Noradrenergic Control of GnRH Release from the Ewe Hypothalamus In Vitro: Sensitivity to Oestradiol

The present study investigates the influence of α₁‐adrenoreceptors in GnRH release in vitro and determines whether oestradiol modulates α₁‐adrenoreceptor‐GnRH interaction. Within 10 min after ewe sacrifice, saggital midline hypothalamic slices were dissected, placed in oxygenated Minimum Essential Media‐α (MEM‐α) at 4°C and within 2 h were singly perifused at 37°C with oxygenated MEM‐α (pH 7.4; flow rate 0.15 ml/min), either with or without oestradiol (24 pg/ml). After 4‐h equilibration, 10‐min fractions were collected for 4 h interposed with a 10‐min exposure at 60 min to specific α₁‐adrenoreceptor agonist (methoxamine) or antagonist (thymoxamine) at various doses (0.1–10 mm ). The α₁‐adrenoreceptor agonist (10 mm ) increased (p < 0.05) GnRH release at 90 min both in presence and absence of oestradiol. However, in presence of oestradiol, α₁‐adrenoreceptor agonist (10 mm )‐induced GnRH release remained elevated (p < 0.05) for at least 60 min. The bioactivity of the released GnRH was studied using a hypothalamus–pituitary sequential double‐chamber perifusion. Only after exposure of hypothalamic slices to α₁‐adrenoreceptor agonist (10 mm ), did the hypothalamic eluate stimulate LH release from pituitary fragments (n = 9, 7.8 ± 12.3–36.2 ± 21.6 ng/ml) confirming that the α₁‐adrenoreceptor agonist stimulated release of biologically active GnRH. In summary, GnRH release from the hypothalamus is under stimulatory noradrenergic control and this is potentiated in the presence of oestradiol.     

GABA Control of GnRH Release from the Ewe Hypothalamus In Vitro: Sensitivity to Oestradiol

The present study examines the involvement of GABA_{A or B} receptors in gonadotrophin‐releasing hormone (GnRH) release in vitro and determines whether oestradiol modulates γ‐aminobutyric acid (GABA)–GnRH interaction. Within 10 min after ewe killing, hypothalamic slices were dissected and placed in oxygenated Minimum Essential Media (MEM)‐α at 4°C; within 2 h, slices were singly perifused at 37°C with oxygenated MEM‐α (0.15 ml/min), with or without oestradiol (24 pg/ml). After 4 h equilibration, fractions were collected for 4 h interposed with a 10 min exposure to specific GABA_{A or B} receptor ligands (0.1–10 mm ). The GABA_{A or B} agonists (muscimol or baclofen) did not greatly influence GnRH release. However, GnRH increased (p < 0.05) after exposure to 10 mm GABA_{A or B} antagonists (bicuculline or CGP52432, respectively). The GABA_A antagonist stimulated greater sustained GnRH release (p < 0.05) in the absence of oestradiol than in its presence. The bioactivity of the released GnRH was studied using a hypothalamus‐pituitary sequential double‐chamber perifusion. Only after exposure of hypothalamic slices to the GABA_A antagonist, did the hypothalamic eluate stimulate luteinizing hormone release from pituitary fragments (p < 0.05) confirming that the GABA_A antagonist stimulated release of biologically active GnRH. In summary, GnRH release from the hypothalamus is predominantly under GABA_A receptor inhibitory control and this is attenuated in the presence of oestradiol.     

Ovarian Responses, Hormonal Profiles and Embryo Yields in Anoestrous Ewes Superovulated with Folltropin®-V after Pretreatment with Medroxyprogesterone Acetate-releasing Vaginal Sponges and a Single Dose of Oestradiol-17β

In ruminants, superovulatory treatments started at the time of follicular wave emergence result in greater and less variable ovulatory responses and embryo yields compared with the treatments begun in the presence of a large growing antral follicle(s) from the previous waves. The progesterone–oestradiol treatment is routinely used for follicular wave synchronization in cattle. The main objective of this study was to characterize the ovarian responses, hormonal profiles and in vivo embryo production in anoestrous Rideau Arcott ewes (May‐June), which were superovulated after pretreatment with medroxyprogesterone acetate (MAP)‐releasing intravaginal sponges and a single dose of oestradiol‐17β (E₂‐17β). Six days after insertion of MAP sponges, eight ewes were given an i.m. injection of 350 μg of E₂‐17β (E₂‐17β‐treated ewes); 10 ewes were given an i.m. injection of vehicle (control ewes). Multiple‐dose Folltropin®‐V treatment, followed by the bolus injection of GnRH (50 μg i.m.), began 6 days after E₂‐17β/vehicle injection. Transrectal ovarian ultrasonography revealed that: (i) the interval between E₂‐17β/vehicle injection and regression of all follicles ≥5 to 3 mm in diameter was shorter (p < 0.01; 2.6 ± 0.4 vs 4.8 ± 0.6 days respectively); and (ii) the interval between injection and emergence of the next follicular wave was longer (p < 0.05; 5.4 ± 0.3 vs 1.2 ± 0.4 days, respectively) in E₂‐17β‐treated than in control ewes. During the 6 days after injection, the mean FSH peak concentration and basal FSH concentration were lower (p < 0.01) in E₂‐17β‐treated ewes. The mean ovulation rate and the number of recovered embryos did not differ (p > 0.05) between the two groups of ewes. However, the number of luteinized unovulated follicles per ewe, and the variability in the number of luteal structures and overall embryo yield were less (p < 0.05) in E₂‐17β‐treated compared with control ewes. In conclusion, the MAP–E₂‐17β pretreatment significantly reduced the variability in ovarian responses and embryo yields, without affecting the embryo production in superovulated anoestrous ewes.     

Blood Lymphocyte Subpopulations, Neutrophil Phagocytosis and Proteinogram During Late Pregnancy and Postpartum in Mares

The aim of this study was to evaluate peripheral blood lymphocyte subpopulations, neutrophil phagocytic capacity and proteinogram characteristics in mares, during the last trimester of pregnancy and in postpartum. Measurement of phagocytosis and quantification of T‐lymphocyte subsets were done by flow cytometry. Quantification of T‐lymphocyte subsets was performed with monoclonal antibodies specific for CD2, CD3, CD4 and CD8 cell markers. Natural killer and B‐cell counts were estimated mathematically. Serum proteinogram was obtained by electrophoresis. No significant differences were observed between gestation and postpartum on CD4⁺, CD8⁺ and NK⁺ lymphocyte subsets, CD4 : CD8 ratio and phagocytosis. The percentage of cells expressing CD3 (64.2 ± 1.8) and CD2 (68.4 ± 1.7) (Mean ± SEM) was reduced during gestation vs postpartum (69.7 ± 1.5 and 73.8 ± 1.4 respectively) (p < 0.05). During pregnancy, CD19⁺ (31.6 ± 1.7) was higher than in postpartum (26.2 ± 1.4) (p < 0.05). Total T cells (2911 ± 227 cells/μl), T helper cells (2144 ± 169 cells/μl) and T‐cytotoxic cells (767 ± 68 cells/μl) were depressed in pregnancy, when compared with postpartum (4093 ± 337 cells/μl; 3004 ± 276 cells/μl; 1089 ± 94 cells/μl respectively) (p < 0.01). Total white blood cell count was reduced during pregnancy (8815 ± 427 cells/μl) with respect to postpartum (10742 ± 446 cells/μl) (p < 0.01), while neutrophil count did not change. Total proteins, albumin, α₁,α₂,β₁, β₂, γ globulins and albumin : globulin did not differ. Our results suggest that the physiological immune depression occurring in mares, during gestation might be due to T‐helper and T‐cytotoxic lymphocytes reduction.     

Combined GnRH and PGF2α Application in Cows with Endometritis Puerperalis Treated with Antibiotics

The investigations were carried out on a total of 70 cows with puerperal endometritis. In addition to intrauterine antibiotic treatment, 30 experimental animals were administered 20 μg GnRH analogue, buserelin, between days 10 and 12 post‐partum followed by 500 μg PGF_2α analogue, cloprostenol, 10 days later. Forty control cows were treated only with intrauterine antibiotics. Blood samples for progesterone determination were collected from the tail vein twice weekly until day 70 post‐partum. The first rise in progesterone level above 3.18 nmol/l occurred significantly earlier in the experimental than in control cows (21.6 ± 9.2 versus 27.8 ± 12.3 days; p ≤ 0.05). The duration of the first cycle post‐partum was 15.0 ± 4.3 days in experimental and 19.7 ± 7.3 days in control animals (p ≤ 0.05). However, no significant differences were observed in the occurrence of first oestrus post‐partum. The involution of the uterus was improved after hormone treatment. At day 42 post‐partum, completion of uterine involution was found in 93.3% of hormone‐treated cows and in 82.5% of those treated with antibiotic only (p ≤ 0.05). Clinical recovery was 96.6% in the experimental and 82.5% in the control group (p ≤ 0.05). First service pregnancy rate was significantly better in hormone‐treated than control cows (51.7 versus 36.4%; p ≤ 0.05). Total pregnancy rate and insemination index values were not significantly improved following GnRH and PGF_2α treatment. The average service period was 89.8 ± 21.2 days in cows after hormone treatment, and 112.6 ± 24.5 days in control cows. The difference was statistically significant (p ≤ 0.05). These results indicate, that the sequential GnRH and PGF_2α application in cows with puerperal endometritis positively affected ovarian function and uterine involution, resulting in improved fertility performance.     

Pharmacokinetics of midazolam administered concurrently with ketamine after intravenous bolus or infusion in dogs

Brown, S.A., Jacobson, J.D., Hartsfield, S.M. Pharmacokinetics of midazolam administered concurrently with ketamine after intravenous bolus or infusion in dogs. J. vet. Pharmacol. Therap. 16 , 419–425. Midazolam, a water-soluble benzodiazepine tranquilizer, has been considered by some veterinary anaesthesiologists to be suitable as a combination anaesthetic agent when administered concurrently with ketamine because of its water solubility and miscibility with ketamine. However, the pharmacokinetics of midazolam have not been extensively described in the dog. Twelve clinically healthy mixed breed dogs (22.2–33.4 kg) were divided into two groups at random and were administered ketamine (10 mg/kg) and midazolam (0.5 mg/kg) either as an intravenous bolus over 30 s (group 1) or as an i.v. infusion in 0.9% NaCl (2 ml/kg) over 15 min. Blood samples were obtained immediately before the drugs were injected and periodically for 6 h afterwards. Serum concentrations were determined using gas chromatography with electron-capture detection. Serum concentrations were best described using a two-compartment open model and indicated a t_½α of 1.8 min and t_½β.p of 27.8 min after i.v. bolus, and t_½α f 1–35 min and t_½β of 31.6 min after i.v. infusion. The calculated pharmacokinetic coefficient B was significantly smaller after i.v. infusion (429 ± 244 ng/ml) than after i.v. bolus (888 ± 130 ng/ml, P = 0.004). Furthermore, AUC was significantly smaller after i.v. infusion (29 800 ±6120 ng/h/ml) than after i.v. bolus (42 500 ± 8460 ng/h/ml, P < 0.05), resulting in a larger Cl_B after i.v. infusion (17.4 ± 4.00 ml/min/kg than after i.v. bolus (12.1 ± 2.24 ml/min/kg, P < 0.05). No other pharmacokinetic value was significantly affected by rate of intravenous administration.     

Cellular coupling of potassium channels with β2 adrenoceptors in mediating myometrial relaxation in buffaloes (Bubalus bubalis)

Choudhury, S., Garg, S. K., Singh, T. U., Mishra, S. K. Cellular coupling of potassium channels with β₂ adrenoceptors in mediating myometrial relaxation in buffaloes (Bubalus bubalis). J. vet. Pharmacol. Therap. 33 , 22–27. Present study unravels the functional presence of potassium channels and their involvement in mediating β₂ adrenoceptors‐induced myometrial relaxation in buffalo myometrium. Isolated myometrial preparations exhibited rhythmic spontaneity with regular pattern of amplitude and frequency. Levcromakalim produced concentration‐dependent inhibitory effect on myometrial spontaneity and relaxant effect and the dose–response curve (DRC) was shifted towards right in the presence of glybenclamaide. In the tissues pretreated with glybenclamide, relaxant effect of albuterol was significantly (P < 0.05–0.001) lower (pD₂ = 6.94, R_max = 96.8 ± 3.3%; n = 5) compared with albuterol alone (pD₂ = 8.55, R_max = 101.1 ± 6.3%; n = 6) and the DRC was shifted to right. In the presence of tetraethyl ammonium (TEA) also, significant (P < 0.001) rightward shift of DRC of albuterol with decrease in maximal effect (pD₂ = 8.05, R_max = 71.2 ± 7.4%; n = 7 vs. control pD₂ = 8.55, R_max = 101.1 ± 6.3%; n = 6) was observed. On the other hand, 4‐aminopyridine (1 mm ) sensitized the myometrial strips and increased the amplitude and frequency/min of myometrial spontaneity but failed to significantly alter the DRC of albuterol. Results of present study suggest the functional presence of K_ATP, BK_Ca and K_V channels in buffalo myometrium, but β₂‐adrenoreceptor agonist‐induced myometrial relaxation seems to be K_ATP and BK_Ca channel‐dependent only. Further, our studies also suggest promising therapeutic potential of potassium channel modulators as tocolytics in buffaloes.     

γ-Amino Butyric Acid Control of Arginine Vasopressin Release from the Ewe Hypothalamus In Vitro: Sensitivity to Oestradiol

The present study aims to ascertain the influence of gamma-amino butyric acid (GABA)(A or B) receptors on arginine vasopressin (AVP) release in vitro and determine whether E(2) modulates GABA-AVP interaction. Within 10 min of ewe killing, saggital midline hypothalamic slices (from the anterior preoptic area to the mediobasal hypothalamus along with the median eminence, 2-mm thick, two per ewe) were dissected, placed in oxygenated minimum essential media (MEM)-alpha at 4 degrees C and within 2 h were singly perifused at 37 degrees C with oxygenated MEM-alpha (pH 7.4; flow rate 0.15 ml/min), either with or without E(2) (24 pg/ml). After 4-h equilibration, 10-min fractions were collected for 4 h interposed with a 10-min exposure at 60 min to a specific GABA(A or B) receptor agonist or antagonist at various doses (0.1-10 mm). GABA(A) (muscimol; no E(2), n = 7 perifusion chambers, with E(2), n = 11) or GABA(B) (baclofen; no E(2), n = 8, with E(2), n = 15) agonists (10 mm) did not influence AVP concentrations. However, AVP release increased (p < 0.05) 20-30 min after exposure to 10 mm GABA(A or B) antagonists (bicuculline, no E(2), n = 7: from 4.6 +/- 0.7 to 33.0 +/- 0.4, with E(2), n = 17: from 11.9 +/- 1.4 to 32.8 +/- 6.0; CGP52432, with E(2), n = 14: from 14.0 +/- 2.6 to 28.8 +/- 3.9 pg/ml). At the end of the collection period, hypothalamic slices responded to KCl (100 mm) with AVP efflux (p < 0.05). GABA(B) but not GABA(A) antagonist-stimulated AVP release was enhanced in the presence of E(2). In summary, AVP release is under the inhibitory influence of GABA input with further potentiation by E(2) through GABA(B) receptors in vitro.     

Effects of Hydrogen Peroxide (H2O2) on Fresh and Cryopreserved Buffalo Sperm Functions During Incubation at 37°C In Vitro

The magnitude of damage to buffalo spermatozoa during incubation with different levels of H₂O₂ was assessed. A total number of 24 ejaculates from four Murrah buffalo bulls were analysed in the study. Each ejaculate was split into two parts (part I and II). Part I was extended in Tris–egg yolk–citrate extender (20% egg yolk:7% glycerol), equilibrated (4 h at 5°C) and cryopreserved in 0.5‐ml French straws and stored in liquid nitrogen. The other part was utilized for fresh semen studies. The sperm in fresh, equilibrated and frozen–thawed semen was separated by centrifugation (1500 g ; 15 min) and were washed with sperm TALP. The sperm cells were re‐suspended in incubation TALP at the rate of 10⁸ sperm cells per millilitre and incubated with 0, 10, 25, and 50 μm H₂O₂ per ml at 37°C. Sperm motility, viability and intact acrosome percentages were assessed at 15‐min intervals up to 60 min of incubation. Lipid peroxidation levels of sperm were assessed at 0 and 60 min of incubation. The results of the experiment revealed that sperm motility decreased drastically during incubation with H₂O₂. Among the different levels of H₂O₂, the 50‐μm H₂O₂‐incorporated group had significantly (p < 0.05) higher malonaldehyde (MDA) level than the other groups. In the 50‐μm H₂O₂‐incorporated group, the MDA levels in fresh, equilibrated and frozen–thawed semen after incubation for 60 min were 961.6 ± 12.7, 991.8 ± 10.3 and 1234.9 ± 9.6 nm per 10⁹ spermatozoa respectively. An inverse relationship was observed between sperm motility, viability, intact acrosome percentages and concentration of H₂O₂ and duration of incubation. The decrease in sperm functions with duration of incubation and concentration of H₂O₂ was significantly (p < 0.05) higher in frozen–thawed than fresh and equilibrated spermatozoa.     

Enhancement of Ovulatory Follicle Development in Maiden Sheep by Short-term Supplementation with Steam-flaked Corn

The effect of a short‐term nutritional supplementation with steam‐flaked corn on metabolism and folliculogenesis was evaluated in 14 maiden sheep. Oestrus was synchronized with two prostaglandin F_2α doses given 10 days apart. From day 11 to 15 of the oestrous cycle induced with prostaglandins, half of the ewes (group 2M) were supplemented with steam‐flaked corn, double the daily maintenance ration of the control sheep (group 1M). Body weight and condition remained unaffected, but the energetic supply increased plasma concentrations of glucose (3.6 ± 0.1 vs 4.3 ± 0.1 mmol/l, p < 0.0001) for the first 4 days and 3‐hydroxybutyrate (0.323 ± 0.58 vs 0.582 ± 0.04 mmol/l, p < 0.005) from day 2 to 4. The profile of insulin secretion was also affected by the treatment, increasing in group 2M to reach significant differences on days 13 and 14 (p < 0.05). From similar values at the start of the food supply, the treatment induced a higher follicular development in group 2M (1.1 ± 1.2 vs 7.4 ± 1.06 total follicles in day 15, p < 0.05), as evidenced by the lineal increase in the number of larger follicles (>4 mm, p < 0.005). Then, the number of follicles >4 mm in size in 2M was around 60% higher on day 16 (7.86 ± 0.45 vs 4.86 ± 0.63, p < 0.005). Thereafter, the mean number of corpora lutea per ewe was around 30% higher in group 2M (1.43 ± 0.2 vs 1.10 ± 0.1, although differences were not found to be statistically significant). These data suggest that the use of diets containing high starch sources, like the steam‐flaked corn, increases folliculogenesis and ovulation rate in sheep and can be applied in short‐term feeding practices.     

Estradiol stimulates glycogen synthesis whereas progesterone promotes glycogen catabolism in the uterus of the American mink (Neovison vison)

Glycogen synthesis by mink uterine glandular and luminal epithelia (GE and LE) is stimulated by estradiol (E₂) during estrus. Subsequently, the glycogen deposits are mobilized to near completion to meet the energy requirements of pre‐embryonic development and implantation by as yet undetermined mechanisms. We hypothesized that progesterone (P₄) was responsible for catabolism of uterine glycogen reserves as one of its actions to ensure reproductive success. Mink were treated with E₂, P₄ or vehicle (controls) for 3 days and uteri collected 24 h (E₂, P₄ and vehicle) and 96 h (E₂) later. To evaluate E₂ priming, mink were treated with E₂ for 3 days, then P₄ for an additional 3 days (E₂→P₄) and uteri collected 24 h later. Percent glycogen content of uterine epithelia was greater at E₂ + 96 h (GE = 5.71 ± 0.55; LE = 11.54 ± 2.32) than E₂+24 h (GE = 3.63 ± 0.71; LE = 2.82 ± 1.03), and both were higher than controls (GE = 0.27 ± 0.15; LE = 0.54 ± 0.30; P < 0.05). Treatment as E₂→P₄ reduced glycogen content (GE = 0.61 ± 0.16; LE = 0.51 ± 0.13), to levels not different from controls, while concomitantly increasing catabolic enzyme (glycogen phosphorylase m and glucose‐6‐phosphatase) gene expression and amount of phospho‐glycogen synthase protein (inactive) in uterine homogenates. Interestingly, E₂→P₄ increased glycogen synthase 1 messenger RNA (mRNA) and hexokinase 1mRNA and protein. Our findings suggest to us that while E₂ promotes glycogen accumulation by the mink uterus during estrus and pregnancy, it is P₄ that induces uterine glycogen catabolism, releasing the glucose that is essential to support pre‐embryonic survival and implantation.     

The effects of L‐659,066, a peripheral α2‐adrenoceptor antagonist,and verapamil on the cardiovascular influences of dexmedetomidine in conscious sheep

Raekallio M. R., Honkavaara J. M., Vainio O. M. The effects of L‐659,066, a peripheral α₂‐adrenoceptor antagonist, and verapamil on the cardiovascular influences of dexmedetomidine in conscious sheep. J. vet. Pharmacol. Therap. 33 , 434–438. We investigated whether administration of L‐659,066, a peripheral α₂‐adrenoceptor antagonist, or verapamil, a calcium‐channel antagonist, would prevent the cardiovascular effects of dexmedetomidine. Eleven sheep received three intravenous treatments with a randomized, cross‐over design: dexmedetomidine (5 μg/kg, DEX); DEX with L‐659,066 (250 μg/kg, DEX + L); and verapamil (0.05 mg/kg) 10 min prior to DEX (Ver + DEX). Haemodynamics were recorded at intervals upto 40 min. Acute increases in mean arterial pressure (MAP) (106 ± 10.7 to 120.8 ± 11.7 mmHg), central venous pressure (CVP) (3.3 ± 3.2 to 14.7 ± 5.0 mmHg) and systemic vascular resistance (SVR) (1579 ± 338 to 2301 ± 523 dyne s/cm⁵), and decreases in cardiac output (CO) (5.36 ± 0.87 to 3.93 ± 1.30 L/min) and heart rate (HR) (88.6 ± 15.3 to 49.7 ± 5.5/min) were detected with DEX. The peak SVR remained lower after Ver + DEX (1835 ± 226 dyne s/cm⁵) than DEX alone, but the other parameters did not significantly differ between these treatments. 2 min after drug delivery, differences between DEX and DEX + L were statistically significant for all measured haemodynamic parameters. With DEX + L, an early decrease in MAP (99.9 ± 6.8 to 89.3 ± 6.6 mmHg) was detected, and DEX + L induced a slight but significant increase in CVP and a decrease in HR at the end of the observation period, while SVR and CO did not significantly change. All animals were assessed as deeply sedated from 2–20 min with no differences between treatments. L‐659,066 has great potential for clinical use to prevent the cardiovascular effects of dexmedetomidine mediated by peripheral α₂‐adrenoceptors, whereas the effects of verapamil were marginal.     

Effects of Ram Introduction After the Second Prostaglandin F2α Injection on Day 11 on the LH Surge Characteristics in Fat-Tailed Ewes

The aim of this study was to evaluate the effects of ram introduction after the second prostaglandin F_2α (PG F_2α) injection on day 11 on the secretion characteristics of pre‐ovulatory LH surge of fat‐tailed ewes. Multiparous Morkaraman ewes (n=12) were divided into three groups by balancing the groups for liveweight (BW) and body condition score (BCS). On the day of second PGF_2α injection (0 h), performance tested rams (n=2) were either introduced to the ewes at 0 h (ram 0 group, n=4) or at 18 h (ram 18 group, n=4) or were not introduced (control group, n=4). Blood samples were collected at 6, 18, 42, 48, 56, 62, 66, 70, 74, 78 and 90 h for the determination of pre‐ovulatory LH surge. BCS and BW during the experimental period were 2.2 ± 0.2 units and 50.9 ± 2.3 kg, 2.4 ± 0.4 units and 49.2 ± 6.2 kg, 2.1 ± 0.3 units and 45.9 ± 4.4 kg, respectively for the ram 0, ram 18 and control groups (p > 0.05). No significant difference was observed in LH surge characteristics for the experimental groups. Peak LH concentrations were also not different between groups (p > 0.05) and they were 12.2 ± 8.3, 29.1 ± 9.9 and 15.8 ± 9.5 μg/l for the ram 0, ram 18 and control groups, respectively. There was, however, a significant correlation between peak LH concentrations and BCS (p < 0.05, R²=0.373). In conclusion, it appears that, compared with ram introduction, variability in body condition of the ewe has much pronounced effect on the amount of LH secreted after the usage of two PGF_2α injections (11 days apart) as a tool for oestrus synchronization.     

EFFECTS OF INTRAVENOUS DEXMEDETOMIDINE ON CARDIAC CHARACTERISTICS MEASURED USING RADIOGRAPHY AND ECHOCARDIOGRAPHY IN SIX HEALTHY DOGS

Dexmedetomidine is a highly specific and selective α2‐adrenergic receptor agonist widely used in dogs for sedation or analgesia. We hypothesized that dexmedetomidine may cause significant changes in radiographic and echocardiographic measurements. The objective of this prospective cross‐sectional study was to test this hypothesis in a sample of six healthy dogs. Staff‐owned dogs were recruited and received a single dose of dexmedetomidine 250 μg/m² intravenously. Thoracic radiography and echocardiography were performed 1 h before treatment, and repeated 10 and 30 min after treatment, respectively. One observer recorded cardiac measurements from radiographs and another observer recorded echocardiographic measurements. Vertebral heart score and cardiac size to thorax ratio on the ventrodorsal projection increased from 9.8 ± 0.6 v to 10.3 ± 0.7 v (P = 0.0007) and 0.61 ± 0.04 to 0.68 ± 0.03 (P = 0.0109), respectively. E point‐to‐septal separation and left ventricle internal diameter in diastole and systole increased from 2.4 ± 1.1 to 6.6 ± 1.9 mm, 32.3 ± 8.1 to 35.5 ± 8.8 mm, and 19.4 ± 6 to 27.0 ± 7.2 mm, respectively (P < 0.05). Fractional shortening and sphericity index decreased from 40.7 ± 5.8 to 24.4 ± 2.9%, and 1.81 ± 0.07 to 1.58 ± 0.04, respectively (P < 0.05). Moderate‐to‐severe mitral regurgitation and mild pulmonic regurgitation occurred in all dogs after dexmedetomidine administration. Findings indicated that dexmedetomidine could cause false‐positive diagnoses of valvular regurgitation and cardiomegaly in dogs undergoing thoracic radiography and echocardiography.     

Oestradiol stimulates the release of AVP and GnRH from the ewe hypothalamus in vitro.

Oestradiol (E(2)) sensitizes the stress and reproductive axes in vivo. Our current aim is to investigate whether E(2) directly influences hypothalamic AVP and GnRH release in vitro. Within 10 min of ewe killing, saggital midline hypothalamic slices (from the anterior preoptic area to mediobasal hypothalamus, 2 mm thick, two per sheep) were dissected, placed in oxygenated MEM-alpha at 4 degrees C and within next 2 h were singly perifused at 37 degrees C with oxygenated MEM-alpha (pH 7.4; flow rate 150 microl/min) alone (vehicle; n = 15), with low (6 pg/ml; n = 14) or high E(2) (24 pg/ml; n = 13). After 5 h equilibration, 10 min fractions were collected for 3 h with exposure to 100 mm KCl for 10 min within the last hour. Concentrations of AVP and GnRH were measured by RIA. Baselines for AVP and GnRH were 7.0 +/- 1.1 and 17.4 +/- 0.8 pg/ml respectively. Basal values with low E(2) were similar to vehicle for AVP (7.5 +/- 1.2 pg/ml) and GnRH (17.5 +/- 1.1 pg/ml). However, high E(2) increased basal AVP (11.7 +/- 1.4 pg/ml; p < 0.05) and GnRH (23.7 +/- 1.4 pg/ml; p < 0.05). After KCl, AVP and GnRH respectively, increased (p < 0.05) to 25.6 +/- 7.5 and 38.2 +/- 5.6 (vehicle), 26.3 +/- 7.5 and 23.6 +/- 2.1 (low E(2)) and 24.1 +/- 5.4 and 41.3 +/- 6.6 pg/ml (high E(2)). After KCl, maximum values of AVP occurred at 20 and GnRH at 30 min. In conclusion, high E(2) concentration augments AVP and GnRH release by direct action on the ewe hypothalamus.     

Effect of eCG dose on ovarian haemodynamics,hormonal profiles and prolificacy rate when oestrus was induced during low-breeding season in Beetal goats

The objectives of the experiment were to determine the effect of two doses of equine chorionic gonadotropin (eCG) in a standard synchronization protocol based on a short-term progesterone (P₄) priming on ovarian structures and haemodynamics, concentrations of steroid hormones and prolificacy rate when oestrus was induced during low-breeding season (LBS) in Beetal dairy goats. We hypothesized that inclusion of eCG in a short-term P₄ priming-based synchronization protocol would increase the blood perfusion to ovarian structures leading to enhance oestrous and ovulatory responses and prolificacy rate in goats. Forty-two multiparous acyclic goats were blocked by body condition and, within block, assigned randomly to receive saline as control (CON), low eCG (L-eCG; 300 IU) or high eCG (H-eCG; 600 IU) dose. Initially, a controlled internal drug release (CIDR) device was placed in the anterior vagina on d −8, followed by removal of CIDR on d −3, concurrent with the administration of PGF_2α and eCG according to their respective treatments. Goats were monitored for oestrous response. B-mode and Doppler ultrasonography was performed with 12-h interval, starting from day −3 until natural breeding (day 0), and then on days 5, 10, 15 and 20 post-breeding to monitor follicular and luteal dynamics and blood flow, respectively. Blood was sampled at 0, 12, 24, 36 and 60 h after CIDR removal to quantify plasma concentrations of estradiol-17β (E₂), whereas plasma concentrations of P₄ were assayed at days 5, 10, 15 and 20 after breeding. Pregnancy and prolificacy rates were determined at day 30 and 150 after breeding, respectively. Data were analysed with mixed-effects models, and orthogonal contrasts were used to evaluate the effect of treatment [Con vs. (½ L-eCG + ½ H-eCG)] and dose of eCG (L-eCG vs. H-eCG). Data are presented in sequence as CON, L-eCG, H-eCG (LSM ± SEM). The oestrous intensity score (152.9 vs. 182.7 vs. 186.5 ± 15.1; p = .02) was greater in eCG-treated goats as compared to CON. Administration of eCG reduced the intervals to standing oestrus (66.2 vs. 41.8 vs. 48.9 h ± 5.5; p = .05), breeding (70.2 vs. 44.4 vs. 45.4 h ± 4.5; p = .03) and ovulation (84.5 vs. 61.2 vs. 63.4 h ± 6.2; p = .05) compared with CON goats. The mean growth rate of pre-ovulatory follicle was greater (1.11 vs. 1.49 vs. 1.45 mm ± 0.08; p = .01) in eCG-treated goats resulting in an increased diameter of pre-ovulatory follicle (6.27 vs. 7.20 vs. 7.31 mm ± 0.07; p < .01) and corpora lutea (6.75 vs. 8.26 vs. 8.07 mm ± 0.42; p = .04) than CON. The mean follicular blood flow did not differ among treatments; however, the mean luteal blood flow was greater in L-eCG-treated goats (0.81 vs. 1.61 vs. 1.07 cm² ± 0.12; p = .001). The mean concentrations of E₂ (4.03 vs. 5.21 vs. 4.78 pg/ml ± 0.42; p = .04) and P₄ (4.85 vs. 6.39 vs. 6.22 ng/ml ± 0.34; p = .04) were greater in eCG-treated goats. The twinning rate did not differ between treatments; nevertheless, prolificacy rate was greater (p = .04) in L-eCG-treated goats. Collectively, our data suggest that the administration of eCG improves the induction of oestrous and ovarian dynamics. Administration of L-eCG enhances prolificacy rate, therefore, a low dose of eCG might be practically beneficial to improve reproduction during LBS in acyclic Beetal dairy goats.     

Coenzyme Q10 and α‐Tocopherol Prevent the Lipid Peroxidation of Cooled Equine Semen

Biotechnology applied for equine semen increases the levels of reactive oxygen species and reduces the natural antioxidant defence, by both dilution and removal of seminal plasma. Therefore, the aims of this study were to evaluate the effect of adding coenzyme Q10 (CoQ10) and α‐tocopherol (α‐TOH) to the cooling extender, singly or in combination, on sperm parameters, and their effectiveness in preventing lipid peroxidation (LPO) of equine semen during cooling at 5°C for 72 h. Ten adult stallions of proven fertility were used, using two ejaculates each, subjecting them to the treatments with the following concentrations: α‐TOH: 2 mm ; CoQ10: 40 μg/ml; and CoQ10 + α‐TOH: 40 μg/ml + 2 mm for control (C) without the addition of antioxidants and for vehicle control (EtOH) with 100 μl ethanol. The CoQ10 group had a higher percentage of total motility (69.1 ± 16.2%) compared to control (62.1 ± 16.2%) and EtOH (58.1 ± 18.6%). CoQ10 + α‐TOH and α‐TOH groups were most effective in preventing LPO compared to controls (1765.9 ± 695.9, 1890.8 ± 749.5, 2506.2 ± 769.4 ng malondialdehyde/10⁸ sptz, respectively). In conclusion, CoQ10 and α‐TOH were effective during the cooling process of equine semen at 5°C for 72 h, providing increased levels of total motility, as well as lower LPO.     

N-acetyl-l-cysteine supplementation improves boar spermatozoa characteristics and subsequent fertilization and embryonic development

The effects of 1.0 mm N‐acetyl‐l ‐cysteine (NAC) supplementation during the incubation of frozen–thawed and preserved boar sperm were studied in addition to subsequent oocyte IVF. Frozen–thawed and preserved boar sperm were supplemented with 1.0 mm NAC and incubated for 60 min to allow capacitation to occur followed by the addition of calcium ionophore 23187 to induce the acrosome reaction. The number of sperm having undergone the acrosome reaction was determined using the Wells–Awa staining technique. DNA damage was detected using single‐cell gel electrophoresis. Membrane lipid peroxidation was estimated by the end point generation of malondialdehyde (MDA). Frozen–thawed sperm was not different in the ability of sperm to undergo the acrosome reaction but did have significantly (p < 0.05) more DNA damage (59.8 ± 1.0) compared to preserved sperm (32.0 ± 1.0%). Supplementing 1.0 mm NAC did not have an effect on the ability of sperm to undergo the acrosome reaction but did have significantly (p < 0.05) less DNA (39.2 ± 1.0%) damage compared to no antioxidant supplementation (52.7 ± 1.0%). Frozen–thawed sperm produced a significantly higher (p < 0.05) concentration of MDA (2.08 ± 0.05 μm MDA/10⁷ cells) compared to preserved sperm (1.82 ± 0.05 μm MDA/10⁷ cells), and non‐supplemented sperm produced a significantly higher (p < 0.05) concentration of MDA (3.62 ± 0.05 μm MDA/10⁷ cells) compared to the 1.0 mm NAC‐supplemented sperm (0.28 ± 0.05 μm MDA/10⁷ cells. Supplementation or semen storage method had no effect on IVF or embryonic development. These results indicate that supplementation with 1.0 mm NAC improved the ability to use frozen–thawed boar sperm during IVF as it reduces the DNA fragmentation and lipid peroxidation of the sperm.     

Pharmacokinetics and dynamics of mycophenolate mofetil after single‐dose oral administration in juvenile dachshunds

Mycophenolate mofetil (MMF) is recommended as an alternative/complementary immunosuppressant. Pharmacokinetic and dynamic effects of MMF are unknown in young‐aged dogs. We investigated the pharmacokinetics and pharmacodynamics of single oral dose MMF metabolite, mycophenolic acid (MPA), in healthy juvenile dogs purpose‐bred for the tripeptidyl peptidase 1 gene (TPP1) mutation. The dogs were heterozygous for the mutation (nonaffected carriers). Six dogs received 13 mg/kg oral MMF and two placebo. Pharmacokinetic parameters derived from plasma MPA were evaluated. Whole‐blood mitogen‐stimulated T‐cell proliferation was determined using a flow cytometric assay. Plasma MPA C_max (mean ± SD, 9.33 ± 7.04 μg/ml) occurred at <1 hr. The AUC_0–∞ (mean ± SD, 12.84±6.62 hr*μg/ml), MRT_inf (mean ± SD, 11.09 ± 9.63 min), T1/2 (harmonic mean ± PseudoSD 5.50 ± 3.80 min), and k/d (mean ± SD, 0.002 ± 0.001 1/min). Significant differences could not be detected between % inhibition of proliferating CD5+ T lymphocytes at any time point (p = .380). No relationship was observed between MPA concentration and % inhibition of proliferating CD5+ T lymphocytes (R = .148, p = .324). Pharmacodynamics do not support the use of MMF in juvenile dogs at the administered dose based on existing therapeutic targets.