Simultaneous evaluation of plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential in bovine spermatozoa by flow cytometry

The present study aimed to develop an objective evaluation procedure to estimate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bull spermatozoa simultaneously by flow cytometry. Firstly, we used frozen-thawed semen mixed with 0, 25, 50, 75 or 100% dead spermatozoa. Semen was stained using three staining solutions: SYBR-14, propidium iodide (PI), and phycoerythrin-conjugated peanut agglutinin (PE-PNA), for the evaluation of plasma membrane integrity and acrosomal integrity. Then, characteristics evaluated by flow cytometry and by fluorescence microscopy were compared. Characteristics of spermatozoa (viability and acrosomal integrity) evaluated by flow cytometry and by fluorescence microscopy were found to be similar. Secondly, we attempted to evaluate the plasma membrane integrity, acrosomal integrity, and also mitochondrial membrane potential of spermatozoa by flow cytometry using conventional staining with three dyes (SYBR-14, PI, and PE-PNA) combined with MitoTracker Deep Red (MTDR) staining (quadruple staining). The spermatozoon characteristics evaluated by flow cytometry using quadruple staining were then compared with those of staining using SYBR-14, PI, and PE-PNA and staining using SYBR-14 and MTDR. There were no significant differences in all characteristics (viability, acrosomal integrity, and mitochondrial membrane potential) evaluated by quadruple staining and the other procedures. In conclusion, quadruple staining using SYBR-14, PI, PE-PNA, and MTDR for flow cytometry can be used to evaluate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bovine spermatozoa simultaneously

Serum iron concentration as a marker of inflammation in young cows that underwent dehorning operation

This study aimed to assess the usefulness of serum iron (Fe) concentration as a marker of inflammation caused by the dehorning operation. Five young Holstein cows aged 205.0 ± 10.7 days and weighing 207.2 ± 24.1 kg underwent the dehorning operation. Blood samples were withdrawn before dehorning (pre) and at time periods of t=0.5, 2, 4, 6, 8, 12, 24, and 48 hr. The serum amyloid A (SAA) concentration was significantly high at t=48 hr (P<0.01). The serum Fe concentration significantly decreased, reaching 90.0 ± 36.4 µg/dl at t=24 hr (P<0.001). Therefore, serum Fe concentration showed significant and negative correlation with SAA concentration (r^2=0.500, P<0.01). In conclusion, serum Fe concentration is a useful marker of inflammation in young cows that have undergone the dehorning operation

Relationship between postnatal days, serum Cu concentration and plasma diamine oxidase activity in Japanese Black calves

The aim of study was to investigate the relationships among serum diamine oxidase (DAO) activity, postnatal days and the plasma copper (Cu) concentration, using calves with or without diarrhea. In healthy calves, the serum DAO activity was significantly higher at 2 postnatal days than at ≥7 postnatal days, and no significant changes were observed after 7 postnatal days. In addition, no significant correlation was found between serum DAO activity and plasma Cu concentration at all postnatal days in healthy calves. Although, the serum DAO activity in 14 diarrheic calves (66.78 ± 14.37 IU/ml) was lower than that in 19 healthy calves (170.33 ± 97.83 IU/ml, P<0.01), plasma Cu concentrations in all calves remained within the normal range

DataSheet_1_Faint chemical traces of conspecifics delay settlement of barnacle larvae.pdf

Larval settlement drives population and community dynamics and evolution of marine invertebrates. While it is well known that larvae sense a variety of biological and environmental factors, there is limited information on how they react to specific factors so as to locate settlement sites. Proteinaceous Waterborne Settlement Pheromone (WSP) was purified from adult barnacles, Amphibalanus amphitrite, in a previous study, but it is unclear how WSP guides barnacle cyprids to the source. Here, a series of settlement assays were conducted to investigate the concentration dependence of recombinant WSP. We report that low concentrations of barnacle WSP decrease the probability of settlement of conspecific cyprids, whereas high concentrations have the opposite effect. We suggest that this is because weak WSP informs cyprids that suitable settlement sites with adult barnacles are distant, inducing them to extend the larval phase. The present study clearly shows that larvae use WSP concentration, rather than presence/absence information. In barnacles, several other conspecific chemical/visual cues also attract larvae. This study opens the door to future research to determine how these cues influence larval behavior in nature.</p

Table_1_Faint chemical traces of conspecifics delay settlement of barnacle larvae.xlsx

Larval settlement drives population and community dynamics and evolution of marine invertebrates. While it is well known that larvae sense a variety of biological and environmental factors, there is limited information on how they react to specific factors so as to locate settlement sites. Proteinaceous Waterborne Settlement Pheromone (WSP) was purified from adult barnacles, Amphibalanus amphitrite, in a previous study, but it is unclear how WSP guides barnacle cyprids to the source. Here, a series of settlement assays were conducted to investigate the concentration dependence of recombinant WSP. We report that low concentrations of barnacle WSP decrease the probability of settlement of conspecific cyprids, whereas high concentrations have the opposite effect. We suggest that this is because weak WSP informs cyprids that suitable settlement sites with adult barnacles are distant, inducing them to extend the larval phase. The present study clearly shows that larvae use WSP concentration, rather than presence/absence information. In barnacles, several other conspecific chemical/visual cues also attract larvae. This study opens the door to future research to determine how these cues influence larval behavior in nature.</p