**6. Metabolic phenotypes: a result of the separate evolutionary strategies developed by mammals to optimize reproductive indexes**

All of data showed above highlight a phenomenon that has not been much explained, al‐ though it is well known by all of investigators in this field. This phenomenon is the strong species-specificity that energy obtainment mechanisms show when comparing separate mammals. Differences are so intense that several metabolic phenotypes can be defined, de‐ pending on the metabolic characteristics showed by each species. In this manner, there are at least two separate metabolic phenotypes regarding mammalian spermatozoa. The first phenotype will be composed by species in which energy substrates, mainly monosacchar‐ ides, will be directed to the practically immediate utilization of all of the assimilated sugars through the appropriate catabolic pathways, especially glycolysis. This specific metabolic phenotype is very common in mammalian sperm, especially in those species, which do not require a long, sperm-survival time-lapse inside the female genital tract such as pig and bull [24, 47]. However, a second phenotype is evident in species where sperm survival inside the female genital tract must be relatively long, such as the dog [15]. In these species, an energy strategy based upon an entirely catabolic metabolism would not be efficient. The optimiza‐ tion of energy Management in relatively long-living sperm like dog would be optimized with the presence of alternative anabolic pathways, such as glycogen synthesis, which al‐ lows for the maintenance of a significant mid-to-long intracellular energy reserve. This re‐ serve would play an important role in the maintenance of "in vivo" sperm survival. In fact, as discussed above, the existence of a fully functional glycogen metabolism has been dem‐ onstrated in sperm from species like dog, boar, horse and ram [5]. Remarkably, dog sperm shows the most active glycogen metabolism of all of the studied species, in this way accu‐ mulating the maximal recorded intracellular levels [5]. As described above, this glycogen plays an important role in the achievement of feasible "in vitro" capacitation [1, 2], reinforc‐ ing thus the importance of this anabolic pathway in dog. The importance of glycogen syn‐ thesis in dog would be surely linked to another important feature, also described above. It is worth noting that dog sperm is the only studied species so far that shows the presence of two separate hexokinase activities. The first of them is similar to hexokinase-I, which is present in all of the studied mammalian sperm. The second, however, is similar in kinetic and immunologic properties to the hepatic and pancreatic isoform glucokinase [16]. The presence of a glucokinase-like activity in dog sperm but not in other species like boar ac‐ quires utmost importance when the precise role that hepatic and pancreatic glucokinase plays is studied. Thus, it is well known that hepatic glucokinase acts as a "metabolistate" that diverts hexoses metabolism to either anabolic or catabolic pathways, depending on fac‐ tors such as the precise physiologic cell status and sugar extracellular levels [9]. If a similar role for the dog sperm glucokinase-like activity is assumed, the inference that this protein also regulates the entry of energy metabolites in either anabolic or catabolic pathways can be also yielded. These assumptions, notwithstanding will depend on both the precise energy necessities and the extracellular concentrations of sugars inside the female genital tract. Moreover, this "metabolistate" seems to be in the basis of above described, observed differ‐ ential effects of fructose and glucose in the serine phosphorylation levels of dog sperm pro‐ teins like protein kinase C [17]. Thus, dog sperm reaches an even more fine regulation of not only their intracellular energy levels, but also their overall functional status. This very fine regulation would surely increase survival ability of these cells.

being a mere energy-producing factory. Mitochondria also play a key role in the control of other very important aspects of eukaryotic cells function, like modulation of apoptosis and the control of calcium metabolism. Thus, it is very probable that mitochondria from sperm of species like boar or mouse exert their most important functions on other cellular function‐ al points than energy management. Unpublished results from our laboratory are strongly pointing out this supposition. Thus, the incubation of boar sperm in a capacitation medi‐ um in the presence of olygomycin A, a specific inhibitor of the electronic chain and the chemiosmosis steps [11], immobilizes boar sperm and prevent them to achieve "in vitro" capacitation. However, this effect was accomplished without any significant changes in the rhythm of O2 production and the intracellular ATP levels (Figures 2, 4, 5 and data not shown from our labvoratory). In contrast, the incubation of boar sperm in a capacitation medium without calcium induces an increase in the velocity parameters of these cells, although the achievement of capacitation is also prevented (data not shown). The effect linked to the lack of extracellular calcium however, is again concomitant with no changes in both the rhythm of O2 production and the intracellular ATP levels (Figures 2, 4 and data not shown). The conclusion from these results is that boar (and probably mice) sperm mitochondria play an important regulatory role in the control of functional aspects such as motility patterns and the achievement of "in vitro" capacitation by ways that are not directly linked to energy production. This opens a new perspective in the manner in which investigators would have to approximate to the understanding of the mitochondria role in the control of sperm func‐ tion. However, much more work is needed in order to achieve a complete view of this

166 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed

**6. Metabolic phenotypes: a result of the separate evolutionary strategies**

All of data showed above highlight a phenomenon that has not been much explained, al‐ though it is well known by all of investigators in this field. This phenomenon is the strong species-specificity that energy obtainment mechanisms show when comparing separate mammals. Differences are so intense that several metabolic phenotypes can be defined, de‐ pending on the metabolic characteristics showed by each species. In this manner, there are at least two separate metabolic phenotypes regarding mammalian spermatozoa. The first phenotype will be composed by species in which energy substrates, mainly monosacchar‐ ides, will be directed to the practically immediate utilization of all of the assimilated sugars through the appropriate catabolic pathways, especially glycolysis. This specific metabolic phenotype is very common in mammalian sperm, especially in those species, which do not require a long, sperm-survival time-lapse inside the female genital tract such as pig and bull [24, 47]. However, a second phenotype is evident in species where sperm survival inside the female genital tract must be relatively long, such as the dog [15]. In these species, an energy strategy based upon an entirely catabolic metabolism would not be efficient. The optimiza‐ tion of energy Management in relatively long-living sperm like dog would be optimized with the presence of alternative anabolic pathways, such as glycogen synthesis, which al‐ lows for the maintenance of a significant mid-to-long intracellular energy reserve. This re‐

**developed by mammals to optimize reproductive indexes**

complex phenomenon.

These two separate metabolic phenotypes would not surely be the only present among mammalian species. Much more work is needed in order to describe and analyze this phe‐ nomenon. In any case, the existence of these metabolic phenotypes would be of the greatest importance. These phenotypes, in fact, will be the reflection of the sperm specialization due to the adoption of separate reproductive strategies among mammals. Thus, these separate evolutive, reproductive strategies will cause the existence of great differences among sperm of separate species not only under a morphological, but also under a metabolic point of view. These differences among species would be, in turn, at the basis of the described differ‐ ences in vital aspects of sperm function, such as motility patterns and capacitation mecha‐ nisms. Finally, these physiological differences would also be reflected in changes in the specific strategy developed to store a particular semen sample from a precise species in opti‐ mal conditions.
