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Opposite variations of two epididymal components and blood plasma testosterone in two breeds of rams
Comp. Biochera. Physiol. Vol. 99A, No. I/2, pp. 173-177, 1991
0300-9629/91 $3.00+ 0.00 © 1991 PergamonPress ple
Printed in Great Britain
OPPOSITE VARIATIONS OF TWO EPIDIDYMAL COMPONENTS A N D BLOOD PLASMA TESTOSTERONE IN TWO BREEDS OF RAMS J. BESAN(~ON,*~"P. DEMEKS,~ J. P. LEMAY~ and R. R. TKEMBLAY§ *Department of Didactics, :~Department of Animal Science; and §Department of Medicine and Physiology, Universit6 Laval, Ste-Foy, Quebec, Canada G1K 7P4 (Received 21 August 1990)
Abstract--l. Testicular volume (T Vol), blood plasma testosterone ('13 concentration, seminal plasma a-glucosidase (=-G) specific activity, L-carnitine (L-C) concentration as well as semen characteristics were compared in eight Finnish Landrace (F) and eight Suffolk (S) rams throughout 21 months. 2. Only T Vol and T exhibited a typical seasonal variation in both breeds, whereas L-C, a-G and live sperm output presented a seasonal profile only in S rams. 3. L-C and a-G variations were opposite to those of T in S rams, while they fluctuated in F rams throughout the entire experiment, as did live sperm output. 4. Only the number of ejaculates and T were significantly higher in F rams (3.50 + 0.08 in 5 min and 7.62 + 0.40 ng/ml) than in S rams (2.30 + 0.05 in 5 min and 5.5 + 0.30 ng/mi); these two characteristics might therefore be considered as two indexes of sexual activity in rams. 5. By contrast, among all characteristics measured, only a-G was significantly higher in S rams than in F rams (1.33 + 0.04 vs 0.77 + 0.03 mU/mg proteins); this result, as well as seasonal =-G profile present in only S rams, allowed us to conclude that ,~-G might be considered as an additional index of seasonal reproduction in rams.
INTRODUCTION
Although adult rams continuously produce spermatozoa and m a i n t a i n a high level of sexual activity throughout the year (Gordon, 1983), their reproductive performance varies according to the season (Ortavant et al., 1964; Lincoln, 1976; Colas, 1981). The change from long days to short days stimulates the increase of blood plasma LH concentration which is primarily involved in testosterone synthesis (Pelletier and Ortavant, 1975; Lincoln and Peet, 1977). However, some phenomena of ram reproduction remain intriguing, such as testicular changes as well as pituitary activity which occur before the solstices (Lincoln and Davidson, 1977; Thimonier, 1981). Investigation into factors involved in epididymal sperm maturation (Bedford, 1975) might be of high interest to obtain further knowledge about ram reproduction. Recently, Besan~;on et al. (1985b) suggested that ram seminal ~-glucosidase specific activity might be considered as a useful marker of epididymal function. Therefore, the objective of the present study is to compare seasonal variations of testosterone concentration, testicular volume and semen characteristics with those of seminal plasma ~-glucosidase specific activity and L-carnitine concentration in order to evaluate whether these two biochemical parameters of the seminal plasma were correlated with the reproductive profile of rams. Observations were conducted in Finnish Landrace and Suffolk rams precisely because of the higher level of sexual activity in the first
breed and of the shorter breeding season in the second one (Schanbacher and Lunstra, 1976). MATERIALS AND METHODS
Animals
Eight Finnish Landrace and eight Suffolk rams born in February 1980 at the Station Agronomique de St-Augnstin, Universit6 Laval, were kept in natural daylight conditions and fed with a 1 kg/ram/d grain mixture containing 12% protein, good quality hay and fresh water ad libitum throughout the 21 months of the study which took place from October 1981 to June 1983. The average weight of these rams was 130kg (Suffolk) and 90kg (Finnish Landrace) and they were in excellent health over the experimental period. Sample collection and storage
Three months prior to the beginning of the study, and throughout the entire study period, semen was collected every two weeks, from each of the 16 rams, with an artificial vagina in the presence of an immobilized non-oestrus ewe as described by Besangon et al. (1985b). On the day of the semen collection, blood samples were taken from the jugular vein between 09.00 and 10.00 a.m., prior to all other manipulation of the animals. Blood plasma was separated by centrifugation at 800 g for 10 min and stored at -20°C. Seminal plasma was obtained after centrifugation of the semen at 30,000 g for 30 rain at 4°C and then stored at - 2 0 ° C . Prior to the assays, blood and seminal plasma were thawed for measurements of testosterone concentration, a-giucosidase specific activity and L-carnitine concentration. Semen characteristics and testicular volume
1"Allcorrespondence should be addressed to: Jacques Besan9on, Department of Didactics, Faculty of Education Sciences, Universit6 Laval, Ste-Foy, Quebec, Canada G I K 7P4.
Evaluation of semen characteristics and testicular volume were assessed as previously reported by Besan~on et al. (1985b). Percentage of live spermatozoa was determined as the ratio of cells excluding the nigrosin-eosin coloration out 173
174
J, B ~ r ~ o N SUFFOLK
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Fig. 1. Testicular volume, blood plasma testosterone concentration and seminal plasma at-glucosidase specific activity and L-carnitine concentration of Finnish Landrace and Suffolk rams over a 21 months period (each point represents month mean values + SEM).
Fig. 2. Semen characteristics of Finnish Landrace and Suffolk rams over a 21 months period (each point represents month mean values + SEM).
of 100 ceils drawn at random from a smear of semen, according to the method developed by Ortavant et al. (1953). Live sperm output was calculated as follows: sperm concentration (sperm no./semen ml)x total volume of semen (ml) ejaculated during a 5 min period x percentage of live spermatozoa.
testosterone concentration appears as measured in ng/ml in Fig. 1. Correlations were calculated across a few parameters.
ct-Glucosidase specific activity, L-carnitine and testosterone concentrations Seminal plasma ~t-glucosidase specific activity and Lcarnitine concentration as well as blood plasma testosterone concentration were measured according to methods previously described by Besanqon et al. (1985b). Statistical analysis All parameters studied were subjected to an analysis of variance using the SAS General Linear Models procedure (1982). The statistical model included the fixed effects of breeds, individual rams in the breed, the month and the interaction of the month with the breed. Variance of the periodical within ram testosterone measures increased with the corresponding mean testosterone concentrations; so all the statistical analysis with testosterone concentrations were performed on log-transformed data, adjusted to avoid negative and nonsense values. The log-transformed data is as follows: x = log[100(l + concentration)]. For simplicity,
RESULTS
Throughout the 21 months of the study, the eight characteristics (Figs 1 and 2) measured in both breeds varied significantly with regard to the months (P <0.01) and individual ram variations within breeds were also significant (P <0.01) for all these characteristics. Their mean level in both breeds throughout the experiment are reported in Table 1. Testicular volume a n d testosterone concentration
Testicular volume as well as blood plasma testosterone concentration exhibited a typical seasonal variation under our latitude in both breeds (Fig. 1): low during spring ( < 200 cm 3 and < 5 ng/ml, respectively) and high during fall ( > 2 3 0 c m 3 and > 13 ng/ml, respectively). Variations of both characteristics were also positively correlated (P <0.01) between themselves in both breeds (r = 0 . 5 3 in Suffolk and r = 0.27 in Finnish Landrace rams). The
Semen ~-glucosidase and L-carnitine in rams
175
Table 1. Mean level+ SEM of the eight characteristics measured in both breeds throughout 21 months of the study Characteristics Testieular volume (cm3) Testosterone (ng/ml) a-Glucosidasc (munits/mg proteins) L-Carnitine (raM) Ejaculates (number in 5 rain) Concentration ( x 109 spermatozoa/ml) Live sperm output ( x 109 × 5 rain) Motility (%)
Suffolk 198.17 + 3.09 5.50 + 0.30* 1.33 + 0.04t 8.02 + 0.17 2.30 + 0.051" 3.78 + 0.07 5.56 _+0.18 59.60 + 0.42
Breeds Finnish Landrace 189.03 + 3.19 7.62 + 0.40 0.77 + 0.03 8.51 _+0.25 3.50 + 0.08 3.91 +_0.08 6.77 + 0.23 61.50 + 0.52
*P < 0.01; "['P <0.05.
mean level of testosterone concentrations however was higher in Finnish Landrace than in Suffolk rams (P < 0.01) but that of testicular volume was not different between breeds (Table 1). Seminal plasma ~-glucosidase activity and L-carnitine concentration In Suffolk rams, seminal plasma a-glucosidase specific activity and L-carnitine concentration were high during winter (>l.5munits/mg proteins and > 9 m M , respectively) and low during summer (<0.8 mU/mg proteins and < 6 mM, respectively) as illustrated in Fig. 1. These variations were negatively correlated (P <0.01) with that of blood plasma testosterone concentration (r = - 0 . 4 1 and r = - 0 . 2 6 , respectively) but were positively correlated (P < 0.01) between themselves (r = 0.61). By contrast, in Finnish Landrace rams, both characteristics fluctuated throughout the 21 months of the study. Their variations were not correlated with those of testosterone concentration but were positively correlated (P < 0.01) between themselves (r = 0.53). The mean level of a-glucosidase specific activity w a s higher in Suffolk than in Finnish Landrace rams (P < 0.05) but that of L-carnitine concentration was not different between breeds (Table 1). Semen characteristics Variations of semen characteristics are illustrated in Fig. 2. The number of ejaculations within the 5 min collection period fluctuated throughout the 21 months of the study in both breeds, without a seasonal profile. However, the mean level of the number of ejaculates was higher (P < 0.05) in Finnish Landrace than in Suffolk rams (Table 1). Amongst variations of the spermatozoa concentration level, the percentage of motility and the live sperm output level [which were not significantly different between breeds (Table 1)], only the last characteristic in Suffolk rams presented a seasonal profile. In the 5min semen collection period, Suffolk rams ejaculated less live sperm in spring than in fall ( < 5 x 109 VS > 6 x 109 live sperm respectively). DISCUSSION
Seasonal variation of testicular volume and that of blood plasma testosterone concentration, which were, in addition, positively correlated between themselves in both breeds, have been previously described
in several studies on these breeds (Katongole et al., 1974; Sanford et al., 1974; Schanbacher and Ford, 1979; D'Occhio et al., 1984; Dufour et al., 1984). Results of present studies regarding semen characteristics are in agreement with a previous study which demonstrated that seasonal variations of ram reproductive activity were more obvious in quantity than in quality (Courot, 1976). The significant higher mean number of ejaculates in Finnish Landrace rams throughout the 21 months of the experiment might be an indication of a higher level of sexual activity in this breed as reported by Schanbacher and Lunstra (1976). Furthermore, these authors observed a higher mean level of testosterone concentration in Finnish Landrace than in Suffolk rams, as was noted in our study. Therefore, a higher level of sexual activity in Finnish Landrace rams might be shown by a higher number of ejaculates in 5 min or a higher blood plasma testosterone concentration. By contrast, neither testosterone nor testicular volume, which varied similarly in both breeds, can indicate a difference between breeds with regard to their seasonal reproduction. The only semen characteristic which exhibited the well known short breeding season in Suffolk rams was the live sperm output. In seminal plasma, ~-glucosidase activity and L-carnitine concentration also exhibited a seasonal profile in Suffolk rams. Furthermore there was no significant difference in mean levels of live sperm output between breeds while seminal plasma c~-glucosidase activity was the only characteristic which was significantly higher in Suffolk than in Finnish Landrace rams. While the signification of seasonal variation of enzyme activity is presently unknown, the results of this study suggest that seminal plasma ~-glucosidase specific activity might be considered as an additional index of seasonal reproduction in rams. Because of the existence of a negative correlation between ~-glucosidase and testosterone in Suffolk rams, a relationship between the seminal plasma enzyme and blood plasma prolactin concentration, of which seasonal variation is also opposite to that of testosterone (Pelletier, 1973; Ravault, 1976), could be anticipated. However, this hypothesis was excluded because a bromoergocryptine treatment in rams of the same breeds did not change ~-glucosidase activity when prolactin concentration diminished drastically (Besanqon et al., 1981). Positive correlations between seminal plasma ~-glucosidase specific activity and L-carnitine concentration in both breeds could support the hypothesis that both variables are indeed of a physiological meaning (Tremblay et al., 1982;
176
J. BESAN~ONet al.
Besan~;on et al., 1985a; Cooper et al., 1988). An array of enzymes, esterases and carbohydratases, for example present in luminal fluid (Murdoch and White, 1968; Jones, 1978) (several of which are secreted by the epididymis), could act on extracellular proteins and glycoproteins to modify the spermatozoa membrane functions with consequences on sperm motility (Ascott and Hoskins, 1978; Lea and French, 1981) or on sperm-egg binding (Hartmann and Hutchison, 1977). If such is the case in several species, one must conclude that ~-glucosidase in rams is not at all representative of the carbohydratases contribution to ram semen maturation because no firm correlation can be established between any semen characteristics reported in Fig. 2 and u-glucosidase content of the seminal plasma. This observation casts serious doubt on ~-glucosidase modulation by androgens in rams because ~-glucosidase, as with L-carnitine, is at its lowest value while plasma testosterone reaches a peak in fall. This is reminiscent of an observation by Brooks (1987) indicating the fact that there are a number of proteins whose synthesis and secretion appears unaffected by the androgen status. Our results do not negate, however, the classical knowledge that sperm maturation is dependant upon a normal androgenic support to the epididymis (Dyson and Orgebin-Crist, 1973; Fournier-Delpech et al., 1984). With respect to carnitine, its opposite variation in seminal plasma to blood plasma testosterone in Suffolk rams or the absence of correlation between both characteristics in Finnish Landrace rams does not support evidence gathered in rats and rabbits suggesting that androgens are required for the carnitine transport into the epididymis (Marquis and Fritz, 1965; Jones and Glover, 1973; Bghmer and Hansson, 1975). A high concentration of carnitine in ram epididymal fluid (Hinton et al., 1979; Besangon et al., 1985a) should be more than incidental on this parameter in total seminal plasma unless the spermatozoa metabolism is significantly modified during breeding seasons. Indeed, Milkowski et al. (1976) have demonstrated that a major function of carnitine present in spermatozoa is to store "acetyl units" for aerobic oxidation and energy production when needed. With motility of spermatozoa being dependant upon the storage of those "acetyl units" as acylcarnitine in spermatozoa (Milkowski et al., 1976; Johansen and Bohmer, 1979), one could postulate that the end result of this process leads to a decrease in seminal plasma carnitine content, as observed in this experiment. Otherwise, it could have been extremely useful to determine blood plasma carnitine in a time-course manner in our rams, because an inverse relationship between circulating testosterone and serum carnitine has been found in bulls (Carter et al., 1980) and those variations in carnitine, between species, might reflect the relative importance of the uptake phenomenon by spermatozoa at different degrees of maturation. Acknowledgements--The authors wish to thank Mrs L. Lacouline, Mr R. Lavigne and Mr A. Cormier for their skillful technical assistance. This research was partially supported by a grant from the Medical Research Council of Canada (MA. 5848) and by the Facult6 des Sciences de l'Agriculture et de l'Alimentation de rUniversit~ Laval.
REFERENCES Ascott T. S. and Hoskins D. D. (1978) Bovine forward motility protein. Partial purification and characterization. J. Biol. Chem. 253, 6744-6750. Bedford J. M. (1975) Maturation, transport and fate of spermatozoa in the epididymis. In Handbook of Physiology: Endocrinology (section 7) (Edited by Hamilton D. W. and Greep R. O.) Vol. V, pp. 303-317. Am. Physiol. Soc., Washington D.C. Besan~on J., Dumas G., Tremblay R. R. and Lemay J. P. (1981) Interactions entre l'~-glueosidase du plasma srminal et la spermatogrnrse du b~lier. Union m~dicale du Canada 110 44, S-18 (Abstr.). Besangon J., Dacheux J. L., Paquin R. and Tremblay R. R. (1985a) Major contribution of epididymis to ~-glucosidase content of ram seminal plasma. Biol. Reprod. 33, 296-301. Besangon J., No~l J. C., Tremblay R. R. and Lemay J. P. (1985b) Effect of physiological (in lamb) or drug-induced (in adult) immaturity of ram spermatozoa on seminal plasma ~-giucosidase activity. Comp. Biochem. Physiol. 82A, 681-686. Behmer T. and Hansson V. (1975) Androgen-dependant accumulation ofcaruitine by rat epididymis after injection of [3H] butyrobetaine in vivo. Moll. Cell Endocrinol. 3, 103-115. Brooks D. E. (1987) Androgen-regulated epididymal secretory proteins associated with post-testicular sperm development. Ann. N.Y. Acad. Sci. 513, 179-194. Carter A. L., Hutson S. M., Stratman F. W. and Haning R. V. (1980) Relationship of carnitine and acylcarnitines in ejaculated sperm to blood plasma testosterone in dairy bulls. Biol. Reprod. 23, 820-825. Colas G. (1981) Variations saisonni~res de la qualit~ du sperme chez le b~lier Ile-de-France. II. F6condance: Relation avec les crit~res qualitatifs observes in vitro. Reprod. Nutr. Dbvelop. 21, 399-407. Cooper T. G., Yeung C. H., Nashan D. and Nieschlag E. (1988) Epididymal markers in human infertility. J. Androl. 9, 91. Courot M. (1976) Semen quality and quantity in the ram. Prec. 1976. Internat. Congr. Sheep Breeding. Western Australia (G. J. Tomes, D. E. Robertson and R. J. Lightfoot) pp. 276-285. D'Occhio M. J., Schanbacher B. D. and Kinder J. E. (1984) Profiles of luteinizing hormone, follicle-stimulating hormone, testosterone and prolactin in rams of diverse breeds: effects of contrasting short (8L:I6D) and long (16L:8D) photoperiod. Biol. Reprod. 30, 1039-1054. Dufour J. J., Fahmy M. H. and MinvielleF. (1984) Seasonal changes in breeding activity, testicular size, testosterone concentration and seminal characteristics in rams with long and short breeding season. J. Anim. Sci. 58, 416-422. Dyson A. L. M. B. and Orgebin-Crist M. C. (1973) Effect of hypophysectomy, castration and androgen replacement upon the fertilizingability of rat epididymal spermatozoa. Endocrinology 93, 391-402. Fournier-Delpech S., Hamamah S., Tananis-Antony C., Courot M. and Orgebin-Crist M. C. (1984) Hormonal regulation of zona-binding ability and fertilizing ability of rat epididymal spermatozoa. Gamete Res. 9, 21-30. Gordon I. (1983) Introduction to controlled breeding in sheep. In Controlled Breeding in Farm Animals (3rd Ed.), pp. 155-167. Pergamon Press, New York. Hartmann J. F. and Hutchison C, F. (1977) Involvement of two carbohydrate-containing components in the binding of uncapacitated spermatozoa to eggs of the golden hamster in vitro. J. Exp. Zool. 201, 383-390. Hinton B. T., Snoswell A. M. and Setchell B. P. (1979) The concentration of carnitine in the luminal fluid of the testis
Semen ct-glucosidase and L-carnitine in rams and epididymis of the rat and some other mammals. J. Reprod. Fertil. 56, 105-11 I. Johansen L. and Bghmer T. (1979) Motility related to the presence of carnitine/acetylcarnitine in human spermatozoa. Int. J. Androl. 2, 202-210. Jones R. (1978) Comparative biochemistry of mammalian epididymal plasma. Comp. Biochem. Biophys. 61B, 365-370. Jones R. and Glover T. D. (1973) The effects of castration on the composition of rabbit epididymal plasma. J. Reprod. Fertil. 34, 405-411. Katongole C. B., Naftolin F. and Short R. V. (1974) Seasonal variations in blood luteinizing hormone and testosterone levels in rams. J. Endocrinol. 60, 101-106. Lea O. A. and French F. S. (1981) Characterization of an acidic glycoprotein secreted by principal cells of the rat epididymis. Biochim. Biophys. Acta 668, 370-376. Lincoln G. A. (1976) Seasonal variations in the episodic secretion of luteinizing hormone and testosterone in the ram. J. Endocrinol. 69, 213-226. Lincoln G. A. and Davidson W. (1977) The relationship between sexual and aggressive behaviour and pituitary and testicular activity during the seasonal cycle of rams, and the influence of photoperiod. J. Reprod. Fertil. 49, 267-276. Lincoln G. A. and Peet M. J. (1977) Photoperiodic control of gonadotropin secretion in the ram: a detailed study of the temporal changes in plasma levels of folliclestimulating hormone, luteinizing hormone and testosterone, following an abrupt switch from long to short days. J. Endocrinol. 74, 335-367. Marquis N. R. and Fritz I. B. (1965) Effects of testosterone on the distribution of carnitine, acetylcarnitine, and carnitine acetyl transferase in tissues of the reproductive system in the male rat. J. Biol. Chem. 240, 2197-2200. Milkowski A. L., Babcock D. F. and Lardy H. A. (1976) Activation of bovine epididymal sperm respiration by caffeine. Its transient nature and relationship to the utilization of acetylcarnitine. Arch. Biochem. Biophys. 176, 250-256.
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Murdoch R. N. and White I. G. (1968) Studies on the distribution and source of enzymes in mammalian semen. Australian J. Biol. Sci. 21, 483-490. Ortavant R., Dupont S., Pauthe H. and Roussel R. (1953) Contribution ~i l'6tude de la diff6rentiation des spermatozoides morts et vivants dam le sperme de taureau. Ann. Zootechnie 2, 1-8. Ortavant R., Maul6on P. and Thibault C. (1964) Photoperiodic control of gonadal and hypophyseal activity in domestic mammals. Ann. N.Y. Acad. Sci. 117, 157-193. Pelletier J. (1973) Evidence for photoperiodic control of prolactin release in rams. J. Reprod. Fertil. 35, 143-147. Pelletier J. and Ortovant R. (1975) Photoperiodic control of LH release in the ram--I, Influence of increasing and decreasing light photoperiod. Acta Endocrinol. 78, 435-441. Ravault J. P. (1976) Prolactin in the ram: seasonal variations in the concentration of blood plasma from birth until three years old. Acta Endocrinol. 83, 720-725. Sanford L. M., Palmer W. M. and Howland B. E. (1974) Seasonal variations of serum levels of LH and testosterone in the ram. Can. J. Anita. Sci. $4, 247-249. S.A.S. (1982) SAS User's Guide: Statistics. SAS Inst., Inc. Cary, NC. Schanbacher B. D. and Ford J. J. (1979) Photoperiodic regulation of ovine spermatogenesis: relationship to serum hormones. Biol. Reprod. 20, 719-726. Schanbacher B. D. and Lunstra D. D. (1976) Seasonal changes in sexual activity and serum levels of LH and testosterone in Finnish Landrace and Suffolk rams. J. Anita. Sci. 43, 644-650. Thimonier J. (1981) Control of seasonal reproduction in sheep and goats by light and hormones. In Reproductive Endocrinology of Domestic Ruminants (Edited by Scaramuzzi R. J., Lincoln D. W. and Weir B. J.), J. Reprod. Fertil. (Suppl. 30), 33-45. Tremblay R. R., Chapdelaine P., Roy R. and Thabet M. (1982) Correlation between L-carnitine and ct-1,4-glucosidase activity in the semen of normal, infertile and vasectomized men. Infertility 5, 61-70.
0300-9629/91 $3.00+ 0.00 © 1991 PergamonPress ple
Printed in Great Britain
OPPOSITE VARIATIONS OF TWO EPIDIDYMAL COMPONENTS A N D BLOOD PLASMA TESTOSTERONE IN TWO BREEDS OF RAMS J. BESAN(~ON,*~"P. DEMEKS,~ J. P. LEMAY~ and R. R. TKEMBLAY§ *Department of Didactics, :~Department of Animal Science; and §Department of Medicine and Physiology, Universit6 Laval, Ste-Foy, Quebec, Canada G1K 7P4 (Received 21 August 1990)
Abstract--l. Testicular volume (T Vol), blood plasma testosterone ('13 concentration, seminal plasma a-glucosidase (=-G) specific activity, L-carnitine (L-C) concentration as well as semen characteristics were compared in eight Finnish Landrace (F) and eight Suffolk (S) rams throughout 21 months. 2. Only T Vol and T exhibited a typical seasonal variation in both breeds, whereas L-C, a-G and live sperm output presented a seasonal profile only in S rams. 3. L-C and a-G variations were opposite to those of T in S rams, while they fluctuated in F rams throughout the entire experiment, as did live sperm output. 4. Only the number of ejaculates and T were significantly higher in F rams (3.50 + 0.08 in 5 min and 7.62 + 0.40 ng/ml) than in S rams (2.30 + 0.05 in 5 min and 5.5 + 0.30 ng/mi); these two characteristics might therefore be considered as two indexes of sexual activity in rams. 5. By contrast, among all characteristics measured, only a-G was significantly higher in S rams than in F rams (1.33 + 0.04 vs 0.77 + 0.03 mU/mg proteins); this result, as well as seasonal =-G profile present in only S rams, allowed us to conclude that ,~-G might be considered as an additional index of seasonal reproduction in rams.
INTRODUCTION
Although adult rams continuously produce spermatozoa and m a i n t a i n a high level of sexual activity throughout the year (Gordon, 1983), their reproductive performance varies according to the season (Ortavant et al., 1964; Lincoln, 1976; Colas, 1981). The change from long days to short days stimulates the increase of blood plasma LH concentration which is primarily involved in testosterone synthesis (Pelletier and Ortavant, 1975; Lincoln and Peet, 1977). However, some phenomena of ram reproduction remain intriguing, such as testicular changes as well as pituitary activity which occur before the solstices (Lincoln and Davidson, 1977; Thimonier, 1981). Investigation into factors involved in epididymal sperm maturation (Bedford, 1975) might be of high interest to obtain further knowledge about ram reproduction. Recently, Besan~;on et al. (1985b) suggested that ram seminal ~-glucosidase specific activity might be considered as a useful marker of epididymal function. Therefore, the objective of the present study is to compare seasonal variations of testosterone concentration, testicular volume and semen characteristics with those of seminal plasma ~-glucosidase specific activity and L-carnitine concentration in order to evaluate whether these two biochemical parameters of the seminal plasma were correlated with the reproductive profile of rams. Observations were conducted in Finnish Landrace and Suffolk rams precisely because of the higher level of sexual activity in the first
breed and of the shorter breeding season in the second one (Schanbacher and Lunstra, 1976). MATERIALS AND METHODS
Animals
Eight Finnish Landrace and eight Suffolk rams born in February 1980 at the Station Agronomique de St-Augnstin, Universit6 Laval, were kept in natural daylight conditions and fed with a 1 kg/ram/d grain mixture containing 12% protein, good quality hay and fresh water ad libitum throughout the 21 months of the study which took place from October 1981 to June 1983. The average weight of these rams was 130kg (Suffolk) and 90kg (Finnish Landrace) and they were in excellent health over the experimental period. Sample collection and storage
Three months prior to the beginning of the study, and throughout the entire study period, semen was collected every two weeks, from each of the 16 rams, with an artificial vagina in the presence of an immobilized non-oestrus ewe as described by Besangon et al. (1985b). On the day of the semen collection, blood samples were taken from the jugular vein between 09.00 and 10.00 a.m., prior to all other manipulation of the animals. Blood plasma was separated by centrifugation at 800 g for 10 min and stored at -20°C. Seminal plasma was obtained after centrifugation of the semen at 30,000 g for 30 rain at 4°C and then stored at - 2 0 ° C . Prior to the assays, blood and seminal plasma were thawed for measurements of testosterone concentration, a-giucosidase specific activity and L-carnitine concentration. Semen characteristics and testicular volume
1"Allcorrespondence should be addressed to: Jacques Besan9on, Department of Didactics, Faculty of Education Sciences, Universit6 Laval, Ste-Foy, Quebec, Canada G I K 7P4.
Evaluation of semen characteristics and testicular volume were assessed as previously reported by Besan~on et al. (1985b). Percentage of live spermatozoa was determined as the ratio of cells excluding the nigrosin-eosin coloration out 173
174
J, B ~ r ~ o N SUFFOLK
et al.
FINNISH LANDRACE
SUFFOLK
TESTICULAR VOLUME 250-
FINNISH LANDRACE EJACULATES
¢E
5
z
4
o 200-
~
3
150-
~ Z
2 I i
TES'~
¢
E ~.= 10. 5
L
3.0 5O
4.0
x 2.0
2.0-~
' ' ' i i LIVE SPERM OUTPUT
,~-GLUCOSIDASE
i
i
i
i
i
c 10.0 8.0 gJ o. ¢n 6.0
1.5-
:~
i
CONCENTRATION ~ IE
15 ¸
,,~
i
;TERONE
1.0x
4.0
L-CARNITINE
MOTILITY 7O
2
6560 55
,],4,4,1,Pl,H,l,l^l'.~i,l,I, P~H,I H,I,'I 'l' IH'H'I,H~H'H.'I' H'H
N '11'l"-i'|;I ']7i"1TI'IT;FF........~TI ! H,'I I' F.'I,H,I,H.t f,H I, H.,H
Fig. 1. Testicular volume, blood plasma testosterone concentration and seminal plasma at-glucosidase specific activity and L-carnitine concentration of Finnish Landrace and Suffolk rams over a 21 months period (each point represents month mean values + SEM).
Fig. 2. Semen characteristics of Finnish Landrace and Suffolk rams over a 21 months period (each point represents month mean values + SEM).
of 100 ceils drawn at random from a smear of semen, according to the method developed by Ortavant et al. (1953). Live sperm output was calculated as follows: sperm concentration (sperm no./semen ml)x total volume of semen (ml) ejaculated during a 5 min period x percentage of live spermatozoa.
testosterone concentration appears as measured in ng/ml in Fig. 1. Correlations were calculated across a few parameters.
ct-Glucosidase specific activity, L-carnitine and testosterone concentrations Seminal plasma ~t-glucosidase specific activity and Lcarnitine concentration as well as blood plasma testosterone concentration were measured according to methods previously described by Besanqon et al. (1985b). Statistical analysis All parameters studied were subjected to an analysis of variance using the SAS General Linear Models procedure (1982). The statistical model included the fixed effects of breeds, individual rams in the breed, the month and the interaction of the month with the breed. Variance of the periodical within ram testosterone measures increased with the corresponding mean testosterone concentrations; so all the statistical analysis with testosterone concentrations were performed on log-transformed data, adjusted to avoid negative and nonsense values. The log-transformed data is as follows: x = log[100(l + concentration)]. For simplicity,
RESULTS
Throughout the 21 months of the study, the eight characteristics (Figs 1 and 2) measured in both breeds varied significantly with regard to the months (P <0.01) and individual ram variations within breeds were also significant (P <0.01) for all these characteristics. Their mean level in both breeds throughout the experiment are reported in Table 1. Testicular volume a n d testosterone concentration
Testicular volume as well as blood plasma testosterone concentration exhibited a typical seasonal variation under our latitude in both breeds (Fig. 1): low during spring ( < 200 cm 3 and < 5 ng/ml, respectively) and high during fall ( > 2 3 0 c m 3 and > 13 ng/ml, respectively). Variations of both characteristics were also positively correlated (P <0.01) between themselves in both breeds (r = 0 . 5 3 in Suffolk and r = 0.27 in Finnish Landrace rams). The
Semen ~-glucosidase and L-carnitine in rams
175
Table 1. Mean level+ SEM of the eight characteristics measured in both breeds throughout 21 months of the study Characteristics Testieular volume (cm3) Testosterone (ng/ml) a-Glucosidasc (munits/mg proteins) L-Carnitine (raM) Ejaculates (number in 5 rain) Concentration ( x 109 spermatozoa/ml) Live sperm output ( x 109 × 5 rain) Motility (%)
Suffolk 198.17 + 3.09 5.50 + 0.30* 1.33 + 0.04t 8.02 + 0.17 2.30 + 0.051" 3.78 + 0.07 5.56 _+0.18 59.60 + 0.42
Breeds Finnish Landrace 189.03 + 3.19 7.62 + 0.40 0.77 + 0.03 8.51 _+0.25 3.50 + 0.08 3.91 +_0.08 6.77 + 0.23 61.50 + 0.52
*P < 0.01; "['P <0.05.
mean level of testosterone concentrations however was higher in Finnish Landrace than in Suffolk rams (P < 0.01) but that of testicular volume was not different between breeds (Table 1). Seminal plasma ~-glucosidase activity and L-carnitine concentration In Suffolk rams, seminal plasma a-glucosidase specific activity and L-carnitine concentration were high during winter (>l.5munits/mg proteins and > 9 m M , respectively) and low during summer (<0.8 mU/mg proteins and < 6 mM, respectively) as illustrated in Fig. 1. These variations were negatively correlated (P <0.01) with that of blood plasma testosterone concentration (r = - 0 . 4 1 and r = - 0 . 2 6 , respectively) but were positively correlated (P < 0.01) between themselves (r = 0.61). By contrast, in Finnish Landrace rams, both characteristics fluctuated throughout the 21 months of the study. Their variations were not correlated with those of testosterone concentration but were positively correlated (P < 0.01) between themselves (r = 0.53). The mean level of a-glucosidase specific activity w a s higher in Suffolk than in Finnish Landrace rams (P < 0.05) but that of L-carnitine concentration was not different between breeds (Table 1). Semen characteristics Variations of semen characteristics are illustrated in Fig. 2. The number of ejaculations within the 5 min collection period fluctuated throughout the 21 months of the study in both breeds, without a seasonal profile. However, the mean level of the number of ejaculates was higher (P < 0.05) in Finnish Landrace than in Suffolk rams (Table 1). Amongst variations of the spermatozoa concentration level, the percentage of motility and the live sperm output level [which were not significantly different between breeds (Table 1)], only the last characteristic in Suffolk rams presented a seasonal profile. In the 5min semen collection period, Suffolk rams ejaculated less live sperm in spring than in fall ( < 5 x 109 VS > 6 x 109 live sperm respectively). DISCUSSION
Seasonal variation of testicular volume and that of blood plasma testosterone concentration, which were, in addition, positively correlated between themselves in both breeds, have been previously described
in several studies on these breeds (Katongole et al., 1974; Sanford et al., 1974; Schanbacher and Ford, 1979; D'Occhio et al., 1984; Dufour et al., 1984). Results of present studies regarding semen characteristics are in agreement with a previous study which demonstrated that seasonal variations of ram reproductive activity were more obvious in quantity than in quality (Courot, 1976). The significant higher mean number of ejaculates in Finnish Landrace rams throughout the 21 months of the experiment might be an indication of a higher level of sexual activity in this breed as reported by Schanbacher and Lunstra (1976). Furthermore, these authors observed a higher mean level of testosterone concentration in Finnish Landrace than in Suffolk rams, as was noted in our study. Therefore, a higher level of sexual activity in Finnish Landrace rams might be shown by a higher number of ejaculates in 5 min or a higher blood plasma testosterone concentration. By contrast, neither testosterone nor testicular volume, which varied similarly in both breeds, can indicate a difference between breeds with regard to their seasonal reproduction. The only semen characteristic which exhibited the well known short breeding season in Suffolk rams was the live sperm output. In seminal plasma, ~-glucosidase activity and L-carnitine concentration also exhibited a seasonal profile in Suffolk rams. Furthermore there was no significant difference in mean levels of live sperm output between breeds while seminal plasma c~-glucosidase activity was the only characteristic which was significantly higher in Suffolk than in Finnish Landrace rams. While the signification of seasonal variation of enzyme activity is presently unknown, the results of this study suggest that seminal plasma ~-glucosidase specific activity might be considered as an additional index of seasonal reproduction in rams. Because of the existence of a negative correlation between ~-glucosidase and testosterone in Suffolk rams, a relationship between the seminal plasma enzyme and blood plasma prolactin concentration, of which seasonal variation is also opposite to that of testosterone (Pelletier, 1973; Ravault, 1976), could be anticipated. However, this hypothesis was excluded because a bromoergocryptine treatment in rams of the same breeds did not change ~-glucosidase activity when prolactin concentration diminished drastically (Besanqon et al., 1981). Positive correlations between seminal plasma ~-glucosidase specific activity and L-carnitine concentration in both breeds could support the hypothesis that both variables are indeed of a physiological meaning (Tremblay et al., 1982;
176
J. BESAN~ONet al.
Besan~;on et al., 1985a; Cooper et al., 1988). An array of enzymes, esterases and carbohydratases, for example present in luminal fluid (Murdoch and White, 1968; Jones, 1978) (several of which are secreted by the epididymis), could act on extracellular proteins and glycoproteins to modify the spermatozoa membrane functions with consequences on sperm motility (Ascott and Hoskins, 1978; Lea and French, 1981) or on sperm-egg binding (Hartmann and Hutchison, 1977). If such is the case in several species, one must conclude that ~-glucosidase in rams is not at all representative of the carbohydratases contribution to ram semen maturation because no firm correlation can be established between any semen characteristics reported in Fig. 2 and u-glucosidase content of the seminal plasma. This observation casts serious doubt on ~-glucosidase modulation by androgens in rams because ~-glucosidase, as with L-carnitine, is at its lowest value while plasma testosterone reaches a peak in fall. This is reminiscent of an observation by Brooks (1987) indicating the fact that there are a number of proteins whose synthesis and secretion appears unaffected by the androgen status. Our results do not negate, however, the classical knowledge that sperm maturation is dependant upon a normal androgenic support to the epididymis (Dyson and Orgebin-Crist, 1973; Fournier-Delpech et al., 1984). With respect to carnitine, its opposite variation in seminal plasma to blood plasma testosterone in Suffolk rams or the absence of correlation between both characteristics in Finnish Landrace rams does not support evidence gathered in rats and rabbits suggesting that androgens are required for the carnitine transport into the epididymis (Marquis and Fritz, 1965; Jones and Glover, 1973; Bghmer and Hansson, 1975). A high concentration of carnitine in ram epididymal fluid (Hinton et al., 1979; Besangon et al., 1985a) should be more than incidental on this parameter in total seminal plasma unless the spermatozoa metabolism is significantly modified during breeding seasons. Indeed, Milkowski et al. (1976) have demonstrated that a major function of carnitine present in spermatozoa is to store "acetyl units" for aerobic oxidation and energy production when needed. With motility of spermatozoa being dependant upon the storage of those "acetyl units" as acylcarnitine in spermatozoa (Milkowski et al., 1976; Johansen and Bohmer, 1979), one could postulate that the end result of this process leads to a decrease in seminal plasma carnitine content, as observed in this experiment. Otherwise, it could have been extremely useful to determine blood plasma carnitine in a time-course manner in our rams, because an inverse relationship between circulating testosterone and serum carnitine has been found in bulls (Carter et al., 1980) and those variations in carnitine, between species, might reflect the relative importance of the uptake phenomenon by spermatozoa at different degrees of maturation. Acknowledgements--The authors wish to thank Mrs L. Lacouline, Mr R. Lavigne and Mr A. Cormier for their skillful technical assistance. This research was partially supported by a grant from the Medical Research Council of Canada (MA. 5848) and by the Facult6 des Sciences de l'Agriculture et de l'Alimentation de rUniversit~ Laval.
REFERENCES Ascott T. S. and Hoskins D. D. (1978) Bovine forward motility protein. Partial purification and characterization. J. Biol. Chem. 253, 6744-6750. Bedford J. M. (1975) Maturation, transport and fate of spermatozoa in the epididymis. In Handbook of Physiology: Endocrinology (section 7) (Edited by Hamilton D. W. and Greep R. O.) Vol. V, pp. 303-317. Am. Physiol. Soc., Washington D.C. Besan~on J., Dumas G., Tremblay R. R. and Lemay J. P. (1981) Interactions entre l'~-glueosidase du plasma srminal et la spermatogrnrse du b~lier. Union m~dicale du Canada 110 44, S-18 (Abstr.). Besangon J., Dacheux J. L., Paquin R. and Tremblay R. R. (1985a) Major contribution of epididymis to ~-glucosidase content of ram seminal plasma. Biol. Reprod. 33, 296-301. Besangon J., No~l J. C., Tremblay R. R. and Lemay J. P. (1985b) Effect of physiological (in lamb) or drug-induced (in adult) immaturity of ram spermatozoa on seminal plasma ~-giucosidase activity. Comp. Biochem. Physiol. 82A, 681-686. Behmer T. and Hansson V. (1975) Androgen-dependant accumulation ofcaruitine by rat epididymis after injection of [3H] butyrobetaine in vivo. Moll. Cell Endocrinol. 3, 103-115. Brooks D. E. (1987) Androgen-regulated epididymal secretory proteins associated with post-testicular sperm development. Ann. N.Y. Acad. Sci. 513, 179-194. Carter A. L., Hutson S. M., Stratman F. W. and Haning R. V. (1980) Relationship of carnitine and acylcarnitines in ejaculated sperm to blood plasma testosterone in dairy bulls. Biol. Reprod. 23, 820-825. Colas G. (1981) Variations saisonni~res de la qualit~ du sperme chez le b~lier Ile-de-France. II. F6condance: Relation avec les crit~res qualitatifs observes in vitro. Reprod. Nutr. Dbvelop. 21, 399-407. Cooper T. G., Yeung C. H., Nashan D. and Nieschlag E. (1988) Epididymal markers in human infertility. J. Androl. 9, 91. Courot M. (1976) Semen quality and quantity in the ram. Prec. 1976. Internat. Congr. Sheep Breeding. Western Australia (G. J. Tomes, D. E. Robertson and R. J. Lightfoot) pp. 276-285. D'Occhio M. J., Schanbacher B. D. and Kinder J. E. (1984) Profiles of luteinizing hormone, follicle-stimulating hormone, testosterone and prolactin in rams of diverse breeds: effects of contrasting short (8L:I6D) and long (16L:8D) photoperiod. Biol. Reprod. 30, 1039-1054. Dufour J. J., Fahmy M. H. and MinvielleF. (1984) Seasonal changes in breeding activity, testicular size, testosterone concentration and seminal characteristics in rams with long and short breeding season. J. Anim. Sci. 58, 416-422. Dyson A. L. M. B. and Orgebin-Crist M. C. (1973) Effect of hypophysectomy, castration and androgen replacement upon the fertilizingability of rat epididymal spermatozoa. Endocrinology 93, 391-402. Fournier-Delpech S., Hamamah S., Tananis-Antony C., Courot M. and Orgebin-Crist M. C. (1984) Hormonal regulation of zona-binding ability and fertilizing ability of rat epididymal spermatozoa. Gamete Res. 9, 21-30. Gordon I. (1983) Introduction to controlled breeding in sheep. In Controlled Breeding in Farm Animals (3rd Ed.), pp. 155-167. Pergamon Press, New York. Hartmann J. F. and Hutchison C, F. (1977) Involvement of two carbohydrate-containing components in the binding of uncapacitated spermatozoa to eggs of the golden hamster in vitro. J. Exp. Zool. 201, 383-390. Hinton B. T., Snoswell A. M. and Setchell B. P. (1979) The concentration of carnitine in the luminal fluid of the testis
Semen ct-glucosidase and L-carnitine in rams and epididymis of the rat and some other mammals. J. Reprod. Fertil. 56, 105-11 I. Johansen L. and Bghmer T. (1979) Motility related to the presence of carnitine/acetylcarnitine in human spermatozoa. Int. J. Androl. 2, 202-210. Jones R. (1978) Comparative biochemistry of mammalian epididymal plasma. Comp. Biochem. Biophys. 61B, 365-370. Jones R. and Glover T. D. (1973) The effects of castration on the composition of rabbit epididymal plasma. J. Reprod. Fertil. 34, 405-411. Katongole C. B., Naftolin F. and Short R. V. (1974) Seasonal variations in blood luteinizing hormone and testosterone levels in rams. J. Endocrinol. 60, 101-106. Lea O. A. and French F. S. (1981) Characterization of an acidic glycoprotein secreted by principal cells of the rat epididymis. Biochim. Biophys. Acta 668, 370-376. Lincoln G. A. (1976) Seasonal variations in the episodic secretion of luteinizing hormone and testosterone in the ram. J. Endocrinol. 69, 213-226. Lincoln G. A. and Davidson W. (1977) The relationship between sexual and aggressive behaviour and pituitary and testicular activity during the seasonal cycle of rams, and the influence of photoperiod. J. Reprod. Fertil. 49, 267-276. Lincoln G. A. and Peet M. J. (1977) Photoperiodic control of gonadotropin secretion in the ram: a detailed study of the temporal changes in plasma levels of folliclestimulating hormone, luteinizing hormone and testosterone, following an abrupt switch from long to short days. J. Endocrinol. 74, 335-367. Marquis N. R. and Fritz I. B. (1965) Effects of testosterone on the distribution of carnitine, acetylcarnitine, and carnitine acetyl transferase in tissues of the reproductive system in the male rat. J. Biol. Chem. 240, 2197-2200. Milkowski A. L., Babcock D. F. and Lardy H. A. (1976) Activation of bovine epididymal sperm respiration by caffeine. Its transient nature and relationship to the utilization of acetylcarnitine. Arch. Biochem. Biophys. 176, 250-256.
177
Murdoch R. N. and White I. G. (1968) Studies on the distribution and source of enzymes in mammalian semen. Australian J. Biol. Sci. 21, 483-490. Ortavant R., Dupont S., Pauthe H. and Roussel R. (1953) Contribution ~i l'6tude de la diff6rentiation des spermatozoides morts et vivants dam le sperme de taureau. Ann. Zootechnie 2, 1-8. Ortavant R., Maul6on P. and Thibault C. (1964) Photoperiodic control of gonadal and hypophyseal activity in domestic mammals. Ann. N.Y. Acad. Sci. 117, 157-193. Pelletier J. (1973) Evidence for photoperiodic control of prolactin release in rams. J. Reprod. Fertil. 35, 143-147. Pelletier J. and Ortovant R. (1975) Photoperiodic control of LH release in the ram--I, Influence of increasing and decreasing light photoperiod. Acta Endocrinol. 78, 435-441. Ravault J. P. (1976) Prolactin in the ram: seasonal variations in the concentration of blood plasma from birth until three years old. Acta Endocrinol. 83, 720-725. Sanford L. M., Palmer W. M. and Howland B. E. (1974) Seasonal variations of serum levels of LH and testosterone in the ram. Can. J. Anita. Sci. $4, 247-249. S.A.S. (1982) SAS User's Guide: Statistics. SAS Inst., Inc. Cary, NC. Schanbacher B. D. and Ford J. J. (1979) Photoperiodic regulation of ovine spermatogenesis: relationship to serum hormones. Biol. Reprod. 20, 719-726. Schanbacher B. D. and Lunstra D. D. (1976) Seasonal changes in sexual activity and serum levels of LH and testosterone in Finnish Landrace and Suffolk rams. J. Anita. Sci. 43, 644-650. Thimonier J. (1981) Control of seasonal reproduction in sheep and goats by light and hormones. In Reproductive Endocrinology of Domestic Ruminants (Edited by Scaramuzzi R. J., Lincoln D. W. and Weir B. J.), J. Reprod. Fertil. (Suppl. 30), 33-45. Tremblay R. R., Chapdelaine P., Roy R. and Thabet M. (1982) Correlation between L-carnitine and ct-1,4-glucosidase activity in the semen of normal, infertile and vasectomized men. Infertility 5, 61-70.