CONTENTS: Flow cytometry has become an important technique in sperm evaluation and is increasingly used both for routine assessment and for research in veterinary science. We have revised the liter ...

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1439-0531.2010.01622.x

Probes and techniques for sperm evaluation by flow cytometry.

We demonstrated previously that a knockout (KO) of the lactate dehydrogenase type C (Ldhc) gene disrupted male fertility and caused a considerable reduction in sperm glucose consumption, ATP production, and motility. While that study used mice with a mixed genetic background, the present study used C57BL/6 (B6) and 129S6 (129) Ldhc KO mice. We found that B6 KO males were subfertile and 129 KO males were infertile. Sperm from 129 wild-type (WT) mice have a lower glycolytic rate than sperm from B6 WT mice, resulting in a greater reduction in ATP production in 129 KO sperm than in B6 KO sperm. The lower glycolytic rate in 129 sperm offered a novel opportunity to examine the role of mitochondrial respiration in sperm ATP production and motility. We observed that in media containing a mitochondrial substrate (pyruvate or lactate) as the sole energy source, ATP levels and progressive motility in 129 KO sperm were similar to those in 129 WT sperm. However, when glucose was added, lactate was unable to maintain ATP levels or progressive motility in 129 KO sperm. The rate of respiration (ZO2) was high when 129 KO or WT sperm were incubated with lactate alone, but addition of glucose caused a reduction in ZO2. These results indicate that in the absence of glucose, 129 sperm can produce ATP via oxidative phosphorylation, but in the presence of glucose, oxidative phosphorylation is suppressed and the sperm utilize aerobic glycolysis, a phenomenon known as the Crabtree effect.

https://bioone.org/journals/biology-of-reproduction/volume-88/issue-4/biolreprod.113.108530/Glycolysis-and-Mitochondrial-Respiration-in-Mouse-LDHC-Null-Sperm1/10.1095/biolreprod.113.108530.pdf

Glycolysis and Mitochondrial Respiration in Mouse LDHC-Null Sperm

Systematic misestimation of cell subpopulations by flow cytometry: a mathematical analysis.

AMP-activated kinase, AMPK, is involved in the maintenance of plasma membrane organization in boar spermatozoa.

Sperm competition often leads to increase in sperm numbers and sperm quality, and its effects on sperm function are now beginning to emerge. Rapid swimming speeds are crucial for mammalian spermatozoa, because they need to overcome physical barriers in the female tract, reach the ovum, and generate force to penetrate its vestments. Faster velocities associate with high sperm competition levels in many taxa and may be due to increases in sperm dimensions, but they may also relate to higher adenosine triphosphate (ATP) content. We examined if variation in sperm ATP levels relates to both sperm competition and sperm swimming speed in rodents. We found that sperm competition associates with variations in sperm ATP content and sperm-size adjusted ATP concentrations, which suggests proportionally higher ATP content in response to sperm competition. Moreover, both measures were associated with sperm swimming velocities. Our findings thus support the idea that sperm competition may select for higher ATP content leading to faster sperm swimming velocity.

/pdf/postcopulatory-sexual-selection-increases-atp-content-in-oquxr8dr8u.pdf

Postcopulatory sexual selection increases atp content in rodent spermatozoa

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 μmol 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.

https://onlinelibrary.wiley.com/doi/pdf/10.2164/jandrol.107.003533

Adenosine Triphosphate Production by Bovine Spermatozoa and Its Relationship to Semen Fertilizing Ability

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.

https://onlinelibrary.wiley.com/doi/pdf/10.2164/jandrol.05112

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

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.

Stuart G Revell

Papers

Adenosine Triphosphate Production by Bovine Spermatozoa and Its Relationship to Semen Fertilizing Ability

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

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