Assessment of T-dependent and T-independent immune responses in cattle using a B cell ELISPOT assay

Understanding the mechanisms that maintain protective antibody levels after immunisation is important for vaccine design. In this study, we have determined the kinetics of plasma and memory B cells detectable in the blood of cattle immunised with model T-dependent or T-independent antigens. Immunisation with the T-D antigen resulted in an expansion of TNP-specific plasma cells post-TNP primary and booster immunisations, which was associated with increased titres of TNP-specific IgG antibodies. Although no TNP-specific memory B cells were detected in the T-D group following the primary immunisation, we detected an increase in the number of TNP-specific memory B cells post-TNP boost. In contrast, no TNP-specific plasma or memory B cells were detected after primary or secondary immunisation with the T-I antigen. We then investigated if immunisation with a third party antigen (tetanus toxin fragment C, TTC) would result in a bystander stimulation and increase the number of TNP-specific plasma and memory B cells in the T-D and/or T-I group. TTC immunisation in the T-D group resulted in a small increase in the number of TNP-specific plasma cells post-TTC primary immunisation and boost, and in an increase in the number of TNP-specific memory B cells post-TTC boost. This bystander effect was not observed in the animals previously immunised with the T-I antigen. In conclusion, the present study characterised for the first time the B cell response in cattle to immunisation with T-D and T-I antigens and showed that bystander stimulation of an established T-D B cell memory response may occur in cattle.     

Flow cytometry in Spermatology: A bright future ahead

Techniques such as mass spectrometry have led to unprecedented knowledge of the proteins that are present in the spermatozoa of humans and other mammals. However, in spite of their high‐throughput and fractioning techniques, most of the techniques in use only offer average values for the entire sperm population. Yet, ejaculate is very heterogeneous, and average values may mask relevant biological information.The application of flow cytometry may overcome this disadvantage, allowing proteomic analysis at the single‐cell level. Moreover, recent advances in cytometry, allowing multiple analyses within a single cell combined with powerful statistical tools, as an expanding subfield in spermatology, are described. The increased use of advanced flow cytometers in andrology laboratories will allow the rapid development of multiparametric, multicolour flow cytometry in andrology that will expand the clinical applications and research possibilities of flow cytometry‐based proteomic approaches, especially in the subfields of clinical andrology and sperm biotechnology.     

Do Computer‐Assisted,Morphometric‐Derived Sperm Characteristics Reflect DNA Status in Canine Spermatozoa?

It is widely accepted that sperm morphology is a strong indicator of semen quality. As the sperm head mainly comprises the sperm DNA, it is have been proposed that subtle changes in sperm morphology may be related to abnormal DNA content. Semen from four mongrel dogs was used to investigate DNA quality by means of the sperm chromatin structure assay (SCSA), and for computerized sperm morphometry (CASMA). Each sperm head was measured for nine primary parameters [head area (A), head perimeter (P), head length (L), head width (W), midpiece width (w), midpiece area (a), distance (d) between the major axes of the head and midpiece, angle (theta) of divergence of the midpiece from the head axis] and four parameters of head shape [FUN1, L/W; FUN2, 4piA/P(2); FUN3, (L - W)/(L + W); FUN4, piLW/4A]. Significant differences were found in all CASMA-derived parameters among dogs (p < 0.001). Linear regression models including sperm head shape factors 1, 3 and 4 predicted the extent of DNA denaturation (p < 0.001). We conclude that the CASMA analysis can be considered a powerful tool to improve the spermiogram.     

Dexmedetomidine and midazolam interaction is synergistic with isoflurane and additive with halothane in terms of MAC reduction

Dexmedetomidine and midazolam have synergistic interaction for the sedative/hypnotic and analgesic effects. The purpose of this study was to assess the type of interaction between dexmedetomidine and midazolam for the immobilizing effect in terms of MAC reduction of either halothane (HAL) or isoflurane (ISO). Fifty‐six rats were randomly allocated into one of eight groups (n = 7): SAL + HAL group received saline solution and halothane, SAL + ISO group received saline solution and isoflurane, DEX + HAL group received an intravenous continuous infusion of dexmedetomidine (0.25 μg kg^–1minute^–1) and halothane, DEX + ISO group received an intravenous continuous infusion of dexmedetomidine (0.25 μg kg^–1 minute^–1) and isoflurane, MID + HAL group received an intravenous bolus of midazolam (1 mg kg^–1) and halothane, MID + ISO group received an intravenous bolus of midazolam (1 mg kg^–1) and isoflurane, DEX +MID + HAL group received dexmedetomidine (0.25 μg kg^–1 minute^–1), midazolam (1 mg kg^–1) and halothane and DEX + MID + ISO group received dexmedetomidine (0.25 μg kg^–1 minute^–1), midazolam (1 mg kg^–1) and isoflurane. The tail clamp method was used for MAC determination. Heart rate, invasive arterial blood pressure, respiratory rate and rectal temperature were continuously monitored. Arterial blood gases were analyzed at the end of each experiment. Data were analyzed using a one‐way anova and a Tukey‐Kramer test for multiple comparisons. A p < 0.01 value was considered statistically significant. MAC values were adjusted to the barometric pressure at sea level. Control MAC_bar values expressed as mean ± SD were 1.31 ± 0.11% for HAL and 1.46 ± 0.05% for ISO. Percentages of MAC reduction were 72 ± 17% for HAL and 43 ± 14% for ISO in DEX groups, 26 ± 11% for HAL and 20 ± 9% for ISO in MID groups, and 90 ± 5% for HAL and 78 ± 5% for ISO in DEX + MID groups. The interaction between dexmedetomidine and midazolam in terms of MAC reduction can be described as additive with halothane and synergistic with isoflurane.     

Redox cycling induces spermptosis and necrosis in stallion spermatozoa while the hydroxyl radical (OH•) only induces spermptosis

Oxidative stress is a major factor explaining sperm dysfunction of spermatozoa surviving freezing and thawing and is also considered a major inducer of a special form of apoptosis, visible after thawing, in cryopreserved spermatozoa. To obtain further insights into the link between oxidative stress and the induction of apoptotic changes, stallion spermatozoa were induced to oxidative stress through redox cycling after exposure to 2‐methyl‐1,4‐naphthoquinone (menadione), or hydroxyl radical formation after FeSO₄ exposure. Either exposure induced significant increases (p < 0.05) in two markers of lipid peroxidation: 8‐iso‐PGF_2α and 4‐hydroxynonenal (4‐HNE). While both treatments induced changes indicative of spermptosis (caspase‐3 activation and decreased mitochondrial membrane potential) (p < 0.01), menadione induced sperm necrosis and a dramatic reduction in motility and thiol content in stallion spermatozoa. Thus, we provided evidence that oxidative stress underlies spermptosis, and thiol content is a key factor for stallion sperm function.     

Changes in Testicular Interstitial Connective Tissue of Hamsters (Mesocricetus auratus) During Ageing and After Exposure to Short Photoperiod

The testicular interstitium of Syrian hamster (Mesocricetus auratus) was studied during ageing and in testicular regression after exposure to a short photoperiod, in relation to the interstitial cells and their connective tissue. This tissue was assessed histochemically using Masson's trichrome technique and the expression of Heat Shock Protein 47 (HSP‐47) and collagen IV (α5) was assessed in Leydig cells. Finally, an ultrastructural analysis of some cells of the testicular interstitium was made. Leydig cells were positive for HSP‐47 and collagen IV (α5). Ageing did not change the parameters studied while the short photoperiod altered the synthetic activity of Leydig cells. The positivity index of these cells for HSP‐47 was significantly higher in the regressed testis, but was lower for collagen IV (α5). During ageing no change were observed. Ultrastructural Leydig cells showed a discontinuous basal lamina that did not change during ageing. The basal lamina was not identified in Leydig cells regressed by exposure to a short photoperiod. In conclusion; the intertubular connective tissue suffers little change with age. By contrast, in the testis regressed after exposure to a short photoperiod the studied parameters related to the intertubular connective tissue were altered. These changes are probably related with the low synthetic activity of regressed Leydig cell.     

Recharge in Volcanic Systems: Evidence from Isotope Profiles of Phenocrysts

Strontium isotope ratios measured from core to rim across plagioclase feldspar crystals can be used to monitor changes in the isotope composition of the magma from which they grew. In samples from three magma systems from convergent margin volcanoes, sudden changes in major element composition, petrographic features, and strontium isotope composition were found to correspond to discrete magmatic events, most likely repeated recharge of more mafic magma with lower ratios of strontium-87 to strontium-86 into a crustally contaminated magma.     

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.     

Does the Microbial Flora in the Ejaculate Affect the Freezeability of Stallion Sperm?

In an attempt to evaluate the possible relationship between the microbial flora in the stallion ejaculate and its ability to freeze, three ejaculates from five stallions were frozen using a standard protocol. Before freezing, an aliquot was removed for bacteriological analysis. Bacterial growth was observed in all the ejaculates studied. The isolated microorganisms were: Staphylococcus spp. and Micrococcus spp. (in all the stallions), β-haemolytic Streptococcus (in stallions 3 and 4), Corynebacterium spp. (in stallions 1, 3–5), Rhodococcus spp. (in stallion number 2), Pseudomonas spp. (in stallion number 1) and Klebsiella spp. (in stallions 1, 3 and 5). The presence and richness of Klebsiella and β-haemolytic Streptococcus in the ejaculate were related to two sperm variables post-thaw, namely the proportion of dead spermatozoa (ethidium+ cells; r = 0.55, p < 0.05) and the amplitude of lateral displacement of the sperm head (ALH, μm; r = −0.56, p < 0.05), respectively. The degree of growth of Corynebacterium spp. in the ejaculate was positively correlated with the percentage of spermatozoa showing high caspase activity post-thaw (r = 0.62, p < 0.05). The presence and number of colonies of β-haemolytic Streptococcus were negatively correlated (r = −0.55, p < 0.05) with low sperm caspase activity. It is concluded that the microbial flora of the equine ejaculate may be responsible for some of the sublethal damage experimented by the spermatozoa during cryopreservation.