Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.     

The Effects of Hoechst 33342 Staining and the Male Sample Donor on the Sorting Efficiency of Canine Spermatozoa

The aim of this study was to evaluate the influence of Hoechst 33342 (H‐42) concentration and of the male donor on the efficiency of sex‐sorting procedure in canine spermatozoa. Semen samples from six dogs (three ejaculates/dog) were diluted to 100 × 10⁶ sperm/ml, split into four aliquots, stained with increasing H‐42 concentrations (5, 7.5, 10 and 12.5 μl, respectively) and sorted by flow cytometry. The rates of non‐viable (FDA+), oriented (OS) and selected spermatozoa (SS), as well as the average sorting rates (SR, sorted spermatozoa/s), were used to determine the sorting efficiency. The effects of the sorting procedure on the quality of sorted spermatozoa were evaluated in terms of total motility (TM), percentage of viable spermatozoa (spermatozoa with membrane and acrosomal integrity) and percentage of spermatozoa with reacted/damaged acrosomes. X‐ and Y‐chromosome‐bearing sperm populations were identified in all of the samples stained with 7.5, 10 and 12.5 μl of H‐42, while these two populations were only identified in 77.5% of samples stained with 5 μl. The values of OS, SS and SR were influenced by the male donor (p < 0.01) but not by the H‐42 concentration used. The quality of sorted sperm samples immediately after sorting was similar to that of fresh samples, while centrifugation resulted in significant reduction (p < 0.05) in TM and in the percentage of viable spermatozoa and a significant increase (p < 0.01) in the percentage of spermatozoa with damage/reacted acrosomes. In conclusion, the sex‐sorting of canine spermatozoa by flow cytometry can be performed successfully using H‐42 concentrations between 7.5 and 12.5 μl. The efficiency of the sorting procedure varies based on the dog from which the sperm sample derives.     

Urinary catheterisation of female dogs: a comparison between three techniques for catheter placement

The objective of this study was to describe a new technique for urinary catheterisation of female dogs using a novel catheterisation device (NCD) and to compare the time taken to place a catheter using this technique with traditional techniques. A secondary objective was to survey participants on which of the techniques they preferred. Female canine cadavers of varying sizes were utilised and veterinary students who had not previously placed a urinary catheter were enrolled. Each participant performed three catheterisation techniques, Visual with speculum (SPEC), Blind Palpation (BP) and catheterisation with NCD on three sizes of dog. Time required using each technique was compared using Kaplan–Meier plots and mixed models Cox Proportional Hazards regression. Median times to catheterisation were 300 s (IQR 261–417 s) with the SPEC method, 420 s (IQR 253–545 s) with the NCD method and 725 s (574–1032s) with the BP method. Both SPEC and NCD methods were significantly faster compared to the BP method, with Hazard Ratios of 3.66 (95% CI 1.94–6.91, P < 0.001) and 3.57 (95% CI 1.87–6.81, P < 0.001), respectively. Six of nine participants found the NCD the easiest technique, 5/9 of the participants found the palpation technique most difficult and 4/9 found the speculum technique most difficult. BP appears to be the technique of least preference and increased time requirement. The novel urinary catheterisation device may provide a simpler method of visualisation of the urethral papilla and may provide a more sterile way of placing the catheter, although further investigation is needed to confirm this.     

Comparison of carprofen and meloxicam for 72 hours following ovariohysterectomy in dogs

OBJECTIVE: To compare the peri- and post-operative (72 hours) analgesic effects of injectable and orally administered carprofen and meloxicam for ovariohysterectomy in dogs. STUDY DESIGN: Prospective, randomized clinical study. ANIMALS: Forty-three dogs undergoing elective ovariohysterectomy. MATERIALS AND METHODS: Dogs were randomly assigned to receive pre-operative carprofen, meloxicam or sterile saline by subcutaneous injection. Pre-anaesthetic medication was intramuscular acepromazine (0.02 mg kg(-1)) and methadone (0.2 mg kg(-1)). Anaesthesia was induced with either thiopentone or propofol injected to effect, and maintained with isoflurane in oxygen. Visual analogue scores (VAS) for pain and sedation were recorded at 1, 2, 3, 4 and 6 hours following tracheal extubation. Oral medication with the same treatment was continued post-operatively for 3 days, with VAS scores for pain being recorded before, and 2 hours after treatment on each day. Differences between group age, body mass, duration of general anaesthesia, time from treatment injection to tracheal extubation and time from treatment injection to first oral treatment were analysed using one-way analysis of variance and Kruskal-Wallis test. Visual analogue scores for pain and sedation were analysed using a re-randomization method. The significance level was set at p < 0.05. RESULTS: Meloxicam-treated subjects had lower mean VAS than the control group at 2 and 6 hours following tracheal extubation. Control group VAS were more varied than meloxicam scores (at 6 hours) and carprofen scores (at 3 and 6 hours). On the first post-operative day, pre- to post-treatment VAS scores decreased significantly after meloxicam. On day 3, scores in the meloxicam-treated group were significantly lower than control values after treatment. Changes in pre- to post-treatment VAS were greater in animals receiving either meloxicam or carprofen compared with those given saline. CONCLUSIONS AND CLINICAL RELEVANCE: Both carprofen and meloxicam provided satisfactory analgesia for 72 hours following ovariohysterectomy in dogs.     

Low-Dose Insemination in Pigs: Problems and Possibilities

Low-dose AI procedures are required by the pig industry to efficiently utilize emerging sperm technologies, such as cryopreservation and sex-sorting. Currently, several different procedures for inseminating with a low or very low number of spermatozoa have been described. Deep intrauterine insemination allows the deposition of the spermatozoa in the depth of the uterine horn, allowing a significant reduction in the number of spermatozoa inseminated with maintenance of optimal reproductive performance. Intra-oviductal laparoscopic insemination has been recently applied in pigs. This technique has proved to be applicable with diluted and sex-sorted spermatozoa. This review discusses several problems encountered during the development of deep intrauterine insemination and intra-oviductal laparoscopic insemination of pigs and provides potential solutions for the practical application of both the technologies.     

Comparison of propofol with ketofol,a propofol‐ketamine admixture,for induction of anaesthesia in healthy dogs

ObjectiveTo compare anaesthetic induction in healthy dogs using propofol or ketofol (a propofol-ketamine mixture).Study designProspective, randomized, controlled, ‘blinded’ study.AnimalsSeventy healthy dogs (33 males and 37 females), aged 6–157 months and weighing 4–48 kg.MethodsFollowing premedication, either propofol (10 mg mL^?1) or ketofol (9 mg propofol and 9 mg ketamine mL^?1) was titrated intravenously until laryngoscopy and tracheal intubation were possible. Pulse rate (PR), respiratory rate (f_R) and arterial blood pressure (ABP) were compared to post-premedication values and time to first breath (TTFB) recorded. Sedation quality, tracheal intubation and anaesthetic induction were scored by an observer who was unaware of treatment group. Mann–Whitney or t-tests were performed and significance set at p = 0.05.ResultsInduction mixture volume (mean ± SD) was lower for ketofol (0.2 ± 0.1 mL kg^?1) than propofol (0.4 ± 0.1 mL kg^?1) (p < 0.001). PR increased following ketofol (by 35 ± 20 beats minute^?1) but not consistently following propofol (4 ± 16 beats minute^?1) (p < 0.001). Ketofol administration was associated with a higher mean arterial blood pressure (MAP) (82 ± 10 mmHg) than propofol (77 ± 11) (p = 0.05). TTFB was similar, but ketofol use resulted in a greater decrease in f_R (median (range): ketofol -32 (-158 to 0) propofol -24 (-187 to 2) breaths minute^?1) (p < 0.001). Sedation was similar between groups. Tracheal intubation and induction qualities were better with ketofol than propofol (p = 0.04 and 0.02 respectively).Conclusion and clinical relevanceInduction of anaesthesia with ketofol resulted in higher PR and MAP than when propofol was used, but lower f_R. Quality of induction and tracheal intubation were consistently good with ketofol, but more variable when using propofol.