet was highly correlated with thawed progressive motility (r = -0.5576, p = 0.0086). Inactive MPO precursor and unknown low molecular weight inactive MPO precursor subunits explain low MPO activity in semen. Major MPO activity was observed in pellet, and post-thaw loss of activity is partially explained by MPO inactivation in extender. Thawed semen motility was negatively correlated with MPO activity in pellet, becoming a potential freezability predictor.

Sperm quality analysis in XX, XY and YY males of the Nile tilapia (Oreochromis niloticus).

In Nile tilapia (Oreochromis niloticus), individuals with atypical sexual genotype are commonly used in farming (use of YY males to produce all-male offspring), but they also constitute major tools to study sex determinism mechanisms. In other species, sexual genotype and sex reversal procedures affect different aspects of biology, such as growth, behavior and reproductive success. The aim of this study was to assess the influence of sexual genotype on sperm quality in Nile tilapia. Milt characteristics were compared in XX (sex-reversed), XY and YY males in terms of gonadosomatic index, sperm count, sperm motility and duration of sperm motility. Sperm motility was measured by computer-assisted sperm analysis (CASA) quantifying several parameters: total motility, progressive motility, curvilinear velocity, straight line velocity, average path velocity and linearity. None of the sperm traits measured significantly differed between the three genotypes. Mean values of gonadosomatic index, sperm concentration and sperm motility duration of XX, XY and YY males, respectively ranged from 0.92 to 1.33%, from 1.69 to 2.22 ×10(9) cells mL(-1) and from 18'04″ to 27'32″. Mean values of total motility and curvilinear velocity 1 min after sperm activation, respectively ranged from 53 to 58% and from 71 to 76 µm s(-1) for the three genotypes. After 3 min of activity, all the sperm motility and velocity parameters dropped by half and continued to slowly decrease thereafter. Seven min after activation, only 9 to 13% of spermatozoa were still progressive. Our results prove that neither sexual genotype nor hormonal sex reversal treatments affect sperm quality in male Nile tilapias with atypical sexual genotype.

Association between myeloperoxidase concentration in equine frozen semen and post-thawing parameters.

Despite improvement of techniques, semen of 20% of stallions remains unfreezable. Recent studies focused on the impact of reactive oxygen species and oxidant enzymes on semen characteristics. Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after cell lysis. It is responsible for the formation of hypochlorous acid, a strong oxidant agent, which could damage spermatozoa. The aim of this study was to determine the relation between MPO concentration and characteristics of frozen semen from stallions. Thirty-five straws from different stallions were analysed. Post-thawing spermatozoal concentration, and progressive and total motility were determined by Computer-Assisted Semen Analysis. Freezability was determined according to post-thawing progressive motility (above or below 15%). Percentage of alive spermatozoa and abnormal forms was determined after Eosin-Nigrosin and Diff-Quick(®) staining, respectively. Post-thawing MPO concentration was measured by enzyme-linked immunosorbent assay (ELISA). Our study shows that frozen thawed semen contains large amounts of free MPO. We also observed that post-thawing MPO ELISA assay can be used as an indicator of equine semen freezability. High MPO concentration samples showed lower total and progressive motility. A higher proportion of abnormal head shape associated with acrosome reaction was observed in our late examinations of the high concentration MPO group. Our results show that MPO adversely affects total and progressive motility of equine semen. A negative correlation between normal motile forms and MPO concentration was also observed. The effect of MPO on dead or abnormal forms remains to be precised.

The use of radial extracorporeal shockwave therapy in the treatment of urethral urolithiasis in the horse: a preliminary study.

BACKGROUND: Radial extracorporeal shockwave therapy (ESWT) is widely used in equine practice for the treatment of orthopedic problems. However, its original use as a lithotripsy device in human and canine urology led us to postulate that it could be used as an alternative to the surgical treatment of urethral calculi in horses. HYPOTHESIS: Radial ESWT can easily and safely fragment calculi in the distal urethra of the horse. ANIMALS: Two postmortem cases and 1 live case of obstructive urinary disease admitted at the Veterinary Teaching Hospital of Liege. METHODS: A radial shockwave device was directly applied to the urethra in an attempt to fragment calculi. An ex vivo trial was performed on the same retrieved calculi to investigate pressure settings in order to obtain complete fragmentation of the calculus. RESULTS: In all cases, radial ESWT was able to fragment the calculus partially, enabling retrieval of the remaining fragments via the urethra. Much higher pressure settings than those used for in vivo partial fragmentation were necessary to obtain complete destruction of the calculi ex vivo. CONCLUSIONS AND CLINICAL IMPORTANCE: This brief report suggests the use of radial ESWT as a safe and useful alternative to more invasive surgical management of urethral calculi in horses.

Activation of tumor necrosis factor-alpha-converting enzyme-mediated ectodomain shedding by nitric oxide.

Ectodomain shedding of cell surface proteins is an important process in a wide variety of physiological and developmental events. Recently, tumor necrosis factor-alpha-converting enzyme (TACE) has been found to play an essential role in the shedding of several critical surface proteins, which is evidenced by multiple developmental defects exhibited by TACE knockout mice. However, little is known about the physiological activation of TACE. Here, we show that nitric oxide (NO) activates TACE-mediated ectodomain shedding. Using an in vitro model of TACE activation, we show that NO activates TACE by nitrosation of the inhibitory motif of the TACE prodomain. Thus, NO production activates the release of cytokines, cytokine receptors, and adhesion molecules, and NO may be involved in other ectodomain shedding processes.

Cellular antioxidant and pro-oxidant actions of nitric oxide.

We describe a biphasic action of nitric oxide (NO) in its effects on oxidative killing of isolated cells: low concentrations protect against oxidative killing, while higher doses enhance killing, and these two effects occur by distinct mechanisms. While low doses of NO (from (Z)-1-[N-(3-ammonio propyl)-N-(n-propyl)-amino]-diazen-1-ium-1,2(2) diolate [PAPA/NO] or S-nitroso-N-acetyl-L-penicillamine [SNAP] prevent killing of rat hepatocytes by t-butylhydroperoxide (tBH), further increasing doses result in increased killing. Similar effects occur with rat hepatoma cells treated with PAPA/NO and tBH or H2O2. Increased killing with higher concentrations of NO donor is due to both NO and tBH, because NO donor alone is without effect. Glutathione (GSH) is not involved in either of these actions. Based on measurements of thiobarbituric acid-reactive substances (TBARS) and effects of lipid radical scavenger (DPPD) and deferoxamine, the protective effect, but not the enhancing effect, involves peroxidative chemistry. Fructose has no effect on tBH killing alone but provides substantial protection against killing by higher concentrations of NO plus tBH, suggesting that the enhancing effect involves mitochondrial dysfunction. Hepatocytes, when stimulated to produce NO endogenously, become resistant to tBH killing, indicative of the presence of an NO-triggered antioxidant defensive mechanism. The finding that the protective effects of low concentrations of NO and the harmful effects of high concentrations of NO are fundamentally different in nature suggest that therapeutic interventions could be designed, which selectively prevent its pro-oxidant activity at high concentrations, thus converting NO from a "Janus-faced" modulator of oxidant injury into a "pure" protectant.

Seasonal changes in the production of two novel and abundant ovarian steroids in the channel catfish (Ictalurus punctatus).

Timely and appropriate changes in steroid plasma titers are necessary for successful reproduction in all vertebrates. Gonadal steroidogenesis of the most intensively cultured teleost species in North America, the channel catfish (Ictalurus punctatus), is poorly understood so a year-long study was conducted to investigate seasonal changes in ovarian steroidogenesis. Incubations of ovarian tissue were conducted monthly with [3H]pregnenolone and the medium was analyzed by high-performance liquid chromatography (HPLC) with radioactivity detection. The suite of steroids produced by the catfish ovary included the expected sex steroids (estradiol and testosterone) and 18 additional ovarian metabolites, including five steroids that have yet to be identified. Androstenedione, 20beta-dihydroprogesterone, 5|P-dihydrotestosterone, estriol, 11beta-hydroxyandrostenedione, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11beta-hydroxytestosterone, and progesterone were characterized by a combination of HPLC and thin-layer chromatography. Two of the most abundant steroids were isolated and analyzed by gas chromatography coupled with mass spectrometry (GC-MS). One of the steroids, 7alpha-hydroxypregnenolone (7P5), is a novel steroid in teleosts and was produced late in vitellogenic growth of the oocyte. Evidence suggests that the enzyme responsible for converting pregnenolone to 7P5, 7alpha-hydroxylase, is a cytochrome P450. The second abundant steroid metabolite was partially characterized by GC-MS as an hydroxylated form of 17-hydroxy-pregnenolone (chi,17P5). This steroid was most abundant when the ovary was regressed and during early vitellogenesis and rapidly decreased prior to spawning. In mammals, 7P5 has been identified as an important neurosteroid; however, the reproductive significance of 7P5 and chi,17P5 in catfish is unknown.