The response of bovine spermatozoa to bicarbonate and its use to assess the influence of added oviductal epithelial proteins on cryopreservation.

The oviduct is a crucial organ for fertilization and has been demonstrated to perform a variety of interactions with spermatozoa ranging from sperm storage, to stabilizing sperm membranes and reducing free radicals. The oviduct is separated into 2 anatomically and physiologically distinct regions: the isthmus, in which sperm are stored, and the ampulla where fertilization occurs. We aimed to investigate whether proteins derived from different regions of the bovine oviduct had beneficial effects on bovine sperm membrane integrity, osmotic resistance, and motility following cryopreservation. The extent to which sperm motility could be activated by bicarbonate was demonstrated and used as a novel approach to postthaw sperm assessment. While oviductal proteins did not increase the degree of postthaw sperm viability, spermatozoa exposed to the isthmic proteins before freezing showed higher osmotic resistance after thawing. The presence of bicarbonate increased the proportion of spermatozoa with high curvilinear (VCL) and straight line velocity (VSL) in all treatment groups. After thawing, spermatozoa exposed to isthmic proteins had higher VCL and VSL than spermatozoa exposed to the ampullar proteins. We conclude that proteins derived from the isthmus can stabilize and protect spermatozoa during cryopreservation.

The precision and accuracy of six different methods to determine sperm concentration.

The development of new technologies and software that are routinely used in laboratories has now allowed for a more diverse novel range of methods to determine sperm concentrations more rapidly. The aim of this study was to compare 3 such novel methods developed in our laboratory, including a new flow cytometry approach, image analysis, and a fluorescent plate reader, with more conventional methods (hemocytometry, spectrophotometry, and Microcell analysis). Fifteen ejaculates were collected from 13 bulls at an artificial insemination center. The semen samples were analyzed for sperm concentration using a spectrophotometer, hemocytometry, and a novel flow cytometry technique based on counting a fixed volume of fluid. The raw ejaculate was also diluted fivefold in a long-term diluent and sent overnight to another laboratory, where sperm numbers were assessed using Microcells, an image analysis system, and a fluorescent plate reader. Each ejaculate was assessed 5 times using each of the methods described in order to determine the coefficient of variation for each method. Comparisons between methods were determined using correlation and limits of agreement. The flow cytometry results showed the lowest coefficient of variation (2.3%), with the plate reader showing the highest coefficient of variation (20.0%). There was no significant difference between any of the methods used, and none of them consistently over- or underestimated numbers when compared against each other. It is concluded that flow cytometry showed the highest repeatability of results. However, the method employed by each laboratory should be determined based on a range of factors, including cost, convenience, sample size, and number of ejaculates to be assessed.

A study of synchronisation between the flagella of bull spermatozoa, with related observations.

Flagellar synchronisation has been observed between bull spermatozoa as they swam in a viscous medium, confined to a glass surface. This process is of interest in understanding the regulation of flagellar oscillation in general. Exact and persisting synchrony between bull spermatozoa occurred only when the spermatozoan heads were tightly coupled mechanically. For these cells, viscous coupling between the flagella was not by itself sufficient to establish synchronisation. Immediately on synchronisation, with the spermatozoan heads superposed, the paired spermatozoa showed rises in conjoint beat frequency, wave velocity and swimming velocity, i.e. in nearly all cases, the new conjoint values were greater than those shown by either of the two singleton spermatozoa. In our interpretation of these results, we put forward hydrodynamic arguments for seeing the primary change as a rise in wave velocity, via a decreased viscous resistance to bend propagation. Mechanistically, the rise in beat frequency is mysterious unless, as we suggest, it is consequential to the rise in wave velocity, and mediated by an as-yet-unknown mechanical feedback process. The rise in swimming velocity is not surprising given the rise in wave velocity but there is evidence for an additional influence due to a subtle re-orientation of the conjoint spermatozoan heads, such that they experienced less frictional drag.

Adenosine triphosphate production by bovine spermatozoa and its relationship to semen fertilizing ability.

This article's objectives are to investigate the relationship between adenosine triphosphate (ATP) production (oxidative phosphorylation and glycolysis) and fertility of bovine spermatozoa, determine the proportion of oxygen consumption devoted to proton leak and that due to nonmitochondrial processes, and discover whether freeze/thawing affects sperm oxygen consumption. Oxygen consumption of bovine spermatozoa was measured using a standard Clark electrode and, for the first time, in an Oxygen Biosensor System (OBS). Total ATP formation by bovine spermatozoa was calculated from the oxygen consumption and lactate production (glycolysis) by the same spermatozoa sample. ATP production varied from 1.99 to 8.09 mumol ATP per 10(8) spermatozoa per hour; glycolysis accounted for 16% to 38% of ATP. Nonmitochondrial oxygen consumption could not be detected in bovine spermatozoa using these methods. A significant proportion (16%-43%) of oxygen consumption was insensitive to oligomycin and was due to "proton leak." There was no significant difference between oxygen consumption of frozen/thawed and fresh spermatozoa for 2 of the 3 bulls tested. However, oxygen consumption of frozen/thawed spermatozoa was significantly higher (P < .05) than fresh spermatozoa for the third bull. When ZO(2) of frozen/thawed spermatozoa from 20 bulls was compared with their 49 day nonreturn rates (NRRs), oxygen consumption was correlated positively with NRR (ie, fresh spermatozoa with a higher ZO(2) were more fertile). Moreover, total ATP production correlated with NNR better than ZO(2). Bulls with a lower NRR produce spermatozoa that are susceptible to damage during the freeze/thawing process, causing an increase in ZO(2), possibly due to mitochondrial membrane damage resulting in more energy being expended in maintaining the proton gradient, or capacitation-like changes causing hyperactivation. Oxygen consumption measured in the OBS may be useful in assessing bovine sperm fertility.