240 G. Stradaioli et al. Animal Reproduction Science 64 2000 233–245
Table 4 Significant correlation coefficients among seminal and biochemical characteristics of Maremmano stallions n
= 96 ejaculates
Spermatozoa count ×10
6
CAT activity
Lactate nmolml
TMMNS
a
×10
9
Raw semen Carnitine nmolml
0.62
∗∗
0.37
∗∗
– –
Acetylcanitine nmolml 0.67
∗∗
– –
0.30
∗∗
Carnitine nmol10
6
spermatozoa –
0.65
∗∗
– –
Acetylcanitine nmol10
6
spermatozoa –
0.48
∗∗
– –
Progressive motility at 24 h –
– 0.36
∗∗
– Progressive motility at 48 h
– –
0.34
∗∗
– Progressive motility at 72 h
– –
0.31
∗∗
– Seminal plasma
Carnitine nmolml 0.60
∗∗
– –
– Acetylcanitine nmolml
0.67
∗∗
– –
–
a
Total number of motile morphologically normal spermatozoa.
∗∗
P 0.01.
AC concentrations were positively correlated with spermatozoa concentration P 0.01. In raw semen AC was also correlated to the total number of motile morphologically nor-
mal spermatozoa P 0.01, while carnitine acetyltransferase activity was correlated to LC and AC. Lactate levels of raw semen were correlated to progressively motile sperma-
tozoa after storage at +4
◦
C P 0.01. In the second ejaculate, significant correlations were also observed among ACLC ratio in raw semen and progressively motile sperma-
tozoa after 48 and 72 h of refrigeration r = 0.47; P 0.01 and r = 0.45; P 0.05, respectively. Furthermore, AC levels were correlated to lactate concentration r = 0.57;
P 0.01.
Blood plasma AC and LC concentrations did not differ significantly among semen col- lection trials data not shown. Blood LC levels were three-fold higher than those of AC
18.25 ± 1.02 versus 5.90 ± 0.35 nmolml, respectively.
4. Discussion
Stallion reproductive characteristics are affected by age and breed Dowsett and Pattie, 1982; Dowsett and Pattie, 1987; Pickett et al., 1989; Dowsett and Knott, 1996. In the
study reported herein, the young Maremmano stallions were maintained in standardized environmental conditions during the 100-day performance test, allowing a more reliable
statistical evaluation of their reproductive characteristics. We have included the semen collection period effects in the statistical analysis, although no significant differences were
observed between seminal characteristics in the November and January trials.
The relevance of comparing the first and the second ejaculate collected 1 h apart has been well established for stallion breeding soundness evaluation Pickett et al., 1976; 1989.
Briefly, repeated semen collections serve to deplete epididymal reserves, and, in particular, the second ejaculate is the richest in motile cells. For these reasons comparison of the two
was one of our objectives.
G. Stradaioli et al. Animal Reproduction Science 64 2000 233–245 241
The gel free volume of the ejaculates was similar to those reported for Quarter Horses Pickett et al., 1976 and smaller than those of Dutch Warmblood stallions Parlevliet et al.,
1994. Spermatozoa concentration and the total number of spermatozoa in the first ejaculate
were about two-fold the second one. These findings are in agreement with data reported by Pickett et al. 1976 and by Parlevliet et al. 1994, both for differences between ejaculates
and value per se, thus suggesting that Maremmano stallions do not differ from other breeds with regard to sperm output.
The percentage of progressively motile and of morphologically normal live spermatozoa increased in the second ejaculate, although they were lower than data reported in other
breeds. The total number of motile morphologically normal spermatozoa was less than half of the value reported in maiden Dutch Warmblood stallions Parlevliet et al., 1994.
The number of subjects which were evaluated in our study was not sufficient to make an exhaustive analysis of the variance; nevertheless, among the characteristics considered, the
major dispersion of the data were linked to the differences between subjects, as depicted by the higher between subject mean square error.
Blood free carnitine content was similar to that previously reported in young thoroughbred horses both at stud and during training Foster et al., 1989.
The seminal plasma LC levels presented herein are in agreement with previous reports using nuclear magnetic resonance analysis Magistrini et al., 1995a,b and high pressure
liquid chromatography Stradaioli et al., 1995. In human beings, seminal plasma LC and AC levels ranged from 200 to 1300 nmolml and
from 60 to 280 nmolml, respectively Menchini-Fabris et al., 1984; Setchell et al., 1994, which do not differ greatly from our results in Maremmano stallions. In the ram, LC levels
resulted highly correlated with sperm concentration and seminal plasma contains five-fold more LC and 40-fold more AC than in the stallion Brooks, 1979. The high carnitine
levels of ram seminal plasma could be due in part to the differences in ejaculate volumes and sperm density between these species. Nevertheless, this phenomenon is also related
to specie differences; indeed, in the rat epididymal plasma LC concentration is 60 mM Bremer, 1983, while in the ram and in the stallion is 19 and 11 mM, respectively Jones,
1978. In bovine frozen semen, diluted with egg yolk citrate and glycerol, LC content ranged from 110 to 230 nmolml Carter et al., 1980, which was lower than in the Maremmano
stallion.
To our knowledge, this is the first report on LC and AC content in stallion raw semen and seminal plasma evaluated in two successive ejaculates. LC and AC levels in the first
ejaculate resulted about two-fold the second one, both in raw semen and seminal plasma. These results are related to the differences in spermatozoa concentration between the two
ejaculates, as demonstrated by the strong correlation between LC and AC. Indeed, both the ejaculates resulted identical when LC and AC are expressed as nmol10
6
spermatozoa. Similarly, French researchers observed that in fractionated semen collection carnitine levels
increase with spermatozoa concentration, thus allowing us to propose carnitine as a marker of epididymal functionality Magistrini et al., 1998.
In our study, the ACLC ratio was higher in raw semen than in seminal plasma; LC was almost 80 in seminal plasma, whereas AC was 55–60 of that revealed in raw se-
men. These findings are in agreement with data reported in human beings and ram where
242 G. Stradaioli et al. Animal Reproduction Science 64 2000 233–245
acetylation levels of carnitine were higher in sperm cells than in seminal plasma Brooks, 1979. These metabolites are in equilibrium within the sperm cell due to carnitine acetyl-
transferase activity, as evident by direct correlation shown in Table 4, which maintains the correct acetyl-CoAfree CoA ratio. The correct ratio acetyl-CoA to free CoA is fundamental
in order to maintain the correct functionality of the Kreb’s cycle and, therefore, a sufficient availability of ATP necessary for spermatozoa motility. Intracellular LC accumulated by
spermatozoa might perform a buffering role, trapping excess mitochondrial acetyl-CoA as AC, and this system would protect the activity of pyruvate dehydrogenase, and other key en-
zymes for mitochondrial respiration, which are inhibited by excess acetyl-CoA Uziel et al., 1988; Abdel-aleem et al., 1995; Jeulin and Lewin, 1996. Moreover, in mammalian sperma-
tozoa, AC may replace the energy storage function of high-energy phosphate compounds Smith et al., 1985.
Pyruvate, lactate and pyruvatelactate ratios were always higher in the second ejaculate. One can speculate that this could be related to the higher content on both unstained and motile
cells in the second ejaculate, as lactate and pyruvate are an intermediate of the glycolytic pathway of live metabolizing spermatozoa. Findings by Leone et al. 1989 that sperm
concentration and spermatozoa motility scores in oligoasthenospermic rats treated with acetyl-l-carnitine were significantly higher P 0.05 than untreated rats, further support
our observation. The positive correlation observed between lactate and the percentage of progressively motile spermatozoa after 24, 48 and 72 h of raw semen storage, in conjunction
with the positive correlation between AC and total number of motile morphologically normal spermatozoa, could indicate that these metabolites influence sperm cell viability. In this
respect, it is interesting to note that, in boar spermatozoa, lactate is the major mitochondrial substrate for ATP production Jones, 1997; Jones and Bubb, 2000; furthermore, lactate and
pyruvate contribute to hold the acetylation state of carnitine through acetyl-CoA formation Casillas, 1973. The correlation between ACLC ratio and spermatozoa motility in the
second ejaculate, observed at 48 and 72 h of storage, further supports this observation.
Both AC and lactate are precursors of the intramitochondrial acetyl-CoA pool, whereas only AC represents a true reservoir of activated acetyl groups Smith et al., 1985. Thus,
endogenous AC could guarantee gamete’s viability in ejaculated spermatozoa Jeulin and Lewin, 1996.
5. Conclusion
