Interactions between straw size and thawing rates on the cryopreservation of agouti (Dasyprocta aguti) epididymal sperm

This study verifies the interactions between straw size and thawing rates and their impact on the epididymal sperm from this species. Caudae epididymidum from 10 agoutis were subjected to retrograde washing using a coconut water extender (ACP‐109c^®). Epididymal sperm were evaluated and extended in ACP‐109c^® plus egg yolk (20%) and glycerol (6%). The samples were packaged in 0.25‐ or 0.50‐ml straws, frozen in liquid nitrogen and thawed at 37°C/1 min or 70°C/8 s, followed by a re‐evaluation. The use of 0.25‐ml straws thawed at 37°C/1 min provided a value of 26.6% for sperm motility. No interactions between straw size and thawing rates were verified on agouti sperm (p > 0.05), but when 0.5‐ml straws were thawed at 70°C/8 s, sperm vigour decreased significantly (p < 0.05). It is recommended that the agouti epididymal sperm cryopreserved in ACP‐109c^® extender should be packaged in 0.25‐ or 0.50‐ml straws and thawed at 37°C/60 s.     

Biological efficacy and stability of diluted ticarcillin-clavulanic acid in the topical treatment of Pseudomonas aeruginosa infections

Topical compounded Timentin^® diluted with an inactive vehicle has been reported to be effective in the treatment of otitis externa caused by Pseudomonas aeruginosa. The aims of this study were to determine the biological efficacy of Timentin^® (ticarcillin and clavulanic acid) when diluted in the carrier vehicle Methopt^® against P. aeruginosa and to determine the efficacy and stability of Timentin^® aqueous stock concentrate solution. Timentin^® stock concentrate was tested against four P. aeruginosa isolates on days 0, 7, 14, 21 and 28; then after 2, 3, 4, 5, 6, 9 and 12 months of storage at 4 or ?20°C. The diluted Timentin^®–Methopt^® solutions were tested against all isolates after 0, 2, 4, 6, 8, 10, 12, 14, 17, 21, 24 and 28 days of storage at 24 or 4°C. Minimal inhibitory concentration (MIC) levels for all strains were determined using the broth microdilution method. The MIC of the stock solution remained relatively constant and acceptable throughout the study when stored at ?20°C and was also acceptable for shorter time periods (6–9 months) when stored at 4°C. The MIC for the diluted Timentin^®–Methopt^® solution remained relatively constant and acceptable throughout the study for all four bacterial strains, with no difference between the solutions stored at 4 or 24°C. The results of this study indicate that storage of the Timentin^® stock solution at ?20°C does not compromise efficacy for at least 12 months and that Timentin^® diluted in Methopt^® was stable for 28 days when stored at either 4 or 24°C.     

In Vitro Evaluation of Liquid‐stored Buffalo Semen at 5°C Diluted in Soya Lecithin Based Extender (Bioxcell®), Tris‐Citric Egg Yolk,Skim Milk and Egg Yolk‐Citrate Extenders

This study was designed to compare the quality of liquid‐stored buffalo bull spermatozoa in soya lecithin based extender Bioxcell^® (BIOX), milk (MILK), tris‐citric egg yolk (TEY) and egg yolk‐citrate (EYC) extender at 5°C. Semen was collected from five Nili‐Ravi buffalo (Bubalus bubalis) bulls of 6–7 years of age with artificial vagina over a period of 3 weeks (two consecutive ejaculates once in a week). Semen ejaculates having more than 60% motility were pooled, split into four aliquots, diluted (37°C; 10 × 10⁶ motile spermatozoa/ml), cooled from 37 to 5°C in 2 h (0.275°C/min) and stored for 5 days. Sperm motility, viability, plasma membrane integrity (PMI) and normal acrosomal ridge were studied at first, third and fifth day of storage. Higher values of progressive sperm motility (%), sperm viability (%), sperm PMI (%) and normal apical ridge (%) were observed in BIOX, MILK and TEY extenders at first, third and fifth day of storage than EYC extender. Progressive sperm motility, sperm viability and sperm PMI in BIOX^® extender were not different from MILK and TEY extenders at 1st and third day storage period. However, at fifth day of storage, the values for these parameters remained significantly higher (p < 0.05) in BIOX^® compared with MILK, TEY and EYC extenders. At fifth day of storage, the semen quality parameters for Bioxcell^® were comparable to those with MILK and TEY extenders at third day of storage. In conclusion, motility, viability and PMI of buffalo bull spermatozoa remained similar in Bioxcell^®, milk and TEY extender at first and third days of storage at 5°C. Yet, the values for the aforementioned parameters in Bioxcell^® were higher compared with milk, TEY and EYC extender at fifth day of storage at 5°C.     

Contagious equine metritis: Antibody response of experimentally infected pony mares

Intrauterine inoculation of pony mares with the bacterium that is the causative agent of contagious equine metritis (CEM) resulted in clinical disease. A humoral immune response could be detected by agglutination and complement fixation (CF), and in some cases precipitating antibody was found by immunodiffusion tests. Agglutinating antibody was the most reliable serological indicator of overt infection and was detected in 8 of 28 mares after initial intrauterine inoculation of 3–4 × 10⁵ bacteria. Seventy percent of mares given a second inoculation and all mares given a third inoculation of 3–4 × 10⁵ bacteria produced detectable agglutinating antibody. Only two of five mares given the third inoculation developed detectable complement-fixing antibody. Only one mare showed evidence of reinfection after a second or third intrauterine inoculation. All of the mares given a single intrauterine inoculum of ≥ 8 × 10⁸ bacteria produced agglutinating antibody 10 to 30 days postinoculation (DPI) and 86% gave a positive CF test 10 to 20 DPI. Only mares with an agglutination titer of 320 or more produced precipitating antibody. Sera were considered positive in agglutination tests if they were reactive at a dilution of greater than 4 and positive in CF tests if they were reactive at a dilution of 4 or greater.Pony serum frozen at ?70°C was anticomplementary (AC). Treatment at 56°C abolished AC activity and revealed enhancing or procomplementary activity with guinea pig complement. Procomplementary activity could be abolished by treatment of heated pony serum with formaldehyde, which increased CF titers ≥ threefold in weakly reactive sera.     

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.     

The effects of decreased body temperature on the onset,duration and action of medetomidine and its antagonist atipamezole in juvenile farmed estuarine crocodiles (Crocodylus porosus)

ObjectiveTo determine the efficacy of medetomidine for immobilisation of captive juvenile crocodiles over a range of temperatures, and its reversibility with atipamezole.Study designProspective experimental study.AnimalsForty male estuarine crocodiles (body weight 2.0 to 4.8 kg).MethodsEach crocodile was randomly assigned to one of four temperature groups: Group 1:32 °C; Group 2:27 °C; Group 3:22 °C; and Group 4:17 °C (n = 10 for each group). Medetomidine (0.5 mg kg^?1) was administered intramuscularly (IM) into the thoracic limb of all crocodiles. After 50 minutes, all animals from each group received 2.5 mg kg^?1 atipamezole IM in the opposite thoracic limb and time to recovery was documented. Heart and respiratory rates and the degree of immobilisation were monitored every 5 minutes until recovery, and behaviour monitored for 7 subsequent days.ResultsOnset of immobilisation occurred at 15 ± 10 minutes in Group 1, and at 30 ± 10 minutes in Groups 2 and 3. In Group 4, animals were not immobilised. Recovery following atipamezole was 10 ± 5 minutes at all temperatures. One-way analysis of variance (anova) demonstrated a significant difference in induction times between groups (p < 0.01) but not in recovery times following atipamezole administration (p < 0.25). Heart and respiratory rates decreased markedly following medetomidine administration and increased markedly following atipamezole reversal.Conclusions and clinical relevanceMedetomidine administered in the thoracic limb of juvenile captive estuarine crocodiles provides profound sedation or immobilisation at temperatures of 22 °C and above. Atipamezole administered in the contralateral thoracic limb results in consistent reversal of the effects of medetomidine and a return to normal behaviour within 15–20 minutes regardless of temperature. Even though immobilisation is not induced at 17 °C, profound reversible sedation does occur reliably and repeatably.     

Comparative in vitro efficacy of antimicrobial shampoos: a pilot study

This study compared the antimicrobial efficacy of shampoos against meticillin‐sensitive Staphylococcus pseudintermedius (MSSP), meticillin‐resistant S. pseudintermedius (MRSP), antibiotic‐sensitive Pseudomonas aeruginosa (PA), multidrug‐resistant P. aeruginosa (MDR‐PA) and Malassezia pachydermatis. Three isolates were incubated for 10, 30 and 60 min with each shampoo diluted in phosphate‐buffered saline. Aliquots were then incubated for 16–18 h on sheep blood agar (bacteria) or for 3 days on Sabouraud’s dextrose agar (Malassezia). The minimal bactericidal concentrations (MBCs) for chlorhexidine products (Malaseb^®, Pyoderm^®/Microbex^® and Hibiscrub^®) were 1:1,024–1:2,048 for MSSP and MRSP, 1:512–1:1,024 for PA and MDR‐PA, and 1:2,048–1:5,096 for Malassezia at all time points. The MBCs for benzoyl peroxide (Paxcutol^®) for MSSP and MRSP were 1:2–1:8 at 10 min, and 1:256 after 30 and 60 min. A 1:2 dilution was effective against Pseudomonas, and 1:512–1:1,024 dilutions were effective against Malassezia at all time points. The MBCs for ethyl lactate (Etiderm^®) for MSSP and MRSP were 1:2 at 10 min, and 1:2–1:16 after 30 and 60 min. A 1:2 dilution was effective against Pseudomonas, and a 1:512 dilution was effective against Malassezia at all time points. Chloroxylenol (Coatex^®) and acetic acid–boric acid (Malacetic^®) were not effective against MSSP, MRSP or Pseudomonas. Both were effective against Malassezia at 1:8–1:16 dilution at 10 min, and at 1:8–1:32 dilution after 30 and 60 min. In conclusion, chlorhexidine appeared to be the most effective topical biocide, and MRSP and MDR‐PA were no less susceptible than antibiotic‐sensitive organisms. These results should, however, be confirmed with larger numbers of isolates.     

Accuracy of the thermodilution method in estimating high flow - an in vitro study

The accuracy of thermodilution for measuring flow rates of 10–40 L/min was evaluated using a commercially available thermodilution cardiac output computer in an in vitro model. Water (36.5–37.5°C) was directed through a mixing chamber via a constant flow pump. Thermodilution estimates of flow using four different volumes (10, 20, 30, 40 ml) of iced water injectate were compared to simultaneous measurements of timed samples of effluent from the mixing chamber. Injectate volume had a significant impact on the accuracy of thermodilution estimation (p < 0.05). Thermodilution overestimated measured flow when 10 and 20 ml of injectate were used to determine flow rates < 20 L/min but underestimated flow when injectate volumes of 30 and 40 ml were used, or when measured flow was > 25 L/min. The discrepancy between thermodilution flow and measured flow increased as rate of fluid flow increased.     

Early Pregnancy Detection of Iraqi Riverine Buffalo (Bubalus bubalis) Using the BioPRYN Enzyme‐Linked Immunosorbent Assay for PSPB and the Progesterone Assay

This study was undertaken to detect pregnancy in Iraqi riverine buffalo (Bubalus bubalis) using three different methods (rectal palpation, plasma progesterone concentration and detection of the presence of pregnancy‐specific protein B (PSPB) with the BioPRYN^® enzyme‐linked immunosorbent assay (ELISA) test. The aim of the study was to identify the most sensitive, early and accurate method for detecting pregnancy. Twenty‐two female riverine buffalo that were 6.0 ± 0.93 years old were used. Four blood samples per buffalo were taken via jugular venipuncture at days 22–24, 32–34, 42–44 and 58–61 post‐mating (PM) to measure the progesterone concentration (ng/ml) and to detect the presence of plasma PSPB. The rectal palpation method was employed to evaluate all buffalo on days 42–44 and 58–61 PM. The BioPRYN^® test differed (p < 0.01) from the other tests with earlier accuracy for detecting pregnant and non‐pregnant buffalo. Eighty‐eight percent of pregnant and 76.9% of non‐pregnant buffalo were distinguished early (days 22–24 PM) using BioPRYN^® and plasma PSPB‐ELISA level (2.09 ± 0.12 ng/ml) in relation to 66.7% and 53.9% detected using the progesterone assay at similar days (4.30 ± 0.40 ng/ml). In conclusion, these results described, for the first time, the early and accurate pregnancy detection of water riverine buffalo using BioPRYN^® technology and provided the plasma levels of PSPB using an ELISA test. These findings will improve the reproductive and productive efficiency of Iraqi riverine buffalo by adapting the recent management and reproductive strategies in Iraq and in the world.     

Comparative in vitro antimicrobial efficacy of commercial ear cleaners

The aim of this study was to compare the antimicrobial efficacy of ear cleaners against Staphylococcus intermedius, Pseudomonas aeruginosa and Malassezia pachydermatis. Single isolates of each organism were incubated in duplicate at 38 °C for 30 min with each ear cleaner diluted 1/2 to1/256 in phosphate‐buffered saline. Positive and negative controls were included. Aliquots were then incubated for 16–18 h on sheep blood agar (bacteria) or for 3 days on Sabouraud's dextrose agar (Malassezia) at 38 °C. The lowest dilutions exhibiting 100% antimicrobial efficacy for S. intermedius were: Cleanaural^® Dog 1/32; Sancerum^® 1/16; Otoclean^® 1/4; EpiOtic^® 1/2; MalAcetic^® 1/2; and Triz Plus^® 1/2. The results for P. aeruginosa were Sancerum^® and Triz Plus^® 1/16; Cleanaural^® Dog and EpiOtic^® 1/8; Otoclean^® 1/4; and MalAcetic^® 1/2. Results for M. pachydermatis were: Cleanaural^® Dog 1/32; Sancerum^®, Otoclean^®, EpiOtic^® and Triz Plus^® 1/8; and MalAcetic^® 1/4. Cleanaural^® Cat, MalAcetic HC^® and Triz EDTA^® did not display any antimicrobial activity at any dilution. Antimicrobial activity appeared to be associated with the presence of isopropyl alcohol, parachlorometaxylenol and a low pH. The results of this study may help clinicians make evidence‐based decisions when selecting ear cleaners for use in individual cases.     

Evaluation of transpulmonary ultrasound dilution cardiac output in piglets: accuracy,precision and trending ability with room temperature injectate

ObjectiveTranspulmonary ultrasound dilution (TPUD) is a minimally invasive technique to measure cardiac output (CO) using a 1 mL kg^–1 isotonic 37 °C saline injectate indicator. The objective was to evaluate the performance of TPUD using a room temperature saline injectate.Study designProspective experimental trial.AnimalsA total of seven anesthetized male Yorkshire piglets.MethodsPiglets aged 1 month and weighing 7.7–9.0 kg were anesthetized with detomidine–ketamine–hydromorphone–isoflurane and a pulmonary artery flow probe (PAFP) placed via a median sternotomy. The thoracic cavity remained open during measurement of CO by PAFP and TPUD. The TPUD indicators of 1 mL kg^–1 0.9% saline at 37 °C and 20 °C were compared during infusions of phenylephrine and dobutamine, blood withdrawal and replacement. Bias, limits of agreement (LoAs) and percentage error (PE) between each iteration of PAFP and TPUD were measured with Bland–Altman plots. Trending ability via concordance, angular bias and radial LoA were compared.ResultsBland–Altman plots showed negligible bias with varying LoAs. PEs of 22% and 38% were found for 37 °C and 20 °C saline injectates, respectively. In the four-quadrant plots, the concordance rate was 94% and 100% for measurements obtained with 37 °C and 20 °C saline injectates, respectively. Angular bias for both were < ±5 °, with radial LoA < ±7 °.ConclusionsTPUD was accurate when using 1 mL kg^–1 of isotonic saline at 37 °C in a range of CO within 0.2–0.8 L minute^–1, and it reliably tracked positive and negative changes in CO. Room temperature (20 °C) indicator was less accurate but equally able to track direction of changes in CO.Clinical relevanceThe use of room temperature injectates allows an easy, readily available clinical application of TPUD CO monitoring while preserving the trending ability of the monitor.     

Evaluation of outflow temperatures generated by a dry heat fluid warmer at low fluid rates

ObjectiveTo evaluate the output temperature of a dry heat fluid warmer at fluid rates typically used in small animal veterinary patients.Study designProspective in vitro study.AnimalsNone.MethodsAmbient temperature lactated Ringer’s (17.9-18.8 °C) was delivered via a dry heat fluid warmer. A temperature probe was used to measure fluid outflow temperature from the compatible giving set at 5, 10, 20, 50, 70, 80, 100, 200, 300, 400 and 500 mL hour^–1. Outflow fluid temperature at plateau (two consecutive readings within 0.1 °C) was compared with baseline fluid temperature (fluid warmer turned off) to calculate temperature changes at each rate. Kruskal–Wallis test was used to compare changes in temperature and time to plateau temperature. Dunn’s post hoc test was used to test for significant differences in temperature compared to 5 mL hour^–1; p value < 0.05.ResultsMedian plateau outflow temperature increased as fluid rate increased, with temperatures of 18.5, 18.6, 18.7, 18.8, 19.4, 19.4, 21.5, 25.3, 28.5, 30.7 and 32.6 °C, at flow rates of 5, 10, 20, 50, 70, 80, 100, 200, 300, 400 and 500 mL hour^–1. Fluid rates > 100 mL hour^–1 showed significant increases from baseline (p = 0.021) There was no difference in temperature change from baseline at fluid rates < 100 mL hour^–1 (p > 0.05). Compared to plateau temperature at 5 mL hour^-1, there was a statistical difference in plateau temperature above 100 mL hour^–1 (p = 0.0207). Maximum outflow plateau temperature was 32.6 °C at 500 mL hour^–1.Conclusions and clinical relevanceA dry heat fluid warmer has significantly decreased efficacy at low fluid rates, with no statistically significant increase in fluid temperature at fluid rates below 100 mL hour^–1 at the end of a compatible fluid line. Inline dry heat fluid warmers are ineffective at fluid rates below 100 mL hour^–1.     

Functional Sperm Parameters and Fertility of Bull Semen Extended in Biociphos-Plus® and Triladyl®

Twenty ejaculates from five dairy AI‐bulls were used to compare, in a split‐sample experiment, the fertility [56 day‐non‐return‐rate (NRR) from more than 14000 AI) and sperm viability post‐thaw of semen diluted with an egg yolk‐ (Triladyl®) or soybean‐based (Biociphos‐Plus®) commercial extender. The in vitro evaluations were divided in two experiments. Experiment 1 (n = 20) included post‐thaw evaluations of motility (subjective and computerized), membrane integrity (CalceinAM/EthD‐1, SYBR‐14/PI, and osmotic resistance test; ORT), and capacitation status (CTC/EthD‐1). Experiment 2 (n = 10) included evaluations of the capacitation‐(CTC/EthD‐1) and acrosome status (FITC‐PSA/EthD‐1) during incubation with/without a challenge with solubilized zona pellucida proteins (SZP). No significant difference in the fertility (69.1 ± 0.8 versus 69.2 ± 0.8) results was found between the two extenders. In experiment 1, the computerized motility evaluations post‐thaw (CASA) showed higher values for Biociphos‐Plus® processed semen for the velocity patterns and lateral sperm head displacement. After 6 h at room temperature (20–22°C) all the CASA motility patterns were significantly higher for Biociphos‐Plus®. The proportion of spermatozoa with intact membranes assessed by CalceinAM was significantly higher in Biociphos‐Plus® (p < 0.001) compared to Triladyl®, but such difference was not seen when using SYBR‐14 or the ORT‐assay. When using the CTC/EthD‐1 assay, a lower proportion of acrosome reacted (AR) spermatozoa post‐thaw (p < 0.01) was found in Biociphos‐Plus® processed semen, as well as a tendency (p < 0.07) for a higher number of uncapacitated spermatozoa. In experiment 2, the proportion of uncapacitated spermatozoa was significantly higher for Biociphos‐Plus® when semen was incubated (38°C and 5% CO₂) without SZP at both 0 (p < 0.001) and 30 min (p < 0.05). Concomitantly, Triladyl® showed a higher percentage of capacitated spermatozoa at 0 (p < 0.01), 30 (p < 0.05) and 120 min (p < 0.05). A higher (p < 0.05) incidence of AR‐spermatozoa was seen in Triladyl® at the beginning of the incubation with SZP. No significant difference between extenders was detected for the acrosome status by the FITC‐PSA‐assay. Incubation with SZP induced acrosome reaction of capacitated spermatozoa in both extenders, which was detected by CTC and FITC‐PSA assays. In conclusion, fertility was not affected by Biociphos‐Plus® when 15 × 10⁶ of spermatozoa per AI dose were inseminated. The finding that higher frequencies of spermatozoa seemed more membrane stable post‐thaw, when frozen in Biociphos‐Plus®, might indicate that this extender better protects the sperm viability compared with Triladyl®.     

Chilled storage of ram semen improves with the addition of egg yolk and glycerol to milk-based extenders

This study aimed at comparing in vitro, ultra‐heat‐treated (UHT) skim milk and INRA‐96^®‐based extenders supplemented or not with 5% egg yolk and/or 2% glycerol on sperm quality parameters along 72 h of preservation at 5°C, using a factorial design. Semen from six healthy mature Merino rams was pooled and extended in each medium using a split sample procedure (six replicates) and chilled. Subjective motility (SM) (%), membrane integrity (MI) (%) and uncapacitated spermatozoa (US) (×10⁶ spermatozoa/AI dose) were used to assess the semen quality at 0, 12, 24, 48 and 72 h of preservation. UHT‐based extenders yielded better (p < 0.05) SM and MI than INRA‐96^®‐based extenders (59.7% vs 57.9%; 60.2% vs 55.8%, respectively) but similar numbers of US (64.2% vs 62.3 × 10⁶ sperm/AI dose, respectively) along the preservation time. Egg yolk–glycerol or just egg yolk as additives improved (p < 0.05) the results compared with the base extenders without additives or just with glycerol. The sperm parameters assessed decline slowly from 0 to 48 h, with a sharp decline (p < 0.05) at 72 h of preservation. In conclusion, UHT and INRA‐96^® were similar as base extenders, and the addition of 5% egg yolk plus 2% glycerol or just 5% egg yolk improved the quality of ram semen preserved at 5°C, at least for 48 h. The combination of egg yolk–glycerol might provide extra protection in case of fluctuation of temperatures below 5°C, commonly seen under field conditions.     

Intramuscular administration of alfaxalone in red‐eared sliders (Trachemys scripta elegans) – effects of dose and body temperature

ObjectiveTo characterise the effects of alfaxalone by intramuscular (IM) injection in red-eared slider turtles and the influence of body temperature on anaesthetic duration and depth.Study designProspective, randomised part-blinded experimental trial.AnimalsTen healthy adult female red-eared sliders.MethodsEach turtle was anaesthetized four times with 10 and 20 mg kg^?1 alfaxalone at 20 and 35 °C respectively. Time to maximal effect and plateau and recovery periods were recorded. Skeletal muscle tone, presence of various reflexes, response to noxious stimuli, and heart rate were assessed.ResultsResults are given for protocols 10 mg kg^?1 20 °C; 20 mg kg^?1 20 °C; 10 mg kg^?1 35 °C and 20 mg kg^?1 35 °C, respectively: mean time (±SD) to maximal effect was 16 ± 8, 19 ± 6, 5 ± 2 and 7 ± 5 minutes; duration of the plateau phase was 13 ± 12, 28 ± 13, 8 ± 5 and 8 ± 5 minutes and recovery time was 76 ± 20, 126 ± 17, 28 ± 9 and 41 ± 20 minutes. Endotracheal intubation was successful in 80%, 100%, 0% and 30% of turtles, respectively. At 35 °C, all animals retained nociceptive sensation in the front limbs, hind limbs and vent, whereas at 20 °C a few turtles lost peripheral nociceptive sensation. Corneal and tap reflexes were retained in all trials. Mean heart rates were 30 ± 2 and 66 ± 4 beats minute^?1 at 20 and 35 °C, respectively.Conclusions and clinical relevanceAlfaxalone administered IM in red-eared sliders provided smooth, rapid induction and uneventful recovery. At 35 °C either dosage provided only short (5–10 minutes) and light sedation. At 20 °C, 10 mg kg^?1 provided sedation suitable for short non-invasive procedures. About 20 mg kg^?1 provided anaesthesia of approximately 20 minutes duration, appropriate for induction of inhalational anaesthesia or for brief surgical procedures with supplemental analgesia.     

Characteristics of Buck Semen with Regard to Ejaculate Numbers, Collection Intervals, Diluents and Preservation Periods

To determine the number of ejaculates which can be collected within a 20‐min period after the smallest number of days of sexual rest, and a good diluent to preserve semen for routine AI, five mature Black Bengal bucks were used in three experiments. In experiment 1, semen from the bucks were collected by using artificial vagina at homosexual mounts as many times as possible during 20 min. The ejaculate numbers 1, 3 and 4 (or 5 when the buck could produce it) were examined for important semen characteristics. The mean ejaculate volume, density, mass activity, sperm motility, sperm concentrations, total spermatozoa/ejaculate, proportion of spermatozoa with normal acrosome, midpiece and tail, and the proportion with normal head morphology varied between 267 and 342 µl, 4.1–4.5 (1–5 scale), 4.1–4.2 (1–5 scale), 77–79%, 4187 × 10⁶–5064 × 10⁶/ml, 1140 × 10⁶–1746 × 10⁶, 91–94% and 99%, respectively, depending on the collection number of the ejaculate. The difference between the ejaculates was significant only with respect to volume (p < 0.05). In experiment 2, semen was collected from the bucks successively during 20 min after 1, 2, 3 and 4 day intervals, and the first ejaculates were evaluated for the above‐mentioned semen characteristics. Semen collected after 2 or more day intervals had significantly higher volume, sperm concentration and total spermatozoa/ejaculate (p < 0.05). In experiment 3, pools of two to three ejaculates were diluted (1 : 5; semen : diluent) in splits with glucose‐citrate‐egg yolk (GCEY), Tris‐fructose‐egg yolk (TFEY) or skim milk (SM) and preserved at +4 to +7°C. Before chilling or after 0 (15 min chilling), 1, 2, 3 and 4 days of preservation, semen was evaluated for motility and proportion of normal spermatozoa with respect to acrosome, midpiece and tail. In data pooled across the bucks, the sperm motility was better in GCEY and TFEY than in SM, and the proportion of normal spermatozoa was higher in SM than in the others (p < 0.05). However, the differences in proportion of normal spermatozoa between diluents were not significant when the data were analysed separately within preservation periods. The sperm motility consistently dropped after 1 day of preservation (p < 0.01); the motility remained 50% or more up to 4 days in TFEY, 3 days in GCEY and only 2 days in SM. The proportion of spermatozoa with normal acrosome, midpiece and tail, which was generally quite high ( 90%), decreased after 3 days of preservation (p < 0.01). We conclude that Black Bengal bucks can be collected three times during 20 min, every 3 days, and that buck semen holds good motility and proportion of normal spermatozoa up to 3 days in GCEY or TFEY at 4 to 7°C.     

An investigation into the suitability of a commercial real‐time PCR assay to screen for Taylorella equigenitalis in routine prebreeding equine genital swabs

Reasons for performing study: Standard bacteriological methods for identifying Taylorella equigenitalis in cervical smears are time consuming. Therefore, a more rapid real‐time PCR assay was evaluated for its suitability in screening swabs. Objective: To compare the results of a commercially available real‐time PCR assay with routine microbiological culture for the identification of T. equigenitalis, the causative organism of contagious equine metritis, in equine genital swab samples, under ‘field trial’ conditions. Materials and methods: Routine prebreeding genital swabs (n = 2072) collected from Thoroughbred mares and stallions during 2009 were examined together with stored T. equigenitalis positive material. Swabs were cultured for T. equigenitalis using standard microbiological techniques. Bacterial lysates were isolated from the swabs and examined for the presence of a 16S DNA fragment of T. equigenitalis, using a commercial multiplex real‐time PCR assay system. Results: There was complete concordance between positive and negative results obtained by the 2 methods. Real‐time PCR also detected T. equigenitalis DNA from swabs that were negative using standard microbiological culture after 6 months' storage at +4°C but from which T. equigenitalis had been isolated following collection. The sensitivities of realtime PCR and bacterial culture were both 10^?3 (equivalent to 3 colony‐forming units). Conclusion and clinical relevance: Routine bacterial culture of T. equigenitalis requires an incubation period of not less than 7 days before a conclusive negative result can be obtained, whereas bacterial extraction and real‐time PCR assay can be completed in less than 6 h. The commercially‐available PCR assay tested provided a rapid and reliable method for the identification of T. equigenitalis from equine genital swabs and could be usefully employed for the screening of mares and stallions for preseason Horserace Betting Levy Board (HBLB) Code of Practice and in other situations such as for bloodstock sales screening requirements, overcoming the current delays imposed by bacterial culture requirements. Its use could be quality assured by the existing HBLB biannual testing scheme for designated laboratories.     

The Effect of Glycerol Concentrations on the Post‐thaw In Vitro Characteristics of Cryopreserved Sex‐sorted Boar Spermatozoa

The objective of this study was to optimize protocols for the cryopreservation of sex‐sorted boar spermatozoa. In the experiment 1, we evaluated the effects of a standard boar sperm cryopreservation procedure (3% final glycerol concentration) on the in vitro characteristics of sex‐sorted sperm frozen at low sperm concentrations (20 × 10⁶ sperm/ml; S20 group). Non‐sorted spermatozoa frozen at 1000 × 10⁶ (C1000 group) and 20 × 10⁶ (C20 group) sperm/ml were used as the freezing control groups. In experiment 2, the effects of different final glycerol concentrations (0.16%, 0.5%, 1.0%, 2.0% and 3.0%) on post‐thaw quality of the S20 and C20 groups were evaluated. In both experiments, the samples were evaluated prior to freezing (5°C) and at 30, 90 and 150 min after thawing. Experiment 1 indicated that freezing sperm at low concentrations decreased (p < 0.05) the total motility (TM) and progressive motility (PM) at 90 and 150 min after thawing regardless of whether the sperm were sorted or not. However, the sperm membrane integrity was not affected at any evaluation step. Inexperiment 2, significant effects on the TM and PM because of increased glycerol concentrations in the S20 and C20 groups were observed only at 90 and 150 min after thawing. The samples frozen in 3% glycerol showed lower (p < 0.05) TM and PM values when compared to those frozen in the presence of 0.5% and 1% glycerol. In both experiments, non‐sorted control samples displayed higher percentages of spermatozoa with damaged DNA than sorted spermatozoa. In conclusion, the optimization of cryopreservation conditions by decreasing the glycerol concentrations can improve post‐thaw motility of sex‐sorted spermatozoa frozen at low concentrations.