Reduced Immunolabelling of a Porcine Oocyte Membrane Protein Reflects Reduced Fertilizability of Porcine Oocytes Following Elevated Ambient Temperature

Decreased fertility in pigs is a common occurrence during summer months. An objective of the current experiments was to evaluate if elevated ambient temperature altered the oocyte plasma membrane including potential receptors for sperm. This would potentially contribute to reduced fertilizability. Treated gilts were exposed in vivo to 32°C for 12 h per day and 20°C for the remaining 12 h per day for 7 days; control gilts were exposed to 22°C for 12 h and 20°C for the remaining 12 h each day. Cumulus–oocyte complexes were also aspirated from ovaries obtained from gilts maintained at thermoneutral ambient temperature and matured in vitro at 38.5°C or 40°C. Relative abundance of a porcine oocyte membrane protein was examined by intensity of immunolabelling of the in vivo and in vitro matured oocytes evaluated with confocal microscopy; fertilizability of the in vitro matured oocytes was evaluated in in vitro fertilization assays. Oocytes obtained from gilts exposed to elevated ambient temperature for 7 days had reduced immunolabelling compared with oocytes from control gilts (p < 0.05). Similarly, oocytes matured in vitro for 44 h at elevated ambient temperature had reduced immunolabelling and reduced fertilizability compared with oocytes matured at 38.5°C (p < 0.01 and p < 0.05). These results suggest porcine oocyte quality is reduced by elevated ambient temperature and immunolabelling of oocytes with antibodies to specific membrane proteins may be effective to evaluate some aspects of oocyte quality.     

Effect of Taurine and Melatonin in the Culture Medium on Buffalo In Vitro Embryo Development

This study was carried out to investigate the effect of supplementing culture medium with different concentrations of taurine and melatonin, on buffalo oocyte in vitro meiotic maturation and embryo development. In experiment 1, oocytes were matured in vitro and the cleaved embryos were cultured in the same following seven culture medium; (i) control (TCM 199 + 10% SS); (ii) control + 0.5 m m taurine; (iii) control + 1 m m taurine; (iv) control + 3 m m taurine; (v) control + 5 μ m melatonin; (vi) control + 10 μ m melatonin and (vii) control + 50 μ m melatonin. In experiment 2, based on the results of experiment 1, to examine the synergistic effect of antioxidants, the oocytes were matured in culture medium (TCM199 + 10% SS), supplemented with both taurine at 1 m m and melatonin at 10 μ m concentration and the cleaved embryos were cultured in the same medium. Supplementation of taurine at 1 m m concentration in the culture medium resulted in a higher (p < 0.05) transferable embryo (TE) yield when compared with control (20.6% vs 14.1%). Supplementation of melatonin at 10 and 50 μ m concentration in the culture medium resulted in a higher (p < 0.05) meiotic maturation rate (90.3% and 88.8% respectively) and TE yield (28.4% and 27.2% respectively), than the other treatments. In experiment 2, the TE yield did not improve by supplementing the culture medium with both taurine and melatonin, when compared with melatonin alone. In conclusion, the results of this study demonstrated that, enriching the culture medium with taurine and melatonin, improves in vitro embryo production efficiency in buffaloes. In particular, a high TE yield was obtained by enriching the culture medium with 10 μ m melatonin.     

The Predictive Value of Semen Analysis in the Evaluation of Stallion Fertility

Pregnancy rates in managed horse populations depend on the innate fertility of the mares and stallions involved and on the quality of breeding management. Of course, because a single stallion usually mates many mares, stallion fertility is a critical factor in the overall success of a breeding program. Unfortunately, accurate evaluation of stallion fertility per se requires a large number of normal mares to be mated and is necessarily retrospective. Rather, the ideal is to predict fertility in advance of the stallion's breeding career, and this is currently attempted by way of a thorough physical examination and a routine analysis of semen quality. However, while such a ‘breeding soundness examination’ identifies stallions that clearly lack the capacity for adequate fertility, it is of limited use for predicting the level of fertility and fails to identify some seriously sub‐fertile animals. Similarly, while various sperm function tests (e.g., sperm head morphometry, the hypoosmotic swelling test, glass wool‐sephadex filtration, progesterone receptor exposure) have been shown to correlate fairly well with fertility in the field, most examine only a single or a narrow range of the attributes that a sperm must possess if it is to fertilize an oocyte in vivo, and are thus more useful for identifying specific causes of sub‐fertility than for predicting the level of fertility. On the other hand, combining the results of the various sperm function tests does improve the reliability of fertility estimation and current research is therefore concentrated on identifying a range of tests that covers as many important sperm attributes as possible but that can be performed rapidly and cheaply. In this respect, flow‐cytometry has proven to be an ideal tool because it allows the objective, rapid and simultaneous analysis of a number of properties in a large number of sperm. Moreover, stains are available for an increasing range of sperm characteristics including viability, capacitation and acrosome status, mitochondrial activity and chromatin integrity. Flow‐cytometric analysis of sperm with appropriate probes thus offers considerable promise for the prediction of stallion fertility.     

Animal‐Level Factors Affecting Ovarian Function in Bos indicus Heifers Treated to Synchronize Ovulation with Intravaginal Progesterone‐Releasing Devices and Oestradiol Benzoate

The primary objective of this study was to investigate the impact of animal‐level factors including energy balance and environmental/management stress, on the ovarian function of Bos indicus heifers treated to synchronize ovulation. Two‐year‐old Brahman (BN) (n = 30) and BN‐cross (n = 34) heifers were randomly allocated to three intravaginal progesterone‐releasing device (IPRD) treatment groups: (i) standard‐dose IPRD [Cue‐Mate^® (CM) 1.56 g; n = 17]; (ii) half‐dose IPRD [0.78 g progesterone (P₄); CM 0.78 g; n = 15]; (iii) half‐dose IPRD + 300 IU equine chorionic gonadotrophin at IPRD removal (CM 0.78 g + G; n = 14); (iv) and a control group, 2× PGF_2α [500 μg prostaglandin F_2α (PGF_2α)] on Day ?16 and ?2 (n = 18). Intravaginal progesterone‐releasing device‐treated heifers received 250 μg PGF_2α at IPRD insertion (Day ?10) and IPRD removal (Day ?2) and 1 mg oestradiol benzoate on Day ?10 and ?1. Heifers were managed in a small feedlot and fed a defined ration. Ovarian function was evaluated by ultrasonography and plasma P₄ throughout the synchronized and return cycles. Energy balance was evaluated using plasma insulin‐like growth factor 1 (IGF‐I) and glucose concentrations. The impact of environmental stressors was evaluated using plasma cortisol concentration. Heifers that had normal ovarian function had significantly higher IGF‐I concentrations at commencement of the experiment (p = 0.008) and significantly higher plasma glucose concentrations at Day ?2 (p = 0.040) and Day 4 (p = 0.043), than heifers with abnormal ovarian function. There was no difference between the mean pre‐ovulatory cortisol concentrations of heifers that ovulated or did not ovulate. However, heifers that ovulated had higher cortisol concentrations at Day 4 (p = 0.056) and 6 (p = 0.026) after ovulation than heifers that did not ovulate.     

Production of Buffalo (Bubalus bubalis) Embryos in vitro: Premises and Promises

Techniques for in vitro production (IVP) of buffalo embryos adopting the procedures developed in cattle have received increasing interest in the recent times. A high oocyte maturation, fertilization and cleavage rate and a low rate of blastocyst yield and calving following transfer of in vitro produced buffalo embryos have been obtained. The efficiency of IVP in buffalo is much lower than that in cattle. Several problems need to be resolved before IVP technology can be used regularly in buffalo breeding. This review attempts to present an overview of the different techniques used in buffalo to produce transferable embryos in vitro, namely in vitro maturation and fertilization of immature oocytes and in vitro development of the resulting cleaved embryos to the blastocyst stage before transfer. The problems associated with IVP, the possible solutions and the new biotechniques linked to IVP are discussed.     

Myofibroblastic fibrosarcoma with multifocal osseous metaplasia at the site of equine influenza vaccination

We describe a fibrosarcoma in a 12-year-old Quarterhorse × Arabian gelding as a sequela to equine influenza vaccination. Shortly after the second vaccination, swelling at the site was noticed by the owner and it continued to increase in size over the following 6 months. Biopsy of the mass indicated a fibrosarcoma had developed at the vaccination site. It was approximately 20 cm in diameter and elevated well above the level of the skin. There was no clinical evidence of metastases to the lungs or local lymph nodes. Surgical resection of the mass was performed and the wound healed by first and second intention. Histopathological examination and immunohistochemical staining confirmed a myofibroblastic fibrosarcoma with multifocal osseous metaplasia. To the authors' knowledge, this is the first equine case of a vaccine-associated fibrosarcoma.     

Indigofera spicata (creeping indigo) poisoning of three ponies

Three ponies continuously grazed a pasture containing an estimated 24% Indigofera spicata (wet weight basis) for 4–6 weeks in April and May 2004. They developed ataxia, paresis, depression, muscle fasciculations, dysphagia, ptyalism and halitosis. Two also developed corneal opacity. One pony recovered with supportive treatment, but the other two were euthanased and necropsied. Neuropathology was not present in either case, but both livers had periacinar and periportal lymphocytic infiltrations and hydropic degeneration of mid‐zonal hepatocytes, with mild to moderate periacinar necrosis also evident in one. The I. spicata contained 2.66 mg 3‐nitropropionic acid (3‐NPA)/g dry matter and 1.5 mg indospicine/g dry matter. Indospicine, but not 3‐NPA, was detected in serum from both of the euthanased ponies and indospicine was detected in heart, liver and muscle from the one pony in which this assay was performed. The clinical syndrome closely resembled ‘Birdsville horse disease’ caused by I. linnaei and was similar to that reported in horses poisoned by the closely related species I. hendecaphylla and to 3‐NPA poisoning of other animals, including humans. 3‐NPA is thought to cause this neurological syndrome. To our knowledge, this is the first authenticated report of I. spicata poisoning in grazing animals. We also report here the first published evidence that 3‐NPA and indospicine exist in naturalised I. spicata in Australia and of the formation of indospicine residues in tissues of animals grazing paddocks infested with I. spicata.     

Conjunctival vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette-Guérin

AIM: To determine the efficacy of conjunctival vaccination of captive brushtail possums (Trichosurus vulpecula) with bacille Calmette-Guérin (BCG), as measured by immunological responses to vaccination and response to intratracheal challenge with Mycobacterium bovis.

METHODS: Nine adult male brushtail possums were vaccinated by the instillation of a suspension of BCG strain Pasteur 1173P2 into the conjunctival sac of each eye. Each drop contained approximately 2.5 × 10⁵ colony forming units (cfu). At 8 weeks post-vaccination (pv) the vaccinated possums and 10 unvaccinated possums were challenged by intratracheal instillation of approximately 100 cfu of M. bovis. Cellular immune responses to bovine purified protein derivative (PPD) antigen were measured using the lymphocyte proliferation assay (LPA). Possums surviving to 50–51 days after challenge were euth anised and subjected to detailed post-mortem examination, including histopathology, to assess protection against tuberculosis. Sections of lung and spleen were cultured for M. bovis.

RESULTS: No conjunctival inflammation or other adverse reactions to the administration of the vaccine were evident macroscopically. The vaccinated group showed a systemic cellular immune response to bovine PPD antigen at 4 and 8 weeks pv, and the response at 8 weeks was significantly greater than at 4 weeks (p<0.05). Conjunctival vaccination induced significant levels of protective immunity, measured as less mass of tuberculous lesions in lung (p<0.05) and less dissemination of disease in vaccinated compared with unvaccinated possums (p<0.05).

CONCLUSIONS: Conjunctival vaccination with BCG induced a significant level of protective immunity against M. bovis infection in possums. This route of vaccination, together with intranasal aerosol vaccination, could be utilised in the delivery of an aerosolised vaccine using a device that sprays the vaccine suspension into the eyes and nose of possums.