European Eel Sperm Diluent for Short‐term Storage

The sperm of European eel shows a high density and the time of spermatozoa motility is very short after activation with sea water. These characteristics make difficult the sperm handling and its quality assessment. Several diluents were previously described for the Japanese eel obtaining over 3 weeks’ conservation times under refrigeration, but they rendered bad results in the European species. In the present study, several diluents were developed taking as basis the P1 medium, and using different dilution ratios (1 : 50, 1 : 100) and two pH (6.5, 8.5). The effect of the addition of bovine serum albumin (BSA, 2% w/v) was also evaluated. At 24 h, undiluted samples already showed significant lower motility and viability than sperm samples diluted in the different media. The results for diluents with pH 6.5 and 8.5 were different. Spermatozoa diluted in media at pH 6.5 cannot be activated at 24 h, while samples diluted in the diluents with pH 8.5 and added with BSA did not show significant differences with respect to the fresh sperm motility until 48 h. The viability (percentage of alive cells) did not show differences until 1 week, independent of the dilution ratio. After 1 week, the motility was approximately 30% in the media containing BSA, which presented no differences for head size of the spermatozoa (perimeter and area) until 72 h and 1 week, respectively. In conclusion, the combination of one medium having similar physico‐chemical characteristics to the seminal plasma, including pH 8.5, and supplemented with BSA can be used in different dilution ratios for the sperm’s short‐term storage, preserving its motility capacity.     

Exogenous DNA Uptake of Boar Spermatozoa by a Magnetic Nanoparticle Vector System

The sperm‐mediated gene transfer method is applicable to transgenesis in many species that use spermatozoa for reproduction recently, which has been shown various results. In the current study, we show that transgenic porcine embryos can be efficiently produced by employing a simple transfection method that uses magnetic nanoparticles (MNPs). The complexes formed between plasmid DNA and MNPs were bounded on ejaculated boar spermatozoa at a higher efficiency compared to methods using DNA alone or lipofection. Using confocal microscopy, rhodamine fluorophore‐labelled MNPs were detected on external surfaces of the spermatozoa membrane, which were bounded on zona pellucida of in vitro maturated oocyte during in vitro fertilization. Electron microscopy revealed that clusters of MNPs were detected in inside of plasma membrane and nucleus of the spermatozoa head. Additionally, we found that magnetofected boar spermatozoa could be fertilized with oocytes in vitro and that the resulting gene of green fluorescent protein was detected in fertilized eggs by genomic PCR analysis. Taken together, these results suggest that MNPs can be used to efficiently introduce a transgene into embryo via spermatozoa.     

The effect of different combinations of local anaesthesia,sedative and non-steroidal anti-inflammatory drugs on daily growth rates of dairy calves after disbudding

AIM: To assess the effect of sedation and local anaesthesia (LA) at disbudding, and the addition of meloxicam or ketoprofen treatment, on weight gain in dairy calves following disbudding.

METHODS: Friesian-Jersey cross calves, from four dairy farms, were enrolled when 3–6 weeks old. All calves (n=271) were disbudded by veterinary personnel and randomly assigned to six groups: 136 were disbudded without sedation or LA, of which 31 received 20 mg meloxicam S/C and 75 received 150 mg ketoprofen I/M. A further 135 were disbudded with sedation (0.25 mg/kg xylazine I/M) and LA, of which 30 also received meloxicam and 75 received ketoprofen. Calves were weighed 3 days before, and 15 and 30 days after, disbudding (Day 0). Daily weight gain was analysed using mixed models and ANOVA.

RESULTS: Complete results were obtained from 263 calves. From Day ?3 to Day 15, the growth rate of calves disbudded without pain relief (0.53 (95% CI=0.47–0.60) kg/day) was less that of calves disbudded with some form of pain relief (0.65 (95% CI=0.62–0.68) kg/d; p=0.004). There was no difference between the effect of meloxicam or ketoprofen (p=1.00). An interaction between use of sedation and LA and additional non-steroidal anti-inflammatory drugs (NSAID) meant that NSAID treatment did not increase growth rates in calves disbudded with sedation and LA but did increase growth rates for calves disbudded without pain relief (p<0.05). From Day 16 to Day 30 there was no effect of NSAID treatment on growth rate, but calves receiving LA and sedation grew faster (0.74 (95% CI=0.69–0.80) kg/day) than calves disbudded without LA and sedation (0.66 (95% CI=0.61–0.71) kg/day; p=0.018). From Day ?3 to Day 30, calves disbudded with sedation and LA grew faster (0.71 (95%CI=0.64–0.77) kg/day) than calves disbudded without sedation and LA (0.60 (95% CI=0.55–0.65) kg/day; p=0.011). However, addition of NSAID to sedation and LA made no further difference to growth rates (p=0.69).

CONCLUSIONS: Dairy calves disbudded with no pain relief had slower growth rates than calves receiving pain relief. From Day 15 to 30 calves given no pain relief, or NSAID alone, grew more slowly than those receiving sedation and LA at disbudding. The addition of NSAID treatment to sedation and LA did not further increase growth rates.

CLINICAL RELEVANCE: This study adds to the evidence that pain management when disbudding is beneficial for calf productivity as well as calf welfare.     

An epizootic of bovine ephemeral fever in New South Wales in 2008 associated with long-distance dispersal of vectors

Objective To report the rapid transmission of bovine ephemeral fever ( BEF) virus from north-western New South Wales south to the Victorian border in January 2008 and to present data that suggests an uncommon meteorological event caused this rapid southward dispersal of vectors. Procedure The locations of reported clinical cases, data from sentinel herds and results from a survey of cattle in the southern affected area were examined to delineate the distribution of virus transmission. Synoptic weather charts for January 2008 were examined for meteorological conditions that may have favoured movement of vectors in a southerly direction. Results Cases of BEF and exposure to BEF virus in NSW were confirmed west of the Great Dividing Range, extending from the Queensland border to Finley, on the far North Coast and around the Hunter Valley. A low-pressure system moved south across the state on 18–19 January 2008, preceding the first cases of BEF in the south of NSW by 1–2 days. Conclusion Heavy rainfall in December 2007 provided a suitable environment for vector breeding, resulting in the initiation of and support for continuing BEF virus transmission in north-western NSW. The movement of a low-pressure system south across central western NSW in mid-January 2008 after the commencement of BEF virus transmission in the north-west of the state provided a vehicle for rapid southward movement of infected vectors.     

Ontogenesis of mRNA levels and binding sites of hepatic alpha-adrenoceptors in young cattle

Catecholamines affect hepatic glucose production through (alpha- and beta2-) adrenoceptors (AR). We studied mRNA abundance and binding of hepatic alpha-AR in pre-term (P0) calves and in full-term calves at day 0 (F0), day 5 (F5) and day 159 (F159) to test the hypothesis that gene expression and numbers of hepatic alpha-AR in calves are influenced by age and associated with beta2-AR and selected traits of glucose metabolism. mRNA levels of alpha1- and alpha2-AR were measured by real time RT-PCR. alpha1- and alpha2-AR numbers (maximal binding, Bmax) were determined by saturation binding of (3H)-prazosin and (3H)-RX821002, respectively. alpha1- and alpha2-AR subtypes were evaluated by competitive binding. alpha1A-AR mRNA levels were lower in P0 than in F0, F5 and F159 and alpha(2AD)-AR mRNA levels were lower in F159 than in P0, F0 and F5, while alpha2C-AR mRNA levels increased from P0 and F0 to F5 and F159. Bmax of alpha1-AR increased from P0 to F5, then decreased in F159. Bmax of alpha2-AR decreased from F0 to F159. Bmax of alpha1-AR was positively associated with mRNA levels of alpha1A-AR (r = 0.7), Bmax of beta2-AR (r = 0.5) and negatively with hepatic glycogen content (r = -0.6). Bmax of alpha2-AR was negatively associated with Bmax of beta2-AR (r = -0.4). In conclusion, mRNA levels and binding sites of alpha1- and alpha2-AR in calves exhibited developmental changes and were negatively associated with hepatic glycogen content.     

Neutrophil function and energy status in Holstein cows with uterine health disorders

The objectives of this study were to investigate the associations between peripheral blood neutrophil (PMN) function, energy status, and uterine health in periparturient dairy cows. Data were collected from 83 multiparous Holstein cows. Blood samples for PMN function determination were collected weekly from 1 week prior to calving (week -1) through 4 weeks after calving and again at 8 weeks after calving. Energy metabolites were measured and dry matter intake (DMI) was determined from weeks -2 to 5 to evaluate energy status of cows during the periparturient period. All cows were examined for uterine health disorders. Blood PMN killing ability was evaluated by determining myeloperoxidase activity and cytochrome c reduction activity in isolated blood PMN's. For cows that were diagnosed with puerperal metritis and subclinical (SC) endometritis and puerperal metritis, blood PMN functions were significantly (P<0.05) impaired during the periparturient period, compared to cows with normal uterine health. Cows with subclinical endometritis and puerperal metritis or SC endometritis also had significantly (P<0.01) higher NEFA and significantly (P<0.001) lower DMI during the periparturient period, and significantly (P<0.05) higher BHBA during early lactation, compared to cows with normal uterine health. Neutrophil function was also significantly (P<0.01) impaired in cows with peripartum negative energy balance, which was characterized by elevated blood levels of NEFA and decreased DMI. Decreased PMN function and energy balance were associated with uterine health disorders and the decreases in PMN function and energy balance occurred prior to parturition and prior to the detection of these uterine disorders.     

Platelet-Rich Plasma in the Treatment of Induced Tendinopathy in Horses: Histologic Evaluation

This study was carried out to evaluate the effect of platelet-rich plasma (PRP) on the treatment of tendinopathy induced in the superficial digital flexor tendon (TFDS) of horses, by using histologic evaluation. Six healthy crossbred geldings aged 8 to 15 years (12 ± 3) were used. The TFDS tendinopathy was provoked in both forelimbs, by intratendinous administration of 2.5 mg collagenase (2.5 mg/mL), and this procedure was considered as the beginning of the experimental phase. At 12 days after induction of the tendinopathy, the animals were subjected to the following treatments: (1) in the lesion caused in the right superficial digital flexor tendon (PRP-treated group), 2.5 mL PRP activated with calcium chloride at 0.0125 mol/L at concentrations from 320,000 to 500,000 platelets/μL, were injected; (2) in the tendinopathy of the left SDFT (control group), 2.5 mL 0.9% saline solution was administrated. Thirty-six days after the treatments, a biopsy of the injured area was performed for histologic evaluation. In both groups, the histologic analysis showed an increase in the fibroblastic density, as well as the presence of neovascularization, lymphocytes, and plasmocytes infiltrate and tissue organization at variable intensity. In the PRP-treated group, the SDFT was more organized, with the collagen fibers and fibroblasts being better arranged on the tendon matrix. The numbers of the fibroblasts and blood vessels did not differ between the groups. Histologic evaluation 36 days after tendinopathy showed that injuries under a single PRP treatment present a more uniform and organized tissue repair when compared with the control group.     

Plasma leptin status in young calves: effects of pre-term birth, age, glucocorticoid status, suckling, and feeding with an automatic feeder or by bucket

Plasma leptin concentrations depend on energy intake and fat stores and are modified by hormones, such as glucocorticoids. We have measured plasma leptin concentrations in pre-term calves (born on day 277 of gestation) during the first week of life, in full-term calves (290 days of gestation), fed similar amounts of nutrients with colostrum or a milk-derived formula, combined with or without dexamethasone treatment (to simulate a high glucocorticoid status), during the first five days of life, and in calves fed with an automatic feeder or by bucket or suckling on dams up to day 28. Leptin concentrations increased (P<0.05) from birth to day 7 in pre-term calves. In full-term calves leptin concentrations were stable from day 1 to day 4 in colostrum-fed animals, but decreased (P<0.05) and were lower (P<0.05) if fed a formula with similar amounts of energy and macronutrients as colostrum. Concentrations increased (P<0.05) from day 1 to day 2 in calves suckling on dams and then remained elevated, but did not change and were lower in calves fed with the automatic feeder or by bucket than in suckling calves. Dexamethasone only slightly elevated leptin concentrations. There was no episodic secretion pattern, and there were no consistent associations of leptin with various metabolites and hormones. In conclusion, plasma leptin in young calves with respect to effects of nutrition (low energy intake) and hormones (glucocorticoids) and in association with metabolic changes behaved differently from what is known in mature cattle.     

Effect of Sperm Cryopreservation on the European Eel Sperm Viability and Spermatozoa Morphology

The main objective of the present work was to study the effect of cryopreservation of European eel sperm both on the sperm viability and the spermatozoa head morphology. Spermatozoa morphology was evaluated with computer-assisted morphology analysis after collection in fresh samples, after adding the freezing medium containing dimethyl sulfoxide as cryoprotectant and, finally, after the cryopreservation process and thawing. Cell viability was assessed, in both fresh and thawed samples, by Hoechst 33258 staining. Computer-assisted sperm analysis (CASA) was used to determine the percentage of motile cells and to measure motility parameters in sperm samples. A significant decrease of head perimeter (12.56%) and area (17.90%) was detected from spermatozoa in fresh to thawed samples, indicating that cells do not recover the original size after the cryopreservation process. CASA was used to measure the percentage of motile cells (51.9%) and spermatozoa motility parameters such as curvilinear, straight line and angular path velocities, as well as beating cross frequency. This technique was employed in the fresh sperm samples but proteins present at the freezing medium (L-alpha-phosphatidylcholine) made impossible to use this last technique in thawed samples. When sperm viability was assessed by Hoechst staining, a significant decrease of approximately 15% (73.10 vs 58.26%) of alive spermatozoa was registered from fresh to thawed samples. The percentage of motile cells measured by CASA in fresh samples (51.9%) was lower than the percentage of alive cells determined by Hoechst stainning, suggesting the existence of different batches of spermatozoa in different stages of development, even during the eight to tenth weeks of treatment, when the highest sperm quality was found.