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Clearance of exogenous carnitine in bovine semen: Potential diagnosis of epididymal function and patency of the ductus deferens
THERlOGENOLOGY CLEARANCE OF EXOGENOUS CARNITINE IN BOVINE SEMEN: POTENTIAL DIAGNOSIS OF EPIDIDYMAL FUNCTION AND PATENCY OF THE DUCTUS DEFERENS H. EilerIy2va C.S. Backus, F.M. Hopkins2 and D.S. Sachan IAgricultural Experiment Station, 2College of Veterinary Medicine and 3Department of Nutrition, The University of Tennessee, P.O. Box 1071 Knoxville, TN 37901-1071 USA Received for publication: October 31, Accepted:
June
13,
2ggo
1991
ABSTRACT Carnitine content in the ejaculate depends mainly on the capability of the epididymis wall to transfer carnitine from the blood and on the patency An elevation of carnitine in semen of ejaculatory ductus systems. subsequent to an intravenous injection of carnitine is expected. Intravenous injections of carnitine (L-isomer and DL-isomers) caused a significant (P t0.05) elevation (more than lo-fold) in blood carnitine. However, carnitine injection failed to increase net secretion of carnitine into the ejaculate and blood elimination half-life was 2.3 hours. Mean concentrations of carnitine in the electroejaculate (3.0 nmoles/ml) were significantly lower than in the ejaculate following natural mating (180 nmoles/ml). Vasectomy decreased net carnitine per ejaculate to about l/5 the prevasectomy value, when ejaculate was collected following natural mating. However, vasectomy did not affect carnitine concentrations in semen collected by electroejaculation. Twenty-one percent of the carnitine in semen originated in the accessory glands and 79% in the epididymides. Carnitine in the electroejaculate was originated almost exclusively in the accessory glands. It was concluded that the diagnostic value of carnitine in semen is limited. Some considerations are: secretion of carnitine is not organ specific, there are large individual variations, there is a negative effect of electroejaculation, and a carnitine loading dose technique is not feasible. However, there is a diagnostic potential in using carnitine assay to detect epididymides occlusion, but only when ejaculate is collected by an artificial vagina. Key words:
carnitine injection, semen, electroejaculation, vasectomy INTRODUCTION
There is a diagnostic value to determinations of carnitine in the azoospermic patient. In men, decreased concentrations of carnitine were consistently found in epididymides occlusion and following vasectomy (l-6). Unfortunately, a large number of men with seminiferous tubular lesions (without obstruction) exhibited similar low values of carnitine. The Acknowledgments The authors thank Dr. Donita Frazier, College of Veterinary Medicine, University of Tennessee for her assistance in the analysis of data. acorrespondence: College of Knoxville, TN 37901-1071 USA. AUGUST19BlVOL.36NO.2
Veterinary
Medicine,
P.O.
Box
1071,
221
THERIOGENOLOGY reason for this is not known, although one possibility is a coexisting azoospermia and decreased capability of the epididymides to concentrate carnitine in the ejaculate. The above studies were based on determinations of endogenous carnitine in semen. It is speculated that 2.1 and 4.29 Lcarnitine would result in an increased transfer of carnitine into the ejaculate, unless there were a metabolic disease of the epididymides or an obstruction of the ejaculatory duct system. This hypothesis is supported by the following facts: in men, most carnitine (60 to 90%) found in the ejaculate is provided by the epididymis, 5% by the prostate and approximately 10% by other accessory glands (5); in the dog, the Carnitine is epididymides contribute about 95% of the carnitine (7). synthesized in the liver and actively transferred into the epididymides from blood (8-ll), and a positive correlation between blood plasma and seminal carnitine concentrations has been reported (12). The objectives of this work were 1) to determine whether or not an intravenous injection of carnitine would increase the net transfer of carnitine into the bovine ejaculate and 2) to investigate the contribution of carnitine by the epididymides. Electroejaculates were used for the Electroejaculated is also used in selected evaluation of bovine semen. clinical conditions in men (13). MATERIALS AND METHODS Experiment 1 Electrae.la 'clu at ion. carnitine injection and vasectomy. Indwelling jugular catheters were placed in six 18-month-old Angus bulls (430 to 518 kg) from the same herd and calving season. The bulls were evaluated clinically and by routine semen analysis. All bulls were classified to be satisfactory potential breeders as defined by the Society of Theriogenology (14). Blood from a jugular vein catheter and semen collected by electroejaculation were collected simultaneously at 0, 20, 60 and 1440 minutes to study the effect of consecutive ejaculations on the carnitine content of semen. Three days later, bulls were injected with 4.2 g i.v. DL-carnitine (Sigma Co. St. Louis, MO) in saline (30 cc) over a 3-minute period, and samples (blood and semen) were collected at 0, 20, 60, 120, 180 minutes and at 24 hours. Bulls were vasectomized, and two weeks later the experiment was repeated. Semen and blood samples were centrifuged within 30 minutes of collection and stored at -20°C until analysis. Free L-carnitine was determined in seminal plasma and blood plasma; L-Carnitine and acylcarnitines were analyzed according to a radioisotopic procedure described by Cederblad and Lindstedt (15), modified by Sachan et al. (16). Experiment 2 Artificial Vaqina. carnitine iniection and vasectomv. In a follow-up experiment, a different group of five bulls was used. Ejaculates were collected at 0, 20, 60, 120 and 180 minutes using the artificial vagina technique. Three days later, bulls were injected with DL-carnitine (4.2 g
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THERIOGENOLOGY i.v. in 30 cc saline) and the experiment was repeated. Semen was collected at 0, 20, 60, 120, and 180 minutes post-carnitine injection. Then, bulls were vasectomized and the experiment was repeated two weeks later. Semen samples were treated as in experiment one. Three different bulls were shamoperated and the experiment was repeated. Experiment 3 DL-carnitine versus L-carnitine inlections. A different group of four bul1s was used in this experiment. This time L-carnitine (2.1 g and 4.2 g) and DL-carnitine (4.2 g and 8.4 g) were injected at two different dosages Semen samples were collected by in a randomized experimental design. electroejaculation at 0, 10 and 30 minutes. Blood was collected at 0, 2, and 30 minutes post injection of carnitine. Three days of rest were allowed between treatments. In this experiment, total carnitine in the seminal plasma was determined. The blood plasma concentrations of L-carnitine were fit to a onecompartment model using an automated curve-stripping procedure followed by nonlinear least squares regression (SAS Institute, Cary NC). Analysis of variance was conducted to determine the effect of treatment on the response When significant differences among treatment means were variables. identified by an F-test, mean separation was accomplished by Newman Keull's test. Significance was reported at the 0.05 level. RESULTS The effect of an intravenous injection of carnitine on the blood plasma concentration of carnitine is shown in Table 1. The pre-injection (O-time) concentration of carnitine in blood plasma was significantly (P ~0.05) lower in the vasectomized than in the intact bull. However, at 20 minutes post injection, blood plasma concentrations of carnitine were not statistically (P >0.05) different between intact and vasectomized bulls. In both groups, the blood plasma concentrations of carnitine declined gradually until 180 minutes post injection. At 24 hours post injection, blood plasma concentrations of carnitine were back to pre-injection concentrations. Calculations showed a similar half-life before and after vasectomy for carnitine. Effect of Electroejaculation The concentration of carnitine in the seminal plasma of bulls that had been collected by electroejaculation before (intact bulls) and after Mean (t SEM) volume of electroejaculate vasectomy is shown in Table 2. throughout the experiment was 5.2 f 0.14 cc. No significant difference in the electroejaculate volumes were detected between intact and vasectomized bulls. Concentrations of carnitine and ejaculate net in the electroejaculate (Table 2) were significantly lower than those in seminal plasma collected with an artificial vagina (Table 3). Mean concentration of carnitine in semen was not significantly (P >0.05) different between vasectomized and nonvasectomized bulls prior to carnitine injection. Furthermore, carnitine injection did not cause significant (P >0.05) elevation of carnitine in the semen of either intact or vasectomized
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THERIOGENOLOGY Table 1.
Effect of an intravenous injection of carnitine on plasma concentrations of carnitine before (intact) and after vasectomy
Time (minutes)
Vasectomized (nmoles/ml)
Intact (nmoles/ml) 37 130 97 77 70 38
2: 1;: 180 1440 Elimination T
(hours)
f 6.7"y f 15.9by f 12.aby f 6.3by k 12.0by -k 9.6ay
2.3 f
0.3y
2.2 f 0.2y
i;Ebers are mean f SEM for six bulls. Different superscripts within the same column indicate a significant (P ~0.05) difference. yyz Different superscripts within the same row indicate significant (P ~0.05) difference between means. Table 2.
Carnitine concentrations in semen of intact and vasectomized bulls collected by electroejaculation at different times before and after an intravenous injection of carnitine
Time (minutes)
2: 1;: 180 1440
Intact Pre Post carnitine carnitine (nmoles/ml) (nmoles/ml) 3.1 + 2.0ay 1.9 f l.aay 4.5 + 2.1ay ND 2.8 iDO.gay
3.0 27.4 21.6 26.3 13.7 3.1
Vasectomized Post Pre carnitine carnitine (moles/ml) (nmoles/ml)
+- 1.7ay -f 10.gaZ f 5.6"' f 14.0a f 7.4a f 1.2ay
Concentration 3.0 + 1.7y 15.9 ? 6.8Y (Mean) 16.6 f 8.8y 82.7 f 35.4y Ejaculate netC
7.7 2.8 4.9
2.4ay
5.5 5.4 30.1 25.0 20.1 5.9
+ 3.3ay k 2.3ay f 14.7ay f 7.6a + 13.2a f 2.9ay
3.3y
15.3
?
f + i ND
5.5ay 1.8"y 3.6ay
6.4 ": 5.5 28.6
+
+
17.2y
79.6
+
7.3y 4O.Oy
Numbers are mean f SEM for six bulls before (intact) and after vasectomy. ayb Different superscripts within the same column indicate a significant (P <0.05) difference between means. yyz Different superscripts within the same row indicate a significant difference (P ~0.05) between means. C Ejaculate net = mean concentrations x ejaculate volume (5.2 cc). ND = Not Determined.
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THERIOGENOLOGY bulls. The lack of statistical significance was probably due in part to the large variability observed among bulls treated with carnitine. Carnitine injection resulted in increased (P ~0.05) concentrations of carnitine in the electroejaculate of intact bulls at 20, and 60 minutes post injection. Collection by an Artificial Vagina The concentration of carnitine in the seminal plasma of bulls that was collected by an artificial vagina before (intact bulls) and after vasectomy is shown in Table 3. Mean (? SEM) volume of ejaculate collected by artificial vagina throughout the experiment was 1.5 ?: 0.26 cc. No significant difference in the ejaculate volumes was detected between the number of ejaculates or treatment groups. Vasectomy (1.4 i 0.16 cc) did not affect ejaculate volume. Both the mean concentration of carnitine in the seminal plasma and the net ejaculate in intact bulls were approximately 4.8 times that of vasectomized bulls. Vasectomy caused a drop in seminal carnitine to 21% that of the prevasectomy level. Therefore, 79% of the seminal carnitine originated in the epididymis and, possibly, a small However, carnitine treatment caused no percentage in the testis. significant (P t0.05) elevation in either mean concentration or net transfer of carnitine in the seminal plasma of either intact or vasectomized bulls that were collected using an artificial vagina. Concentrations of carnitine in the semen of sham-operated bulls at O-time, and at 20, and 60 minutes were 201 f 19, 220 f 29 and 179 f 21 nmoles/ml, respectively. No significant difference was found between vasectomized and intact bulls. DL-Carnitine Versus L-Carnitine Net carnitine ejaculates were statistically the same regardless of Lcarnitine or DL-carnitine injection (Table 4). Two minutes after carnitine injection, blood concentrations of L-carnitine increased between 10 and 20 fold, depending on the size of the carnitine dosage. There was no statistical difference (P >0.05) between bulls injected with L-carnitine 2.1 g and DL-carnitine 4.2 g. There was also no difference between bulls injected with L-carnitine 4.2 g and DL-carnitine 8.4 g. DISCUSSION Intravenous injection of carnitine did not increase the elimination of carnitine from the semen of bulls. This was unexpected, since a variety of substances (17), including small steroidal molecules had been shown to pass into the ejaculate within a few minutes, when injected intravenously (18,19). The dosage of L-carnitine injected intravenously was estimated to be roughly 20 times the amount of carnitine in the systemic blood. Carnitine injection caused an acute increase in the blood concentration of carnitine, approximately 4 to 20 times that of the baseline value. However, there was not a significant elevation in seminal carnitine during this period. This suggests that an acute elevation in blood concentration of carnitine is not critical for determining the net secretion of carnitine into the ejaculate.
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THERIOGENOLOGY Table 3. Carnitine concentrations in semen of intact and vasectomized bulls collected by artificial vagina at different times beforeqand after an intravenous injection of carnitine Intact PrePostcarnitine carnitine (nmoles/ml) (nmoles/ml)
Time (minutes)
0
260 f 29ay
:8 120 180
146 f 4Jay 147 f 44ay 160 f 47ay
:;y 224 139 164
Concentration (Mean) Ejaculate netc
180 f 4Oy 270 f 6Oy
185 f 31ay
i: f f ?:
Vasectomized PrePostcarnitine carnitine (nmoles/ml) (nmol es/ml
$3: 19ay 35ay 4aay
74 f 32az 56 + 2Oaz
)
87 f 14az 46 f
20 f. 1oaz 22 ?: 4az 13 f 3az
1Jaz 41 f 19az 25 f 13az 35 t 12az
178 f 36y
37 f 142
47 f 15z
267 f 54y
56 f 21z
71 f 23z
Nu bers are mean f SEM for five bulls
apI: Different (P (0.05) y*z Different (P ~0.05) C Ejaculate
Table 4.
superscripts within the same column indicate a significant difference between means. superscripts within the same row indicate a significant difference between means. net = mean concentration x Mean ejaculate volume (1.5~~).
Effects of different doses of L-carnitine carnitine content in semen and blood
Compartment and group
Carnitine L 2.1 g
Ejaculate net (nmoles) Controls 78 f 16aY Treated
61 f 19ay
Treatment DL 4.2 g
and DL-carnitine
on
(isomer, dose) L 4.2 g
DL 8.4 g
78 f 16aY
78 f 16aY
78 f i6aY
31 f eiay
92 + 49ay
39 f 16ay
Blood (nmoles/ml) O-time
2.2 f 0.89aY
1.9 f 0.17ay
2.4 f 0.3Jay
2.4 f 0.20ay
2-minutes
20 f 1.62bY
20 f 3.22by
51 f 1.12bz
53 f 4.5bz
30-minutes
8 f 0.39cy
8 f 0.50cy
24 f 8.Jcz
15 f 3.4cz
Nu bers are means t SEM carnitine for four bulls. a,b Different superscripts within the same column indicate a significant difference (P ~0.05) between means. Y,z Different superscripts within the same row indicate a significant difference (P <0.05) between means. d Ejaculate net = mean concentration X mean ejaculate volume (7.3 cc).
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THERIOGENOLOGY Carnitine, unlikely other substances, is actively absorbed from the lumen of the tail of the epididymis as concentrations increase (10,20). Factors determining the steady state, including transfer/absorption rates of carnitine It is from the epididymis, have not been determined in this experiment. suspected that binding capacity of blood plasma proteins affects transfer of molecules into the bovine ejaculate (18,21). This has not been determined for carnitine. Vasectomy experiments have indicated that most carnitine in the electroejaculate (but not in normal ejaculate) of intact bulls originates in the accessory glands and that during the normal ejaculate (Table 3), neither the epididymides nor the accessory glands secreted additional carnitine when carnitine concentrations in blood were elevated. Carnitine content in the vasectomized bull was l/5 that of the intact bull. This observation suggests that active transport mechanisms favor retention of carnitine in the lumena of epididymides. This is consistent with results in the rat (10,20). The lack of net gain of carnitine into the ejaculate was not related to a possible competition between D-isomers and L-isomers (22,23) in the DL-isomer injected animals, since there was no difference in seminal carnitine when either Lcarnitine or DL- carnitine was injected at an equimolar concentration of Lisomer. Regardless of metabolic differences among species, the elimination halflife of carnitine in the blood of bulls (2.3 hours this work) is similar to its half-life in humans (1.5 hours) (24). Approximately 21% of the carnitine persisted in the normal ejaculate following vasectomy. The relative contribution of each accessory gland is not known in the bovine. In men, the prostate contributes approximately 5% of the In the dog, the carnitine, and other accessory glands around lD% (5). prostate contributes 5% (7). In man, 58% to 95% of seminal carnitine originates in the epididymis (1,3,4,25) and 81% in the bull (this work). Both the concentration and net transfer of carnitine in the semen of bulls collected by electroejaculation were significantly smaller than that collected with an artificial vagina. The reason for this difference cannot be explained in this experiment, since two different groups of bulls were used. However, electroejaculation has been shown to decrease the concentrations of exogenous markers in the bovine ejaculate (18) and in human ejaculate (13). This was probably due to selective stimulation of accessory gland secretions by Electroejaculate also eletroejaculation, causing a dilution effect (26). provided little or no fluid from epididymides. The reason for the relatively large variability in the content of carnitine in semen is not known, but a possible explanation could be urine contamination during electroejaculation, different body weights of bulls, environmental factors, sexual activity prior to collection (6) and individual variation. A physiological testing procedure for epididymal function and patency of the ejaculatory ducts based on the study of the clearance of exogenous carnitine in the electroejaculate is desirable. However, the applicability of such a diaanostic aooroach is limited in the bovine by the lack of a net gain of exogenois carnit'i'ne in the ejaculate, by the various sources of carnitine in the semen, by the large variability in carnitine content and by the
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THERIOGENOLOGY negative effects of electroejaculation. However, there is a diagnostic potential in using carnitine assay to detect epididymides occlusion, but only when ejaculate is collected by an artificial vagina. REFERENCES
1. Casano, R., Orlando, C., Caldini, A.L., Barni, T., Natili, A. and Serio,
M. Simultaneous measurement of seminal L-carnitine, a,l-4-glucosidase, and glycerylphosphorylcholine in azoospermic and oligozoospermic patients. Fertil. Steril. G:324-328 (1987).
2. Tomamichel, G.R. and Bandhauer, K. Seminal carnitine content obstructive azoospermia correlation with the anatomic level obstruction. J. Androl. 2:328-330 (1986).
in of
Peterson, R.N., 3. Frenkel, G., Davis, J.E. amd Freund, M. Glycerylphosphorylcholine and carnitine in normal human semen and in postvasectomy semen: Differences in concentrations. Fertil. Steril. a:84087 (1974). 4. Wetterauer, U. and Heite, H.J. Carnitne in seminal plasma: Its significance in diagnostic andrology. Arch. Androl. 4:137-143 (1980). 5. Menchini-Fabris, G.F., Canale, il., Izzo, P.L., Olivieri, L. and Bartelloni, M. Free L-carnitine in human semen: it's variability in different andrologic pathologies. Fertil. Steril. g:263-267 (1984). 6. Soufir, J.C., Ducot, B., Marson, J. Jouannet, P., Feneux, D., Soumah, A. and Spira, A. Levels of seminal free L (-) carnitine in fertile and infertile men. Int. J. Androl. 1:188-197. 7. Olson, P.N., Behrendt, M.D., Amann, R.P., Weiss, D.E., Bowen, R.A., Nett, T.M. and McGarry, J.D. Concentrations of carnitine in the seminal fluid of normospermic, vasectomized, and castrated dogs. Am. J. Vet. Res. @:1211-1215 (1987). 8. Brooks, D.E. Carnitine in the male reproductive tract and its relation to the metabolism of the epididymis and spermatozoa. In: Frenkel, R.H. and McGary, J.D. (eds.), Carnitine Biosynthesis, Metabolism and Functions. Academic Press, New York, 1990, pp. 219-234. 9. Casillas, E.R. The distribution of carnitine in male reproductive tissues and its effect on palmitate oxidation by spermatozoa1 particles. Biochem. Biophys. Acta. m:545-551 (1972). 10. Hinton, 8.T. and Hernandez, H. Selective luminal absorption of Lcarnitine from the proximal regions of the rat epididymis. J. Androl. &:300-305 (1985).
11. Juelin, C., Soufir, J.C., Marson, J., Paquignon, M. and Dacheux, J.L.
The distribution of carnitine and acetylcarnitine in the epididymis and epididymal spermatozoa of the boar. J. Reprod. Fertil. D:523-529 (1987).
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THERIOGENOLOGY 12. Rosecrans, R.R., Jeyendran, R.S., Perez-Pelaez, M. and Kennedy, W.P. Comparison of biochemical parameters of human blood serum and seminal plasma. Andrologia 19:625-628 (1987). 13. Hirsch, I.H., Rosecrans, R.R., Sedor, J. and Jevendran, R.S. Biochemical analysis of human electroejaculates. 14th Annual Meeting. Proc. Am. Sot. Androl. p. 32 abstr. (1989). 14. Ball, L., Ott, R.S., Mortimer, R.G. and Simons, J.C. Manual for Breeding Society of Theriogenology, vol. 12. Soundness Evaluation of Bulls. Hastings, NB, 1983, pp. l-63. A method for the determination of 15. Cederblad, 6. and Lindstedt, S. carnitine in the picomole range. Clin. Chem. Acta. g:235-243 (1972). Ameliorating effects of 16. Sachan, D.S., Rhew, T.H. and Ruark, R.A. carnitine and its precursors on alcohol-induced fatty liver. J. Clin. Nutr. 3:738-744 (1984). 17. Mann, T. and Lutwak-Mann, C. Passage of chemicals into human and animal semen: mechanisms and significance. Toxicology U:l-14 (1982). 18. Eiler, H., Armstrong-Backus, C.S. and Smalling, J.D. Effect of electroejaculation on progesterone and cortisol excretion in bovine semen. Am. J. Vet. Res. 48:492-495 (1987). 19
Nature of the clearance of exogenous Eiler, H. and Graves, C.N. testosterone and progesterone in ejaculated bovine semen. J. Androl. 2:205-210 (1981).
20. Hinton, B.T. and Setchell, B.P. Concentration and uptake of carnitine in the rat epididymis: a micropuncture study. In: Frenkel, R.A. and McGary, J.D. (eds.), Carnitine Biosynthesis, Metabolism, and Functions. Academic Press, New York, 1980, pp. 237-250. 21. Eiler, H. and Armstrong-Backus, C.S. Passage of exogenous L-thyroxine and triiodothyronine into bovine ejaculate. Am. J. Vet. Res. 4&:439-443
(1987).
22. Borum, P.R.
Carnitine.
Ann. Rev. Nutr.
3:233-259 (1983).
23. Rebouche, C.J. and Paulson, D.J. Carnitine metabolism and function in humans. Ann. Rev. Nutr. 6:41-66 (1986). 24. Cederblad, G. Fat metabolism following an intravenous bolus dose of a fat emulsion and carnitine. Clin. Physiol. 4:159-168 (1984). 25. Golan, R., Soffer, Y., Katz, S., Weissenberg, R., Wasserzug, 0. and Lewin, L.M. Carnitine and short-chain acylcarnitines in the lumen of the human male reproductive tract. Int. J. Androl. 6:349-357 (1983) Compartmental analysis of sources of the 26. Seidel, G.E. and Foote, R.H. bovine ejaculate. Biol. Reprod. 2:189-196 (1970).
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ABSTRACT Carnitine content in the ejaculate depends mainly on the capability of the epididymis wall to transfer carnitine from the blood and on the patency An elevation of carnitine in semen of ejaculatory ductus systems. subsequent to an intravenous injection of carnitine is expected. Intravenous injections of carnitine (L-isomer and DL-isomers) caused a significant (P t0.05) elevation (more than lo-fold) in blood carnitine. However, carnitine injection failed to increase net secretion of carnitine into the ejaculate and blood elimination half-life was 2.3 hours. Mean concentrations of carnitine in the electroejaculate (3.0 nmoles/ml) were significantly lower than in the ejaculate following natural mating (180 nmoles/ml). Vasectomy decreased net carnitine per ejaculate to about l/5 the prevasectomy value, when ejaculate was collected following natural mating. However, vasectomy did not affect carnitine concentrations in semen collected by electroejaculation. Twenty-one percent of the carnitine in semen originated in the accessory glands and 79% in the epididymides. Carnitine in the electroejaculate was originated almost exclusively in the accessory glands. It was concluded that the diagnostic value of carnitine in semen is limited. Some considerations are: secretion of carnitine is not organ specific, there are large individual variations, there is a negative effect of electroejaculation, and a carnitine loading dose technique is not feasible. However, there is a diagnostic potential in using carnitine assay to detect epididymides occlusion, but only when ejaculate is collected by an artificial vagina. Key words:
carnitine injection, semen, electroejaculation, vasectomy INTRODUCTION
There is a diagnostic value to determinations of carnitine in the azoospermic patient. In men, decreased concentrations of carnitine were consistently found in epididymides occlusion and following vasectomy (l-6). Unfortunately, a large number of men with seminiferous tubular lesions (without obstruction) exhibited similar low values of carnitine. The Acknowledgments The authors thank Dr. Donita Frazier, College of Veterinary Medicine, University of Tennessee for her assistance in the analysis of data. acorrespondence: College of Knoxville, TN 37901-1071 USA. AUGUST19BlVOL.36NO.2
Veterinary
Medicine,
P.O.
Box
1071,
221
THERIOGENOLOGY reason for this is not known, although one possibility is a coexisting azoospermia and decreased capability of the epididymides to concentrate carnitine in the ejaculate. The above studies were based on determinations of endogenous carnitine in semen. It is speculated that 2.1 and 4.29 Lcarnitine would result in an increased transfer of carnitine into the ejaculate, unless there were a metabolic disease of the epididymides or an obstruction of the ejaculatory duct system. This hypothesis is supported by the following facts: in men, most carnitine (60 to 90%) found in the ejaculate is provided by the epididymis, 5% by the prostate and approximately 10% by other accessory glands (5); in the dog, the Carnitine is epididymides contribute about 95% of the carnitine (7). synthesized in the liver and actively transferred into the epididymides from blood (8-ll), and a positive correlation between blood plasma and seminal carnitine concentrations has been reported (12). The objectives of this work were 1) to determine whether or not an intravenous injection of carnitine would increase the net transfer of carnitine into the bovine ejaculate and 2) to investigate the contribution of carnitine by the epididymides. Electroejaculates were used for the Electroejaculated is also used in selected evaluation of bovine semen. clinical conditions in men (13). MATERIALS AND METHODS Experiment 1 Electrae.la 'clu at ion. carnitine injection and vasectomy. Indwelling jugular catheters were placed in six 18-month-old Angus bulls (430 to 518 kg) from the same herd and calving season. The bulls were evaluated clinically and by routine semen analysis. All bulls were classified to be satisfactory potential breeders as defined by the Society of Theriogenology (14). Blood from a jugular vein catheter and semen collected by electroejaculation were collected simultaneously at 0, 20, 60 and 1440 minutes to study the effect of consecutive ejaculations on the carnitine content of semen. Three days later, bulls were injected with 4.2 g i.v. DL-carnitine (Sigma Co. St. Louis, MO) in saline (30 cc) over a 3-minute period, and samples (blood and semen) were collected at 0, 20, 60, 120, 180 minutes and at 24 hours. Bulls were vasectomized, and two weeks later the experiment was repeated. Semen and blood samples were centrifuged within 30 minutes of collection and stored at -20°C until analysis. Free L-carnitine was determined in seminal plasma and blood plasma; L-Carnitine and acylcarnitines were analyzed according to a radioisotopic procedure described by Cederblad and Lindstedt (15), modified by Sachan et al. (16). Experiment 2 Artificial Vaqina. carnitine iniection and vasectomv. In a follow-up experiment, a different group of five bulls was used. Ejaculates were collected at 0, 20, 60, 120 and 180 minutes using the artificial vagina technique. Three days later, bulls were injected with DL-carnitine (4.2 g
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AUGUST 1991 VOL. 36 NO. 2
THERIOGENOLOGY i.v. in 30 cc saline) and the experiment was repeated. Semen was collected at 0, 20, 60, 120, and 180 minutes post-carnitine injection. Then, bulls were vasectomized and the experiment was repeated two weeks later. Semen samples were treated as in experiment one. Three different bulls were shamoperated and the experiment was repeated. Experiment 3 DL-carnitine versus L-carnitine inlections. A different group of four bul1s was used in this experiment. This time L-carnitine (2.1 g and 4.2 g) and DL-carnitine (4.2 g and 8.4 g) were injected at two different dosages Semen samples were collected by in a randomized experimental design. electroejaculation at 0, 10 and 30 minutes. Blood was collected at 0, 2, and 30 minutes post injection of carnitine. Three days of rest were allowed between treatments. In this experiment, total carnitine in the seminal plasma was determined. The blood plasma concentrations of L-carnitine were fit to a onecompartment model using an automated curve-stripping procedure followed by nonlinear least squares regression (SAS Institute, Cary NC). Analysis of variance was conducted to determine the effect of treatment on the response When significant differences among treatment means were variables. identified by an F-test, mean separation was accomplished by Newman Keull's test. Significance was reported at the 0.05 level. RESULTS The effect of an intravenous injection of carnitine on the blood plasma concentration of carnitine is shown in Table 1. The pre-injection (O-time) concentration of carnitine in blood plasma was significantly (P ~0.05) lower in the vasectomized than in the intact bull. However, at 20 minutes post injection, blood plasma concentrations of carnitine were not statistically (P >0.05) different between intact and vasectomized bulls. In both groups, the blood plasma concentrations of carnitine declined gradually until 180 minutes post injection. At 24 hours post injection, blood plasma concentrations of carnitine were back to pre-injection concentrations. Calculations showed a similar half-life before and after vasectomy for carnitine. Effect of Electroejaculation The concentration of carnitine in the seminal plasma of bulls that had been collected by electroejaculation before (intact bulls) and after Mean (t SEM) volume of electroejaculate vasectomy is shown in Table 2. throughout the experiment was 5.2 f 0.14 cc. No significant difference in the electroejaculate volumes were detected between intact and vasectomized bulls. Concentrations of carnitine and ejaculate net in the electroejaculate (Table 2) were significantly lower than those in seminal plasma collected with an artificial vagina (Table 3). Mean concentration of carnitine in semen was not significantly (P >0.05) different between vasectomized and nonvasectomized bulls prior to carnitine injection. Furthermore, carnitine injection did not cause significant (P >0.05) elevation of carnitine in the semen of either intact or vasectomized
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223
THERIOGENOLOGY Table 1.
Effect of an intravenous injection of carnitine on plasma concentrations of carnitine before (intact) and after vasectomy
Time (minutes)
Vasectomized (nmoles/ml)
Intact (nmoles/ml) 37 130 97 77 70 38
2: 1;: 180 1440 Elimination T
(hours)
f 6.7"y f 15.9by f 12.aby f 6.3by k 12.0by -k 9.6ay
2.3 f
0.3y
2.2 f 0.2y
i;Ebers are mean f SEM for six bulls. Different superscripts within the same column indicate a significant (P ~0.05) difference. yyz Different superscripts within the same row indicate significant (P ~0.05) difference between means. Table 2.
Carnitine concentrations in semen of intact and vasectomized bulls collected by electroejaculation at different times before and after an intravenous injection of carnitine
Time (minutes)
2: 1;: 180 1440
Intact Pre Post carnitine carnitine (nmoles/ml) (nmoles/ml) 3.1 + 2.0ay 1.9 f l.aay 4.5 + 2.1ay ND 2.8 iDO.gay
3.0 27.4 21.6 26.3 13.7 3.1
Vasectomized Post Pre carnitine carnitine (moles/ml) (nmoles/ml)
+- 1.7ay -f 10.gaZ f 5.6"' f 14.0a f 7.4a f 1.2ay
Concentration 3.0 + 1.7y 15.9 ? 6.8Y (Mean) 16.6 f 8.8y 82.7 f 35.4y Ejaculate netC
7.7 2.8 4.9
2.4ay
5.5 5.4 30.1 25.0 20.1 5.9
+ 3.3ay k 2.3ay f 14.7ay f 7.6a + 13.2a f 2.9ay
3.3y
15.3
?
f + i ND
5.5ay 1.8"y 3.6ay
6.4 ": 5.5 28.6
+
+
17.2y
79.6
+
7.3y 4O.Oy
Numbers are mean f SEM for six bulls before (intact) and after vasectomy. ayb Different superscripts within the same column indicate a significant (P <0.05) difference between means. yyz Different superscripts within the same row indicate a significant difference (P ~0.05) between means. C Ejaculate net = mean concentrations x ejaculate volume (5.2 cc). ND = Not Determined.
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THERIOGENOLOGY bulls. The lack of statistical significance was probably due in part to the large variability observed among bulls treated with carnitine. Carnitine injection resulted in increased (P ~0.05) concentrations of carnitine in the electroejaculate of intact bulls at 20, and 60 minutes post injection. Collection by an Artificial Vagina The concentration of carnitine in the seminal plasma of bulls that was collected by an artificial vagina before (intact bulls) and after vasectomy is shown in Table 3. Mean (? SEM) volume of ejaculate collected by artificial vagina throughout the experiment was 1.5 ?: 0.26 cc. No significant difference in the ejaculate volumes was detected between the number of ejaculates or treatment groups. Vasectomy (1.4 i 0.16 cc) did not affect ejaculate volume. Both the mean concentration of carnitine in the seminal plasma and the net ejaculate in intact bulls were approximately 4.8 times that of vasectomized bulls. Vasectomy caused a drop in seminal carnitine to 21% that of the prevasectomy level. Therefore, 79% of the seminal carnitine originated in the epididymis and, possibly, a small However, carnitine treatment caused no percentage in the testis. significant (P t0.05) elevation in either mean concentration or net transfer of carnitine in the seminal plasma of either intact or vasectomized bulls that were collected using an artificial vagina. Concentrations of carnitine in the semen of sham-operated bulls at O-time, and at 20, and 60 minutes were 201 f 19, 220 f 29 and 179 f 21 nmoles/ml, respectively. No significant difference was found between vasectomized and intact bulls. DL-Carnitine Versus L-Carnitine Net carnitine ejaculates were statistically the same regardless of Lcarnitine or DL-carnitine injection (Table 4). Two minutes after carnitine injection, blood concentrations of L-carnitine increased between 10 and 20 fold, depending on the size of the carnitine dosage. There was no statistical difference (P >0.05) between bulls injected with L-carnitine 2.1 g and DL-carnitine 4.2 g. There was also no difference between bulls injected with L-carnitine 4.2 g and DL-carnitine 8.4 g. DISCUSSION Intravenous injection of carnitine did not increase the elimination of carnitine from the semen of bulls. This was unexpected, since a variety of substances (17), including small steroidal molecules had been shown to pass into the ejaculate within a few minutes, when injected intravenously (18,19). The dosage of L-carnitine injected intravenously was estimated to be roughly 20 times the amount of carnitine in the systemic blood. Carnitine injection caused an acute increase in the blood concentration of carnitine, approximately 4 to 20 times that of the baseline value. However, there was not a significant elevation in seminal carnitine during this period. This suggests that an acute elevation in blood concentration of carnitine is not critical for determining the net secretion of carnitine into the ejaculate.
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THERIOGENOLOGY Table 3. Carnitine concentrations in semen of intact and vasectomized bulls collected by artificial vagina at different times beforeqand after an intravenous injection of carnitine Intact PrePostcarnitine carnitine (nmoles/ml) (nmoles/ml)
Time (minutes)
0
260 f 29ay
:8 120 180
146 f 4Jay 147 f 44ay 160 f 47ay
:;y 224 139 164
Concentration (Mean) Ejaculate netc
180 f 4Oy 270 f 6Oy
185 f 31ay
i: f f ?:
Vasectomized PrePostcarnitine carnitine (nmoles/ml) (nmol es/ml
$3: 19ay 35ay 4aay
74 f 32az 56 + 2Oaz
)
87 f 14az 46 f
20 f. 1oaz 22 ?: 4az 13 f 3az
1Jaz 41 f 19az 25 f 13az 35 t 12az
178 f 36y
37 f 142
47 f 15z
267 f 54y
56 f 21z
71 f 23z
Nu bers are mean f SEM for five bulls
apI: Different (P (0.05) y*z Different (P ~0.05) C Ejaculate
Table 4.
superscripts within the same column indicate a significant difference between means. superscripts within the same row indicate a significant difference between means. net = mean concentration x Mean ejaculate volume (1.5~~).
Effects of different doses of L-carnitine carnitine content in semen and blood
Compartment and group
Carnitine L 2.1 g
Ejaculate net (nmoles) Controls 78 f 16aY Treated
61 f 19ay
Treatment DL 4.2 g
and DL-carnitine
on
(isomer, dose) L 4.2 g
DL 8.4 g
78 f 16aY
78 f 16aY
78 f i6aY
31 f eiay
92 + 49ay
39 f 16ay
Blood (nmoles/ml) O-time
2.2 f 0.89aY
1.9 f 0.17ay
2.4 f 0.3Jay
2.4 f 0.20ay
2-minutes
20 f 1.62bY
20 f 3.22by
51 f 1.12bz
53 f 4.5bz
30-minutes
8 f 0.39cy
8 f 0.50cy
24 f 8.Jcz
15 f 3.4cz
Nu bers are means t SEM carnitine for four bulls. a,b Different superscripts within the same column indicate a significant difference (P ~0.05) between means. Y,z Different superscripts within the same row indicate a significant difference (P <0.05) between means. d Ejaculate net = mean concentration X mean ejaculate volume (7.3 cc).
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AUGUST 1991 VOL. 36 NO. 2
THERIOGENOLOGY Carnitine, unlikely other substances, is actively absorbed from the lumen of the tail of the epididymis as concentrations increase (10,20). Factors determining the steady state, including transfer/absorption rates of carnitine It is from the epididymis, have not been determined in this experiment. suspected that binding capacity of blood plasma proteins affects transfer of molecules into the bovine ejaculate (18,21). This has not been determined for carnitine. Vasectomy experiments have indicated that most carnitine in the electroejaculate (but not in normal ejaculate) of intact bulls originates in the accessory glands and that during the normal ejaculate (Table 3), neither the epididymides nor the accessory glands secreted additional carnitine when carnitine concentrations in blood were elevated. Carnitine content in the vasectomized bull was l/5 that of the intact bull. This observation suggests that active transport mechanisms favor retention of carnitine in the lumena of epididymides. This is consistent with results in the rat (10,20). The lack of net gain of carnitine into the ejaculate was not related to a possible competition between D-isomers and L-isomers (22,23) in the DL-isomer injected animals, since there was no difference in seminal carnitine when either Lcarnitine or DL- carnitine was injected at an equimolar concentration of Lisomer. Regardless of metabolic differences among species, the elimination halflife of carnitine in the blood of bulls (2.3 hours this work) is similar to its half-life in humans (1.5 hours) (24). Approximately 21% of the carnitine persisted in the normal ejaculate following vasectomy. The relative contribution of each accessory gland is not known in the bovine. In men, the prostate contributes approximately 5% of the In the dog, the carnitine, and other accessory glands around lD% (5). prostate contributes 5% (7). In man, 58% to 95% of seminal carnitine originates in the epididymis (1,3,4,25) and 81% in the bull (this work). Both the concentration and net transfer of carnitine in the semen of bulls collected by electroejaculation were significantly smaller than that collected with an artificial vagina. The reason for this difference cannot be explained in this experiment, since two different groups of bulls were used. However, electroejaculation has been shown to decrease the concentrations of exogenous markers in the bovine ejaculate (18) and in human ejaculate (13). This was probably due to selective stimulation of accessory gland secretions by Electroejaculate also eletroejaculation, causing a dilution effect (26). provided little or no fluid from epididymides. The reason for the relatively large variability in the content of carnitine in semen is not known, but a possible explanation could be urine contamination during electroejaculation, different body weights of bulls, environmental factors, sexual activity prior to collection (6) and individual variation. A physiological testing procedure for epididymal function and patency of the ejaculatory ducts based on the study of the clearance of exogenous carnitine in the electroejaculate is desirable. However, the applicability of such a diaanostic aooroach is limited in the bovine by the lack of a net gain of exogenois carnit'i'ne in the ejaculate, by the various sources of carnitine in the semen, by the large variability in carnitine content and by the
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THERIOGENOLOGY negative effects of electroejaculation. However, there is a diagnostic potential in using carnitine assay to detect epididymides occlusion, but only when ejaculate is collected by an artificial vagina. REFERENCES
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