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SUMMARYFlagellar 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-yetunknown 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. Supplementary material available online at

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 mmol 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.Key words: Fertility, semen analysis, sperm, oxygen consumption. J Androl 2008;29:449-458A rtificial insemination (AI) accounts for approximately 80% of breedings in the United Kingdom dairy herd. However, the efficiency of AI is highly variable, with an average of 1.7 doses of semen being required per cow to establish a pregnancy. There is a continuing need for more reliable markers of sperm fertilizing ability. A simple, relatively inexpensive method that correlates with nonreturn rate (NRR; sperm fertility measured by the percentage of cows that do not require reinsemination) could reduce the number of low-fertility bulls that enter the costly proofing program of a stud. These could then be replaced with bulls of higher fertility. The test could also be used routinely to assess the fertility of semen before and/or after it is processed for AI. Current methods to evaluate semen quality include tests of motility, morphology, membrane integrity and membrane strength (osmotic resistance test), chromatin structure, and gamete interactions (Rodriguez-Martinez,...

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ABSTRACT: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 longterm 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.