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
No congenital or acquired abnormalities of the genital tract were detected in the animals. Two stallions were excluded from the performance test before the January semen collection
trial, due to muscle skeletal pathology. Since there were no major significant differences in seminal and biochemical characteristics between the November and January trials, data
were also pooled for the first and second ejaculates, irrespective of the trial.
The seminal characteristics of the Maremmano stallions are shown in Table 1. No sig- nificant differences were observed between November and January trials. The spermatozoa
concentration, the total number of spermatozoa and the total number of motile morpho- logically normal spermatozoa resulted significantly lower in the second ejaculate than in
the first one P 0.001; also the percentage of live abnormal spermatozoa and abnormal mid-piece and tail were lower in the second ejaculate P 0.01, whereas the percentage
of progressively motile cells and the percentage of morphologically normal live sperma- tozoa were higher in the second ejaculate P 0.001. Fig. 1 shows the percentage of
progressively motile spermatozoa in first and second ejaculates at the time of collection 0 h and after 24, 48 and 72 h of refrigeration at +4
◦
C; at 0 h the values differ significantly between ejaculates P 0.001.
238 G. Stradaioli et al. Animal Reproduction Science 64 2000 233–245
Table 1 Seminal characteristics LS means of Maremmano stallions n = 25 in the first and second ejaculates and during
the two experimental periods MSE: mean square error
a
Seminal characteristics Ejaculate
Period Main effects
First n = 48
b
Second n = 48
b
November n = 50
b
January n = 46
b
Within subject MSE
Between subject MSE
Gel-free volume ml 56.7
52.8 57.9
51.6 19.6
26.5 Sperm concentration 10
6
ml 249.2
∗∗∗
133.4 202.6
180.1 106.5
169.2 Total sperm concentration 10
9
12.8
∗∗∗
6.4 10.7
8.4 3.7
9.3 Progressive motility
48.6
∗∗∗
52.6 49.8
51.4 3.8
29.4 Morphologically
normal unstained 43.2
∗∗∗
47.0 45.0
45.3 5.4
26.8 Total number of motile
morphologically normal spermatozoa 10
9
3.3
∗∗∗
2.0 3.0
2.3 1.2
4.2 Abnormal head shape
1.1 1.0
0.9 1.3
0.7 1.4
Abnormal mid-piece and tail 13.4
∗∗
11.1 11.3
13.2 3.7
17.9 Curved mid-piece and tail
10.4 9.9
10.6 9.7
2.8 10.0
Detached head and tail 5.4
5.4 6.5
4.2 3.2
11.2 Total live abnormal
spermatozoa 24.9
∗∗
22.0 22.8
24.1 5.0
20.0 Total dead spermatozoa
31.9 30.9
32.2 30.6
7.1 20.8
a
Effect for period was never significant.
b
Number of ejaculates.
∗∗
P 0.01;
∗∗∗
P 0.001.
Biochemical analysis data, pyruvatelactate and ACLC ratios in raw semen are reported in Table 2. In the raw semen the LC and AC were significantly higher in the first ejaculate
than in the second P 0.001, whereas, pyruvate and pyruvatelactate ratios were higher in the second ejaculate P 0.05.
Fig. 1. Percentage of motile spermatazoa at collection and after 24, 48, and 72 h of storage at 4
◦
C in first and second ejaculates 0 h P 0.001.
G. Stradaioli et al. Animal Reproduction Science 64 2000 233–245 239
Table 2 Free carnitine, acetylcarnitine, pyruvate and lactate levels, pyruvatelactate, acetylcarnitinecarnitine ratio and CAT
activity in raw semen LS means of Maremmano stallions n = 25 in the first and second ejaculates and during the two experimental periods MSE: mean square error
a
Ejaculate Period
Main effects First
n = 48
b
Second n = 48
b
November n = 50
b
January n = 46
b
Within subject MSE
Between subject MSE
Carnitine nmolml 1067.0
∗∗∗
559.5 676.0
950.6 323.8
515.4 Acetylcarnitine nmolml
139.0
∗∗∗
78.6 98.2
119.5 51.1
76.7 Acetylcarnitinecarnitine
0.165 0.162
0.191 0.136
0.071 0.100
Carnitine nmol10
6
spermatozoa 5.244
5.255 4.289
6.210 2.557
5.035 Acetylcarnitine nmol10
6
spermatozoa 0.753
0.751 0.656
0.848 0.318
0.512 Pyruvate nmolml
0.076
∗
0.161 0.090
0.147 0.102
0.081 Lactate nmolml
2.396 3.285
2.919 2.762
2.087 2.152
Pyruvatelactate 0.031
∗
0.051 0.041
0.041 0.020
0.029 CAT
c
nmolmin10
6
spermatozoa 0.431
0.506 0.402
0.535 0.217
0.270
a
Effect for period was never significant.
b
Number of ejaculates.
c
Carnitine acetyltransferase.
∗
P 0.05;
∗∗∗
P 0.001.
Seminal plasma AC and LC concentrations Table 3 resulted significantly higher in the first ejaculate P 0.001; moreover, the ejaculate obtained in the January trial presented
higher LC values than the November trial P 0.05. The pyruvatelactate ratio was higher in the second ejaculate P 0.05.
Significant correlation coefficients among seminal and biochemical characteristics of Maremmano stallions are reported in Table 4. Both raw semen and seminal plasma LC and
Table 3 Free carnitine, acetylcarnitine, pyruvate and lactate levels, pyruvatelactate, acetylcarnitinecarnitine ratio in sem-
inal plasma LS means of Maremmano stallions n = 25 in the first and second ejaculates and during the two experimental periods MSE: mean square error
Ejaculate Period
Main effects First
n = 48
a
Second n = 48
a
November n = 50
a
January n = 46
a
Within subject MSE
Between subject MSE
Carnitine nmolml 863.2
∗∗∗
479.2 544.4
∗
798.1 312.3
452.8 Acetylcarnitine nmolml
75.24
∗∗∗
49.59 56.09
68.75 21.44
33.07 Acetylcarnitinecarnitine
0.137 0.139
0.132 0.144
0.118 0.110
Pyruvate nmolml 0.085
0.141 0.104
0.122 0.108
0.102 Lactate nmolml
2.063 2.932
2.199 2.797
1.887 1.764
Pyruvatelactate 0.037
∗
0.061 0.055
0.043 0.029
0.035
a
Number of ejaculates.
∗
P 0.05;
∗∗∗
P 0.001.
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
