Effect of Overnight Staining on the Quality of Flow Cytometric Sorted Stallion Sperm: Comparison with Tradtitional Protocols

Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sorted and compared with spermatozoa from the same ejaculate that had been sexed on the previous day. All sperm parameters evaluated remained unchanged when fresh sorted and refrigerated sorted semen were compared. Pre‐sorting storage at 5°C did not alter sperm velocities nor kinetics, viability or membrane permeability, production of reactive oxygen species, mitochondrial membrane potential or DNA fragmentation index of the sorted sample. The findings open for the possibility of using semen from stallions housed far from the sorting facilities. Processed and stained sperm could be shipped refrigerated on the previous day, sorted and inseminated on the next day.     

Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality

Studies have demonstrated the importance of mitochondria to sperm functionality, as the main source of ATP for cellular homoeostasis and motility. However, the role of mitochondria on sperm metabolism is still controversial. Studies indicate that, for some species, glycolysis may be the main mechanism for sperm energy production. For ram sperm, such pathway is not clear. Thus, we evaluated ram sperm in response to mitochondrial uncoupling and glycolysis inhibition aiming to assess the importance of each pathway for sperm functionality. Statistical analysis was performed by the SAS System for Windows, using the General Linear Model Procedure. Data were tested for residue normality and variance homogeneity. A p < .05 was considered significant. Groups treated with the mitochondrial uncoupler Carbonyl cyanide 3 chlorophenylhydrazone (CCCP) showed a decrease in the percentage of cells with low mitochondrial activity and high mitochondrial membrane potential. We also observed that the highest CCCP concentration promotes a decrease in sperm susceptibility to lipid peroxidation. Regardless the lack of effect of CCCP on total motility, this substance induced significant alterations on sperm kinetics. Besides the interference of CCCP on spermatic movement patterns, it was also possible to observe such an effect in samples treated with the inhibitor of glycolysis (2‐deoxy‐d ‐glucose, DOG). Furthermore, treatment with DOG also led to a dose‐dependent increase in sperm susceptibility to lipid peroxidation. Based on our results, we suggest that the glycolysis appears to be as important as oxidative phosphorylation for ovine sperm kinetics as this mechanism is capable of maintaining full motility when most of the cells have a low mitochondrial membrane potential. Furthermore, we found that changes in the glycolytic pathway trough glycolysis inhibition are likely involved in mitochondrial dysfunction and sperm oxidative unbalance.     

Production benefits from pre- and post-lambing anthelmintic treatment of ewes on commercial farms in the southern North Island of New Zealand

Abstract

AIMS: To measure the magnitude and variability in production responses to anthelmintic treatments administered to adult ewes around lambing.

METHODS: Ewes carrying twin lambs, from sheep and beef farms (eight in Year 1 and six in Year 2) in the Wairarapa region of New Zealand, were enrolled in 14 trials (part of an experiment carried out on one farm in one year). Experiment 1 compared ewes treated 2–4?weeks pre-lambing with a controlled release capsule (CRC) containing abamectin, albendazole, Se and Co, to ewes injected pre-lambing with a long-acting Se plus vitamin B₁₂ product, and to untreated ewes. Experiment 2 included these treatments, plus a CRC administered at pregnancy scanning. Experiment 3 included the same treatments as Experiment 1, plus administration of a CRC containing albendazole, Se and Co, injectable moxidectin or oral derquantel plus abamectin, all administered pre-lambing, or oral derquantel plus abamectin administered 4–6?weeks after lambing. Variables compared were ewe liveweight at weaning and pre-mating, lamb liveweight at weaning, total weight of lamb weaned per ewe and ewe dag score at weaning.

RESULTS: Ewes treated with a CRC pre-lambing were heavier than untreated ewes (mean 3.2?kg) at weaning in 12/14 trials, and pre-mating (mean 2.8?kg) in 9/14 trials (p<0.001). Compared with mineral-treated ewes the mean difference was 2.8?kg pre-lambing (9/14 trials) and 1.7?kg pre-weaning (6/14 trials). Lambs reared by treated ewes were heavier (mean 1.55?kg) at weaning in 6/14 trials (p<0.001), but there was no effect of CRC treatment on total weight of lambs weaned per ewe (p=0.507). Variation in weight of lamb weaned per ewe was largely explained by differences in lamb survival from birth to weaning (p<0.001), with no effect of CRC treatment (p>0.65).

Treatment of ewes with a CRC at pregnancy scanning was neither better nor worse than a pre-lambing treatment (p=0.065).

There was no difference in the response from treatment with either of the two CRC or moxidectin. Treatment with short-acting oral anthelmintics resulted in no consistent benefit.

CONCLUSIONS: Anthelmintic treatments administered to ewes around lambing resulted in variable responses between farms and years, which in some trials were negative for some variables, and some of the variability was due to the mineral component of the CRC. The widespread perception amongst farmers and veterinarians that anthelmintic treatment of ewes around lambing will always result in positive benefits is not supported.     

Pathological findings in the bulbourethral glands of bulls

The bulbourethral glands of 323 Bos indicus or B. indicus crossbred bulls more than 1 1/2 years old were examined in an abattoir study. Bulbourethral adenitis was diagnosed grossly and confirmed by histological examination in 4 (1.2%). Unilateral chronic interstitial inflammation was seen in 2 cases; one of these was associated with a degenerative-type seminal vesiculitis. In the others adenitis was bilateral; in one case it was associated with a concretion and foreign (plant) material in the principal duct of the left bulbourethral gland; in the other bilateral case, numerous calculi were present and microscopically, a chronic active and diffuse inflammation was observed. Chemical analysis of the calculi showed calcium oxalate and tricalcium phosphate to be the most important components. Corynebacterium spp was isolated from the lesion with multiple calculi but attempts to isolate Chlamydia spp, Mycoplasma spp and Brucella abortus from the 4 adenitis cases were unsuccessful. Congenital abnormalities such as glandular fusion (2.2%) or unilateral aplasia (0.6%) were also observed. Cysts were the most common finding (19.2%), and duct dilation was frequent (7.1%). The significance of these findings in relation to fertility is considered.     

Acceptance by stoats (Mustela erminea) of 1080 (sodium monofluoroacetate) in small-volume baits and its effect on behaviour and time to death

AIMS: To assess whether stoats (Mustela erminea) would eat small baits containing 0.1% sodium monofluoroacetate (1080); whether they would die from it; how long it would take to kill them; and to document the behaviour of 1080-intoxicated stoats.

METHODS: Stoats were offered 1-g baits of two semi-fluid formulations containing 0.1% 1080, presented in open dishes, and their subsequent behaviour was monitored by video and direct observation. Muscle samples from stoats that died were analysed for 1080 residues.

RESULTS: There was no significant difference between two types of bait with regard to acceptance, mortality, and time to death, and behavioural effects were similar; consequently, results from the two types of bait were combined. Twelve of 14 stoats offered the baits ate them voluntarily, and a 13th licked bait off its fur; all 13 died between 1 h 15 min and 4 h 7 min (mean 2 h 38 min) later. At first (range 29 min — 2 h 7 min, mean 1 h 1 min), their behaviour appeared to be normal. Ataxia and hyperactivity were the first behavioural signs of poisoning, and lasted 2 min — 1 h 40 min (mean 26 min). This was followed by recumbency with convulsions and rapid breathing (range 16 min to 2 h, mean 58 min), then recumbency with limited activity and progressively shallow breathing prior to death (range 1–51 min, mean 33 min). Stoats became non-responsive to a light being turned on, or to touch once recumbency became sustained.

Residues of 1080 were found in muscle tissue of all 13 dead stoats, at concentrations ranging from 0.075 μg/g in a 287-g male that died 4 h 7 min after eating only 0.74 g of bait, to 2.5 μg/g in a 254-g female that died 2 h 42 min after taking a whole 1-g bait.

CONCLUSION: Stoats will voluntarily take small (1-g) baits containing a lethal dose of 1080 at 0.1%, and die from it comparatively rapidly for a mammalian carnivore.     

Effect of bandaging on second intention healing of wounds of the distal limb in horses

Objective   To evaluate the effect of a non-occlusive dressing incorporated in a 3-layer bandage on second intention healing of wounds of the distal portion of the limb.
Study Design   Untreated wounds in 33 adult horses used in four studies using the same wound-healing model conducted over 5 years.
Methods   Standardised, full-thickness wounds were made in the skin overlying the dorsomedial aspect of the mid-metacarpus; 17 horses were bandaged with a non-occlusive dressing covered by gauze-coated cotton wool that was compressed with adhesive tape; 16 horses were left unbandaged. Wounds were photographed weekly for 9 weeks and the images were analysed electronically.
Results   There were significant effects associated with bandage (P < 0.0001), week (P < 0.001), and bandage by week interaction (P < 0.0001). There was no difference in wound area at the first time-point after wound creation (P = 0.38). After week 1, there was a difference between bandaged and unbandaged wounds in wound area at each measurement until the end of the study. Bandaged wounds showed greater and more prolonged retraction. Unbandaged wounds retracted for 2 weeks before beginning to contract, whereas bandaged wounds continued to retract for 3 weeks. In bandaged wounds excess granulation tissue required regular trimming, but not in unbandaged wounds. There was no difference between groups in the total days to healing or the overall rate of healing.
Conclusions   These results should be treated with caution until validated with contemporaneous, controlled studies. Covering a wound with a non-occlusive dressing in a 3-layer bandage led to greater wound retraction, modulated the rate of wound contraction and promoted excessive granulation tissue. If excessive granulation tissue is excised regularly, bandaging has no effect on total time to healing.     

Effect of N‐acetyl‐L‐cysteine Supplementation in Semen Extenders on Semen Quality and Reactive Oxygen Species of Chilled Canine Spermatozoa

The objective of this study was to evaluate the quality of chilled dog semen processed with extenders containing various concentrations of N‐acetyl‐L‐cysteine (NAC). Ejaculates from five dogs were collected, pooled and evaluated for concentration, motility, rapid steady forward movement (RSF‐movement), viability, acrosomal integrity and by the hypo‐osmotic swelling test (HOST). In addition, superoxide anion (O₂^‐?) production, hydroxyl radicals (OH^?) and total reactive oxygen species (tROS) were determined. The pool was divided into five aliquots, which were diluted to a final concentration of 66.66 × 10⁶ spermatozoa/ml with Tris‐glucose‐egg yolk extender containing one of the following concentrations of NAC (0, 0.5, 1, 2.5 or 5 mm ). The semen aliquots were chilled and preserved at 4°C. Semen quality was evaluated after rewarming at 72 h. Sperm motility was significantly higher with the 0.5 mm concentration compared with the control group (p = 0.001). Rapid steady forward movement was higher with the 0.5 and 1 mm concentrations compared with the control and 5 mm group (p < 0.001). Viability and HOST percentages were not significantly altered. Compared with the control, the 5 mm concentration showed significantly reduced percentages of spermatozoa with normal acrosomes (p = 0.049). None of the ROS values at 72 h were significantly affected by the presence of NAC in semen extenders, although all NAC concentrations showed lower O₂^‐? and OH^? values compared with the control. Only the concentrations of 1 and 5 mm inhibited the significant increase of tROS values after 72 h, compared with the fresh semen value. In conclusion, NAC supplementation of semen extenders is beneficial to semen motility of canine spermatozoa during chilling with the 0.5 mm concentration being the most effective, although no significant ROS inhibition was observed at 72 h.