G. Stradaioli et al. Animal Reproduction Science 64 2000 233–245 235
thiolase, a reduction of acetyl-CoA by carnitine may relieve such an inhibitory effect Wang et al., 1991; Abdel-aleem et al., 1995; Jeulin and Lewin, 1996. Both extra and intracellular
acetylcarnitine provide readily available acetyl groups for spermatozoa motility Milkowsky et al., 1976; Bruns and Casillas, 1990. In mature spermatozoa, high intracellular l-carnitine
concentrations increase the utilization of pyruvate and lactate Carter et al., 1980; Jones and Murdoch, 1996, thus holding the maximal “acetylation-state” of carnitine.
In human beings, seminal l-carnitine content is correlated with spermatozoa count and progressive motility Menchini-Fabris et al., 1984; Borman et al., 1989 and a reduction of
the acetylcarnitinel-carnitine ratio has been observed in asthenospermic patients Golan et al., 1984; Bartellini et al., 1987. Moreover, significant reduction of seminal carnitine
concentrations has been reported in azoospermic patients affected by bilateral agenesis of the vas deferens and epididymal obstruction Menchini-Fabris et al., 1984; Casano et al.,
1987, as well as during severe testicular failures Lewin et al., 1981. Recently, a reduction of seminal plasma carnitine has been reported in infertile men Zöpfgen et al., 2000. The
positive correlation observed among seminal parameters and seminal carnitine concentra- tion allows proposing carnitine as a “good quality” semen marker Menchini-Fabris et al.,
1984.
Our preliminary observations on breeding stallions demonstrated a positive correlation among sperm quality parameters, such as spermatozoa count, motility, in vitro storage
ability and seminal plasma carnitine activity Stradaioli et al., 1995; Chiodi et al., 1997. In addition, a reduced seminal plasma acetylcarnitine content in two necrospermic infertile
stallions has also been reported Sighieri et al., 1991.
The aim of the study reported herein was to evaluate seminal carnitine and acetylcar- nitine content and carnitine acetyltransferase activity as markers of the semen quality and
spermatozoa storage of young Maremmano stallions in breeding soundness examination.
2. Materials and methods
2.1. Animals, sample collection and seminal analysis The study was carried out over a 2-year period on Maremmano stallions n = 25, 42 ± 5
months of age, which were assigned for physical soundness and pedigree to the 100-day performance test. The animals were maintained in standardized environmental conditions
and training plans. After a 2-month adaptation period, the stallions underwent breeding soundness evalua-
tion. Physical examination of the reproductive tract, including transrectal ultrasound eval- uation of the accessory sexual glands Toshiba Sonolayer, SAL32A, 5 MHz linear probe,
was performed. The stallions were subjected to semen collection in November and January; in each trial
two ejaculates were collected 1 h apart Pickett et al., 1975, using an oestrus jump mare and the Colorado model artificial vagina Animal Reproduction System, Chino, CA, USA.
Immediately after collection, the colour and gel free volume of the ejaculate were de- termined. Progressive motility of the spermatozoa was assessed using a phase contrast
microscope TMS, Nikon, Japan at 200× by diluting the semen samples 1:3 to 1:8,
236 G. Stradaioli et al. Animal Reproduction Science 64 2000 233–245
depending on the concentration, with non-fat dry skim milk-glucose extender E–Z Mixin, Animal Reproduction System, Chino, CA, USA at 37
◦
C. The spermatozoa concentration was measured with the Bürker haemocitometer, corrected for dilution by calculation. Suf-
ficient 37
◦
C E–Z Mixin–amikacin 1000 unitsml seminal extender was then added to a specific amount of semen to achieve a final spermatozoa concentration of 20 × 10
6
ml. The diluted semen was thoroughly mixed and then subdivided into three aliquots, which
were then slowly cooled in a +4
◦
C refrigerator Varner et al., 1988. After 24, 48 and 72 h, the samples were incubated for 30 min at 37
◦
C prior to spermatozoa progressive motility evaluation.
In addition, a smear of the native semen, stained with eosin–nigrosin Blom, 1950, was prepared. The morphology and viability of 500 spermatozoa were assessed under
bright-field illumination at 1000× Optiphot 2, Nikon, Japan according to the criteria outlined by Bielanski et al. 1982. The spermatozoa were clustered in five classes: normal,
abnormal head shape, abnormal mid-piece and tail, curved mid-piece and tail, detached head and tail. Each sperm cell was placed in only one of the above classes; cells having more
than one of the aforementioned morphologic features were classified according to the more proximal one. The percentage of morphologically normal, including cytoplasmic droplets
and eccentric tail implant and abnormal live spermatozoa was calculated. The volume of the ejaculate was multiplied by the concentration 10
6
spzml, the percentage of progressive motile spermatozoa and the percentage of morphologically normal live spermatozoa to
obtain the total number of motile morphologically normal spermatozoa Parlevliet et al., 1994.
Raw semen was centrifuged at 600×g for 15 min and the 1 ml seminal plasma aliquots, after filtration through a 45 mm disposable syringe filter Durapore, Nalgene, were stored
at −20
◦
C until analysis. Raw semen samples 1 ml was also stored. An amount of 200 ml of 35 HClO
4
were added to duplicates of raw semen and seminal plasma for lactate and pyruvate analysis.
Blood samples were collected by jugular venopuncture into a heparinized vacutainer before semen collection and plasma was stored at −20
◦
C until analysis. 2.2. Biochemical analysis
2.2.1. Free l-carnitine and acetylcarnitine In order to measure free l-carnitine LC and acetylcarnitine AC, 1 ml of 5 cold
HClO
4
was added to 0.5 ml of blood plasma. The samples were centrifuged at 3000×g for 10 min at +4
◦
C and analysis was carried out on the supernatant. Absolute methanol 10 ml were added to the 1 ml aliquot duplicates of raw semen and
seminal plasma for LC and AC analysis and centrifuged; the methanol extracts were brought to dryness in a flow of N
2
and recovered with 1 ml bidistilled water. LC and AC concen- trations were measured using spectrophotometric methods as indicated by Pearson et al.
1974. 2.2.2. Pyruvate and lactate
The perchlorised raw semen and seminal plasma samples were centrifuged at 5000×g for 1 h. The extracts were neutralized by addition of 0.25–0.3 ml of KHCO
3
3 M. The clear
G. Stradaioli et al. Animal Reproduction Science 64 2000 233–245 237
supernatant was analyzed by an automatic analyzer according to the spectrophotometric methods reported by Noll 1984 and Lamprecht and Heinz 1984.
2.2.3. Carnitine acetyltransferase activity Carnitine acetyltransferase activity in raw semen was measured radioenzymatically at
37
◦
C as previously described by Chiodi et al. 1994. The medium pH 7.4, which contained 2 mgml of raw semen protein, was composed as follows: 0.25 mM of EDTA Sigma, St.
Louis, MO, USA, 100 mM of Hepes Sigma, St. Louis, MO, USA, 0.08 wv Triton X-100 Sigma, St. Louis, MO, USA, 1 mgml of antimycin A Sigma, St. Louis, MO, USA,
1 mgml of Rotenone Sigma, St. Louis, MO, USA, 0.5 mM acetyl-1-
14
C- Coenzyme A 0.6 Cimol Amersham Pharmacia Biotech, Buckinghamshire, UK and 12 mM LC
Sigma tau s.p.a., Pomezia, Rome, Italy. Incubation was carried out with 0.3 ml of the aforementioned medium for 2 min before and 2 min after addition of LC. The reaction was
stopped with the addition, under stirring, of 0.3 ml of 2 × 8 Dowex resin Fluka Chemie A.G., Switzerland diluted 1:1 wv. Following the addition of the resin the samples were
placed in an ice bath for 5 min, shaken up three times and then centrifuged at 3000×g for 10 min. The incorporation of acetyl-1-
14
C- in AC was evaluated on a 0.3 ml aliquot in a scintillation vial and radioactivity was determined by liquid scintillation counting.
2.3. Statistical analysis Statistical analysis of seminal and biochemical data was performed using a repeated mea-
sure design. The first ejaculate versus the second one, and the interaction between semen collection trials November versus January were considered as main factors; differences
between means were compared with the LSD procedure SPSS, 1997. Coefficient of cor- relation was performed with a two tails Pearson model SPSS, 1997.
3. Results
