Evaluation of Cheetah and Leopard Spermatozoa Developmental Capability after Interspecific ICSI with Domestic Cat Oocytes

The ICSI procedure is potentially of great value for felids, and it has not been extensively studied in these species. The objectives of this work were to determine the best conditions for ICSI in the domestic cat (DC) to generate interspecific embryos by injecting cheetah (Ch) and leopard (Leo) spermatozoa. Firstly, DC oocytes were matured with insulin–transferrin–selenium (ITS) or without it (MM) and cultured using atmospheric (21%) or low (5%) oxygen tension after ICSI. The group ITS‐5%O₂ showed the highest blastocyst rate (p < 0.05), 20.9% vs 8.7%, 7% and 6.5%, for MM‐21%O₂, MM‐5%O₂ and ITS‐21%O₂, respectively. The best conditions were used to generate the interspecific embryos, together with ionomycin activation (Io) after ICSI. Interspecific embryos resulted in high rates of blastocysts that were not positively affected by Io activation: 32.6% vs 21% for Ch and Ch‐Io, 9.8% vs 21% for Leo and Leo‐Io, and 20% vs 17.4% for DC and DC‐Io. We also evaluated DNA‐fragmented nuclei of experiment 1 and 2 blastocysts, using TUNEL assay. The fragmented nucleus proportion was higher in the ITS‐5%O₂ group, 67.6%. Surprisingly, interspecific blastocysts showed the lowest fragmented nucleus proportion: 27% and 29.9% for Ch and Leo, respectively. We concluded that ITS and 5%O₂ improve blastocyst formation in DC, although with a concomitant increase in DNA fragmentation. Most importantly, cheetah and leopard spermatozoa were able to generate blastocysts without artificial activation, which suggests that developmental capacity of wild felid spermatozoa can be evaluated by interspecific ICSI. This technique should be used to assist wild felid reproduction.     

Mechanical testing of a steel-reinforced epoxy resin bar and clamp for external skeletal fixation of long-bone fractures in cats

AIMS: To provide veterinarians with confidence when using a commercially available epoxy resin in external skeletal fixators (ESF), testing was conducted to determine exothermia during curing of the epoxy resin compared to polymethylmethacrylate (PMMA), the hardness of the epoxy resin as a bar over 16 weeks, and the strength of the epoxy resin bar compared with metal clamps in similarly constructed Type 1a ESF constructs simulating the repair of feline long bone fractures.

METHODS: Exothermia of the epoxy resin during curing was tested against PMMA with surface temperatures recorded over the first 15 minutes of curing, using four samples of each product. The hardness of 90 identical epoxy resin bars was tested by subjecting them to cyclic loads (1,000 cycles of 20.5?N, every 7 days) over a 16-week period and impact testing 10 bars every 2 weeks. Ten bars that were not subjected to cyclic loads were impact tested at 0 weeks and another 10 at 16 weeks. Strength of the epoxy resin product, as a bar and clamp composite, was tested against metal SK and Kirschner-Ehmer (KE) clamps and bars in Type 1a, tied-in intramedullary pin, ESF constructs with either 90° or 75° pin placement, subjected to compressive and bending loads to 75?N.

RESULTS: The maximum temperature during curing of the epoxy resin (min 39.8, max 43.0)°C was less than the PMMA (min 85.2, max 98.5)°C (p<0.001). There was no change in hardness of the epoxy resin bars over the 16 weeks of cyclic loading (p=0.58). There were no differences between the median strength of the epoxy resin, SK or KE ESF constructs in compression or bending when tested to 75?N (p>0.05). Stiffness of constructs with 75° pin placement was greater for SK than epoxy resin constructs in compression (p=0.046), and was greater for KE than epoxy resin constructs in bending (p=0.033).

CONCLUSIONS: The epoxy resin tested was found to be less exothermic than PMMA; bars made from the epoxy resin showed durability over an expected fracture healing timeframe and had mechanical strength characteristics comparable to metal bar and clamp ESF constructs.

CLINICAL RELEVANCE: The epoxy resin ESF construct tested in this study can be considered a suitable replacement for SK or KE ESF constructs in the treatment of feline long-bone fractures, in terms of mechanical strength.     

The effect of three different doses of sodium pentosan polysulphate on haematological and haemostatic variables in adult horses

OBJECTIVE: To evaluate the effects of three different doses of sodium pentosan polysulphate (PPS) on haematological and haemostatic variables in adult horses. DESIGN: Eight adult standardbred horses were used. All horses received a single injection of 0, 3, 6, and 10 mg/kg of PPS at the beginning of each treatment week for 4 weeks so that by the end of the study all horses had received all four doses of PPS. Blood samples were collected at 0, 1, 2, 3, 4, 6, 8, 12, 24, 48, and 168 h after each weekly injection of PPS. Variables measured were packed cell volume, haemoglobin, red blood cell count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, white cell count, neutrophil count, lymphocyte count, eosinophil count, monocyte count, serum protein, fibrinogen, prothrombin time, and activated partial thromboplastin time (PTT). Data were analysed using an ANOVA. Significance was set at P < 0.05. RESULTS: There was a dose-dependent increase in PTT. A significant increase in PTT occurrred in all treatment groups when compared to horses receiving 0 mg/kg in which there was no change over time. The PTT values all returned to baseline by 48 h after treatment. The mean neutrophil count was higher 3 h after treatment when compared to time 0. Horses receiving 3 mg/kg of PPS had a higher lymphocyte count 4 h after injection, and those receiving 6 and 10 mg/kg had higher counts at 3,4,6 and 8 h after injection when compared to time 0. At 8 h after injection horses receiving 6 and 10 mg PPS had higher lymphocyte counts than horses not receiving PPS. CONCLUSIONS: PPS causes a dose-dependent prolongation of PTT in horses. At the dose rates currently recommended for treatment of joint problems in horses this increase was small and remained elevated from baseline for up to 24 h. Based on these findings doses of PPS up to 3 mg/kg should not be administered to horses within 24 h of high stress activities or where physical injury may occur.     

Heterologous In Vitro Fertility Evaluation of Cryopreserved Iberian Red Deer Epididymal Spermatozoa with Zona-intact Sheep Oocytes and its Relationship with the Characteristics of Thawed Spermatozoa

A heterologous in vitro system, using zona‐intact sheep oocytes, was used to evaluate the relationship between sperm factors of Iberian red deer thawed epididymal sperm and the percentage of cleaved oocytes. Epididymal spermatozoa were recovered from six males, diluted with freezing extender and cryopreserved. After thawing sperm motility (SM) and acrosome and membrane integrities were evaluated. Again, these parameters were assessed after incubation in freezing extender at 37°C for 2 h. After cryopreservation the values for SM and acrosome and membrane integrities were high (～80, 80 and 70% respectively). However, these values significantly decreased after incubation (～59, 62 and 47% respectively). Red deer thawed epididymal sperm fertilized zona‐intact sheep oocytes, although the percentage of cleaved oocytes was low (～22%). No relationship was found between sperm parameters assessed after thawing and the percentage of cleaved oocytes. Likewise, any sperm parameter evaluated after incubation was assessed in relation to the percentage of cleaved oocytes. However, acrosome and membrane integrities were near to significance (p = 0.06 and p = 0.09 respectively). Then, we conducted a reduced model with these two variables and both were related to the percentage of cleaved oocytes (p = 0.02 and p = 0.04 respectively). Thus, acrosome and membrane integrities were related to the percentage of cleaved oocytes negatively and positively respectively. It was concluded that the classical parameters assessed in deer thawed sperm samples can be good predictors of the ability to fertilize zona‐intact sheep oocytes.     

Corpus Luteum–Endometrium–Embryo Interactions in the Dairy Cow: Underlying Mechanisms and Clinical Relevance

Conception rates of dairy cows are currently declining at an estimated 1% every year. Approximately, 35% of embryos fail to prevent luteolysis during the first three weeks of gestation. Interactions between the corpus luteum, endometrium and embryo are critical to the successful establishment of pregnancy and inadequacies will result in the mortality of the embryo. For example, as little as a one day delay in the post-ovulatory rise of progesterone has serious consequences for embryo development and survival. Recently, we found that LH support, degree of vascularization and luteal cell steroidogenic capacity were not the major factors responsible for this luteal inadequacy, but are nevertheless essential for luteal development and function. Progesterone acting on its receptor in the endometrium stimulates the production of endometrial secretions on which the free-living embryo is dependent. However, their exact composition and effects of inadequate progesterone remains to be determined. The embryo is recognized through its secretion of interferon tau (IFNT), which suppresses luteolytic pulses of prostaglandin F_2α. In the cow, it is most likely that IFNT inhibits oxytocin receptor up-regulation directly and does not require the prior inhibition of oestrogen receptor α (ESR1). Unravelling the precise luteal-endometrium and embryo interactions is essential for us to understand pregnancy establishment and development of strategies to reverse the declining fertility of dairy cows.     

The pathogenesis and treatment of osteomyelitis and laminitis in a stallion after the prolonged topical application of formalin to the distal phalanx

A 12-year-old Quarterhorse stallion was presented with a severe lameness in the left forefoot. There was a 3 cm diameter cavity in the sole that extended to the solar surface of the distal phalanx. Radiographs revealed an osteomyelitis and a sequestrum which probably developed following the prolonged topical application of 10 percent formalin. The sequestrum was removed and the infected bone curetted under general anaesthesia. The horse was shod with heart bar shoes on both front feet 7 days after the surgery. Eight months later, radiographs showed marked rotation of the distal phalanx despite continual shoeing with heart bar shoes. Surgical resection of the dorsal wall of the hoof at the toe removed the pressure applied by the laminar wedge, and combined with the stabilising and supporting action of the heart bar shoe, permitted realignment of the distal phalanx approximately 30 degrees closer to the normal skeletal axis by 26 days post-operatively. This case highlights some of the recent developments in the treatment of laminitis and suggests that effective treatment is possible if the value of the animal warrants the time and investment.