Motility Characteristics and Fertilizing Capacity of Boar Spermatozoa Stained with Hoechst 33342

Flow cytometry sorting of spermatozoa using fluorescence dye Hoechst 33342 is the only effective sex selection methodology validated in numerous laboratories. This study was carried out to determine the effect of Hoechst 33342 on the motility and fertility of stained boar spermatozoa. Experiment 1 evaluated motility parameters (percentage of motile spermatozoa, velocity, angularity and oscillation) of boar spermatozoa stained with Hoechst 33342 by a computer‐aided sperm analysis (CASA) instrument. Spermatozoa (30 million/ml) were divided into five treatment groups and stained during 1 h at 35°C with 9, 18, 27, 60 and 90 μM of H33342. There were no differences in sperm motility patterns nor percentages of motile spermatozoa incubated in the presence of 9, 18 or 27 μM. Percentage of motile spermatozoa and motility parameters decreased significantly (p < 0.05) at 60 μM of Hoechst 33342. Spermatozoa were immotile at concentration of 90 μM. In experiment 2, pregnancy rates, farrowing rates and litter size from sows (n = 275) artificially inseminated (AI) with either Hoechst 33342 stained (27 μM) or unstained (control) spermatozoa were determined. Sows inseminated with stained spermatozoa had no significant lower pregnancy rate (88.33%) as compared with controls (90.32%). Staining neither affected farrowing rates (85.0 vs 87.7%) nor total number of piglets born (10.56 ± 0.32 vs 10.47 ± 0.24, stained and controls, respectively). No phenotypical abnormalities were registered among the newborn piglets. The data suggest that incubating spermatozoa with Hoechst 33342 at levels required for X‐ and Y‐bearing chromosome sperm sorting, does not impair sperm viability or their fertility after AI.     

The Effect of Oxidative Stress on Thawed Bulk‐Sorted Red Deer Sperm

The aims of this study were to assess the effects of the sex‐sorting process on post‐thaw sperm quality as well as on induced oxidative stress damage (H₂O₂ 0 mm  = H000; H₂O₂ 50 mm  = H050; H₂O₂ 100 mm  = H100) and the protective action of reduced glutathione (GSH) and Trolox, when comparing sorted (BSS) and non‐sorted (NS) red deer spermatozoa incubated at 37°C. Sperm samples from three stags were collected by electroejaculation and frozen. Immediately after thawing, sperm motility was higher (p < 0.05) for NS (59% ± 3.3) than BSS (36.9% ± 5.8) sperm. Furthermore, the percentage of apoptotic sperm was higher (p < 0.05) for BSS (21.6% ± 5.0) than NS sperm (14.6% ± 1.2). The presence of H₂O₂ increased DNA damage in NS (H000 = 4.1% ± 0.9; H050 = 9.3% ± 0.7; and H100 = 10.9% ± 2.3), but not in BSS sperm. However, in the presence of oxidant, GSH addition improved (p < 0.05) sperm motility in both groups of sperm samples as compared to their controls (NS: 44.5 ± 4.8 vs 21.1 ± 3.9 and BSS: 33.3 ± 8.1 vs 8.9 ± 1.8). These results demonstrate that the sperm‐sorting process induces sublethal effects, albeit selecting a sperm population with a chromatin more resistant to oxidative stress than that in non‐sorted sperm. Moreover, addition of GSH at 1 mm may be a good choice for maintaining the quality of stressed sperm samples, unlike Trolox, which inhibited sperm motility.     

Muscle enzymes in the diagnosis of ovine weaner nutritional myopathy

SUMMARY The diagnostic performance of plasma tests for muscle enzymes was measured in sheep from flocks affected by clinical and sub-clinical nutritional myopathy. Parallel combinations of tests for creatine kinase (CK), alanine amino transferase (ALT), aspartate amino transferase (AST) and lactate dehydrogenase had higher diagnostic sensitivity than CK alone. The enzymes ALT and AST showed the highest correlation with the degree of muscle damage. A parallel combination of tests for plasma CK and ALT as well as tests for plasma α-tocopherol and red cell glutathione peroxidase are recommended for the diagnosis of nutritional myopathy and a decision on the appropriate treatment. The number of false negative results based on a diagnosis from the microscopic examination of single muscles was higher than for the parallel combination of tests. The number of false negatives was highest for the vastus intermedius and lowest for the tensor fascia lata. Diagnosis using a panel of blood tests has the advantages of overcoming problems of inadequate muscle sampling, a larger number of sheep in the flock can be tested and a more rapid diagnosis can be obtained.     

A critical evaluation of serum methylmalonic acid and vitamin B12 for the assessment of cobalt deficiency of growing lambs in New Zealand

AIM: To derive reference ranges for serum methylmalonic acid (MMA) for the diagnosis of cobalt/vitamin B₁₂-responsiveness in lambs and critique existing serum vitamin B₁₂ reference ranges.

METHODS: Individual animal data from earlier supplementation trials, involving 225 ewes, 106 suckling lambs, 301 lambs during the suckling and post-weaning periods and 414 weaned lambs, for which weight gain to supplementation was observed, were used to derive relationships between serum vitamin B₁₂ and MMA, and liveweight gain.

RESULTS: Serum MMA concentrations were rarely elevated above the norm of <2 µmol/L when serum vitamin B₁₂ concentrations were >375 pmol/L, and not elevated into the range where a liveweight response to supplementation occurred (>10 µmol/L) unless serum vitamin B₁₂ concentrations were below 200 pmol/L. Suckling lambs were able to maintain high growth rates despite elevated serum MMA concentrations (>20 µmol/L).

CONCLUSIONS: The current reference ranges used in New Zealand for serum vitamin B₁₂ are set conservatively high. Serum MMA concentrations appear to allow better differentiation of a responsive condition than vitamin B₁₂ concentrations. Serum MMA concentrations <13 µmol/L indicate responsiveness to supplementation whilst concentrations <7 µmol/L indicate unresponsiveness. In the range 7–13 µmol/L, variation in response was observed and predictability of response is less certain, but supplementation is advisable.

CLINICAL RELEVANCE: The current reference ranges for vitamin B₁₂ responsiveness are conservatively high and lead to over-diagnosis of vitamin B₁₂ deficiency in ill-thriftiness of sheep.     

Caspase Activation,Hydrogen Peroxide Production and Akt Dephosphorylation Occur During Stallion Sperm Senescence

To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h decrease in the percentages of motile and progressive motile sperm occurred. The decrease observed in sperm functionality after 6 h of incubation was accompanied by a significant increase in the production of hydrogen peroxide and the greatest increase in caspase 3 activity. Additionally, the percentage of phosphorylated Akt reached a minimum after 6 h of incubation. These results provide evidences that sperm death during in vitro incubation is largely an apoptotic phenomena, probably stimulated by endogenous production of hydrogen peroxide and the lack of prosurvival factors maintaining Akt in a phosphorylated status. Disclosing molecular mechanisms leading to sperm death may help to develop new strategies for stallion sperm conservation.     

The Importance of Having Zinc During In Vitro Maturation of Cattle Cumulus–Oocyte Complex: Role of Cumulus Cells

The aim of this study was to investigate the influence of zinc (Zn) on the health of cumulus–oocyte complex (COC) during in vitro maturation (IVM). Experiments were designed to evaluate the effect of Zn added to IVM medium on: DNA integrity, apoptosis, cumulus expansion and superoxide dismutase (SOD) activity of cumulus cells (CC). Also, role of CC on Zn transport during IVM was evaluated on oocyte developmental capacity. DNA damage and early apoptosis were higher in CC matured with 0 μg/ml Zn compared with 0.7, 1.1 and 1.5 μg/ml Zn (p < 0.05). Cumulus expansion did not show differences in COC matured with or without Zn supplementation (p > 0.05). Superoxide dismutase activity was higher in COC matured with 1.5 μg/ml Zn than with 0 μg/ml Zn (p < 0.05). Cleavage and blastocyst rates were recorded after IVM in three maturation systems: intact COCs, denuded oocytes with cumulus cells monolayer (DO + CC) and denuded oocytes (DO). Cleavage rates were similar when COC, DO + CC or DO were matured with 1.5 μg/ml Zn compared with control group (p > 0.05). Blastocyst rates were significantly higher in COC than in DO + CC and DO with the addition of 1.5 μg/ml Zn during IVM (p < 0.01). Blastocyst quality was enhanced in COC and DO + CC compared with DO when Zn was added to IVM medium (p < 0.001). The results of this study indicate that Zn supplementation to IVM medium (i) decreased DNA damage and apoptosis in CC; (ii) increased SOD activity in CC; (iii) did not modify cumulus expansion and cleavage rates after in vitro fertilization; (iv) improved subsequent embryo development up to blastocyst stage; and (v) enhanced blastocyst quality when CC were present either in intact COC or in coculture during IVM.     

Positive Effects of Melatonin Treatment on the Reproductive Performance of Young Border Leicester Rams Mated to Merino Ewes in Spring: Preliminary Observations

Poor reproductive performance of Merino ewe flocks when mated to Border Leicester rams during spring may be due to seasonality of the Border Leicester breed. Two approaches were taken to test this assumption. Six young (12 months old) or six mixed‐age (12, 24 and ≥36 months old) Border Leicester rams were either treated or not treated with melatonin implants (2 × 2 design) 6 weeks before the four groups of rams were each put with approximately 300 Merino ewes for an 8‐week mating period. Implants were inserted in early September (experiment 1). The second approach was to yard or not yard ewes and mixed‐age rams on several occasions during the first 3 weeks of the mating period (experiment 2). Pregnancy rate and twinning percentage were assessed by ultrasonography. In experiment 1, melatonin treatment in young rams increased (p < 0.001) pregnancy rate from 5.0% to 92.6%, but mixed‐age rams did not respond (90.7% vs 89.5% for melatonin and non‐melatonin treatments, respectively). Twinning rate was similar (p > 0.05) for ewes mated to either melatonin or non‐melatonin‐treated young rams (36.8% vs 40.0%, respectively), whereas melatonin significantly improved (p < 0.05) twinning rate in those ewes mated to mixed‐age rams (49.1% vs 36.1%). After 6 weeks of melatonin treatment, scrotal circumference was greater (p < 0.05) in both young and mixed‐aged rams than in untreated counterparts. In experiment 2, yarding of ewes and rams overnight on several occasions early in the mating period reduced (p < 0.001) pregnancy rate compared with non‐yarded counterparts (89.5% vs 65.5%). Twinning rate was not affected (37.7% vs 36.1%, respectively). In summary, melatonin treatment of Border Leicester rams significantly improved flock reproductive performance in spring due to improved pregnancy rates with young rams and improved litter size with mixed‐age rams.     

Prevalence of dental and mandibular disorders in culled sheep in Spain

At the slaughterhouse, 1465 culled sheep heads were studied in order to identify disorders of the mandibular cheek teeth. Of these, 227 (15.5%) had evidence of osteomyelitis. The lesional profile showed that the mandible was affected in a similar proportion on both sides (46.7% on the right side vs 50.7% on the left side), mainly in the middle region (55.3%) and with most of the lesions closed without fistulisation (89.4%). In addition, swelling was palpable, with an increase in thickness in the area of the affected body (2.65 ± 0.065 cm vs 1.74 ± 0.030 cm). In 78.6% of the animals, the regional lymph nodes were enlarged. Concerning the mandibular cheek teeth, more than half of the animals lacked at least one tooth (57.0%), with the first premolar being the most frequently missing tooth (34.8%) and the third molar the least (8.2%). The impaction of food around the teeth was very common with the posterior molars being more frequently affected. In the knowledge of the authors, this is the first study that analyses the prevalence of mandibular molar and premolar disorders in sheep, and these are revealed as an important condition affecting culled sheep.     

Disease signs reported in south-eastern Australian dairy cattle while grazing Brassica species

Objective
To estimate the relative frequency of various disease signs in dairy cattle fed brassicas in south-eastern Australia, to estimate the incidence of disease signs within affected groups and to identify risk factors for commonly reported signs in brassica-fed dairy cows.
Design
A case series study and a case-control study.
Procedures
Case data was collected using an incident reporting system. Herd managers and veterinarians recorded details about disease incidents in brassica-fed dairy cattle in summer and autumn 1995 using a standard questionnaire. Potential risk factors for photosensitisation were assessed using a case-control study.
Results
Disease signs were reported in 66 groups of brassica-fed dairy cattle. Photosensitisation and bloat were the more frequently reported signs. While high incidences were reported in some groups, the incidence of disease signs was low within most affected groups. Photosensitisation occurred more frequently among groups of cows fed brassica crops treated with nitrogenous fertilisers or which were low yielding. Risks of other disease signs were greatest while the first one quarter of the crop was grazed.
Conclusions
In the study population, most disease outbreaks occurring in brassica-fed cattle were of low incidence. However, some high incidence outbreaks occurred. Results from this study suggest that important risk factors exist for disease signs in brassica-fed dairy cattle. Further studies are required if these risk factors are to be fully identified. This would allow the development of preventative strategies for high incidence disease outbreaks while feeding brassicas to dairy cattle.