der Siidd. Versuchs- und Forschungsanstalt fur Milchwirtschaft Weihenstephan der Technischen Universitat Miinchen

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1 Zbl. Vet. Med. A, 23, Verlag Paul Parey, Berlin und Hamburg ISSN l/astm-coden: ZVRAAX Aus dem Institut fur Physiologie der Siidd. Versuchs- und Forschungsanstalt fur Milchwirtschaft Weihenstephan der Technischen Universitat Miinchen Testosterone, Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) in Peripheral Plasma of Bulls: Levels from Birth through Puberty and Short Term Variations H. KARG, T. GIM~NEZ, M. HARTL, B. HOFFMANN, E. SCHALLENBERGER and D. SCHAMS With 4 figures and one table (Received for publication Jaiznary 19, 1976) Introduction There is still considerable controversy concerning the hormonal control of sexual maturation in the bull, especially as far as possible interactions of LH and testosterone are concerned (RAWLINGS et al., 1972; ODELL et al., 1970; SWANSON et al., 1971; THIBIER, 1975). In order to further elucidate the hormonal changes and interrelationships occurring during puberty and sexual maturity in the bull, testosterone, LH and FSH were determined in plasma obtained by longitudinal sampling of individual animals. A preliminary report of this study has been reported elsewhere (GIM~NEZ et al., 1974). In order to especially delineate the short term fluctuations in testosterone and LH secretion (KATONGOLE et al., 1971) animals were serially sampled at a high frequency for short time periods. Material and Methods Animals and blood sampling All animals were males of the Brown Swiss breed kept under identical management conditions. In general in this breed sexual maturity occurs at the age of 8-10 months. In experiment I (longitudinal or puberty study), jugular vein plasma was collected from four bulls by venepuncture 3 x weekly between and from birth until the 18th month of age. The 4 bulls were born at different seasons (Nydo , Lado , Kabine , Kaliro ). In experiment I1 jugular vein plasma was obtained by means of an indwelling catheter at 15 minute intervals over a period Zbl. Vet. Med., Reihe A, Bd. 23, Heft 10 54

2 794 KARG, GIM~NEZ, HARTL, HOFFMANN, SCHALLENBERGER and SCHAMS of 8 hours from five males at 4 different ages, two animals representing each age group. In a further study, blood was collected in the same way from bull Gilch at the age of 14.5 months and a 34 month old bull Kaliro during a period of 48 and 32 hours, respectively. All catheters were inserted 1 day before the beginning of the experiment. Blood was cooled (4 C) immediately and centrifuged within one hour. Plasma samples were kept frozen at - 20 OC until assayed. Determination of testosterone Testosterone was analyzed by radioimmunoassay (RIA). The extraction (from 0.5 ml. plasma using 5 ml. ether) as well as the RIA were performed similarly to the method described by HOFPMANN and HAMBURGER (1973) for the determination of progesterone. The antiserum used was obtained after immunization of rabbits against testosterone-3-carboxy-methoxylamine-bovine serum albumin. With regard to specificity this antiserum showed a significant cross reaction (75 0) only with 5 a-dihydrotestosterone (DHT). However, no differences were observed between the testosterone values in peripheral plasma before and after separation of DHT by thin layer chromatography (P > 0,40, n = 8). The accuracy of the method was determined by measurement of added amounts of testosterone to charcoal-stripped calf plasma. Recovery values for these plasma samples containing 0.2 to 0.4 ng. gave a regression coefficient of 0.996, with a coefficient of variation (CV) for the individual samples ranging between 4.5 /o and 21.2 O/o, The reproducibility (interassay variability) was controlled by determining testosterone in various plasma pools during the whole experimental series, yielding a CV from 18.4 /o (0.24 I 0.04 ng. testosterone/ml. plasma, n = 8) to 5.6 /o (2.99 k 0.16 ng. testosterone/ml. plasma, n = 1 I). The lower limit of sensitivity was calculated to be ng. Determination of LH and FSH LH was measured by RIA as described earlier (SCHAMS and KARG, 1969; SCHAMS and KARG, 1970). A significant increase in LH (LH-peak) was considered to be a rise in LH measurable in at least two consecutive plasma samples (positive or negative slope) and exceeding the mean LH concentration plus the standard deviation, calculated for each period of uninterrupted blood collection for each individual animal. Bovine FSH was determined by a heterologous RIA (SCHAMS and SCHAL- LENBERGER, 1976). A highly purified sheep FSH preparation with a biological activity 31 times NIH-FSH-S 1 was used for iodination. An antiserum against ovine FSH was obtained after immunization of guinea pigs. This antiserum showed no cross reaction with bovine prolactin (NIH-P-B 2) or bovine growth hormone (NIH-GH-B 15) and only a slight cross reaction with bovine LH and TSH, which could be eliminated by the addition of bovine TSH and LH to each test tube during the assay. All results are expressed in terms of crude bovine pituitary extracts (Ambinon and NIH-FSH-B,). The biological activity of Ambinon is equivalent to times NIH-FSH-S,, and that of NIH- FSH-B, is equal to 0.49 times NIH-FSH-S,. The radioimmunological potency of NIH-FSH-B, was estimated to be 3.6 times higher than that of Ambinon. Results Experiment I Plasma levels for testosterone, LH and FSH (mean values I SD for each 3 week sampling period) are shown in Figs. 1 and 2. Absolute values and the

3 Testosterone, Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) 795 ng/ml NYDO 1 LAD WEEKS OF AGE l'l'l'l'l'l'l'l'i'i'l'l' Fig. 1. Concentrations of testosterone, LH and FSH (mean values? SD for each 3 week sampling period) in peripheral plasma in 2 bulls from birth through maturation secretion pattern of testosterone were similar in the bulls Nydo, Lado and Kabine. With individual differences, 4 periods of testosterone secretion became obvious in these 3 animals. As shown in Table 1 testosterone values were below ngml KALIRO looo/ 600 L WEEKS OF AGE I II' I ' I' I' I ' I I I' I ' I' I' I T Fig. 2. Concentration3 of testosterone, LH and FSH (mean values If: SD for each 3 week sampling period) in peripheral plasma in 2 bulls from birth through maturation 54'

4 796 KARG, GIMBNEZ, HARTL, HOFFMANN, SCHALLENBERGER and SCHAMS 1 ng./ml. plasma in period I, showed an initial increase in period I1 and remained constant or even decreased in period 111. In the IVth period a final increase in testosterone concentrations was observed. With p < the difference between the testosterone values in period I and I1 and period 111 and IV was highly significant. In the bull Kaliro testosterone coiicentrations were low until the 39th week of age. However, an initial increase (period 11) to 1.1 ng./ml. plasma was also observed in this animal from the 28th to the 30th week and a second one (period IV) after the 39th week of age. Table 1 Phas-s of appearance of testosterone in peripheral plasma of three bulls from birth through puberty (mean values f SD; ng./ml.) 1 Bull I Nydo I Lado I Kabine I 7 range of range of range of secretory (w:9, ks, testosterone conc. age testosterone conc. age testosterone conc. phase (weeks) (weeks] ng / rnl (SO1 ng / rnl (SD) ng / rnl (SO) I The LH concentrations were similar in all animals, with values ranging between 0.60 and 2.75 ng./ml. plasma during the experimental period. While values were rather constant with increasing age in bulls Nydo and Lado (regression equations y = x and y = x), a significant gradual decrease in LH was observed in bulls Kabine and Kaliro [regression equations: y = x (Pb < 0.005) and y = s (Pb < 0.005)]. No significant correlations between testosterone and LH in peripheral plasma were observed. In contrast to LH, FSH-concentrations were elevated in correlation (p < ) with the initial increase of testosterone during periods I and IV in bulls Nydo, Kabine and Kaliro, while they showed a wave-like trend throughout the experiment, with a tendency to flatten with increasing age in bull Lado. Experiment II The testosterone concentrations determined in the 4 and 8 week old calves were constantly low (mean: 0.28 f 0.20 ng./ml.; n = 127), and similarly in one of the 20 week old calves (mean: 0.39 zk 0.12 ng./ml.; n = 33). In the other 20 week old animal and in both cases at the age of 40 weeks, testosterone values showed almost identical daily fluctuations with values above 6 ng./ml. plasma occurring between h. and h. and again between h. and h. No changes in the LH-levels in relation to age were observed. Significant increases in LH (LH-peaks), as defined above, occurred at all age-periods and in any case preceded the observed afternoon increase in testosterone in the 20 and 40 week old animals. Mean FSH levels in plasma were significantly higher (P< ) in the 20 and 40 week old bulls (346 f 64 ng./ml.; n = 132) than in the 4 and 8 week old calves (214 F 55 ng./ml.; n = 132). In fig. 3 an example for each age group is given. The testosterone and LH levels determined in bull Gilch at the age of 14 months and in the 34 months old bull Kaliro, during a period of 48 and 32 hrs.: respectively, are shown in Fig. 4. While the profile in bull

5 Testosterone, Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) 797 WEEKS OF AGE nplml I I2 16 TIME OF DAY t HOUR? ) Fig. 3. Changes in testosterone-, LH- and FSH-concentrations durin a period of 8 hrs. in peripheral plasma of bull calves at the age of 4, 9, 20 anc! 40 weeks Kaliro was rather constant and almost diurnal, a more irregular pattern was observed in bull Gilch. The mean LH levels in plasma calculated for Gilch and Kaliro were 1.01 k 0.21 ng./ml. (n = 193) and 0.85 k 0.37 ng./ml. (n = 126) plasma, respectively. In both animals each LH-peak (range of peak-levels 1.3 to 2.6 ng./ml.) was followed by a rise of testosterone within 1 hour. However, similarly distinct increases of testosterone were also observed in 5 instances (from 1.4 to 13.2; 5.2 to 11.3; 4.0 to 10.0; 1.0 to 3.1 and 1.2 to 4.4 ng./ml.) where, by the definition suggested, no preceding significant increase in LH could be detected. During the experiment FSH levels were very constant around 276 (* 29) ng./ml. plasma (n = 126) in bull Kaliro and showed irregular fluctuations in bull Gilch, especially during nglml 8 L KALIRO 1 ogc: 34 months ) 18 1.a ( oge:14,5monthsl 8 L I , I I I IS Time of doy ( hours I Fig. 4. Changes in testosterone- and LH-concentrations during a period of 32 and 48 hrs., respectively, in peripheral plasma in bulls Kaliro and Gilch

6 798 KARG, GIMENEZ, HARTL, HOFPMANN, SCHALLENBERGER and SCHAMS the first 15 hrs. of the experimental period (mean: 195 k 180 ng./ml.; n = 193). No significant correlations could be established between the levels of FSH and those of testosterone or LH in the whole course of experiment 11. Discussion The results derived from the longitudinal study which concern basic shifts in hormone secretion have clearly shown that from birth until the end of puberty basal levels of LH remained rather constant or even decreased in peripheral plasma, while testosterone, in agreement with observations by LINDNER (1959) and RAWLINGS et al. (1972), showed an initial increase beginnung at the 20th week of age. The results suggest that this increase occurs in a biphasic pattern in relation to age; due to the different times of birth, seasonal effects can be ruled out. Evidence that various processes during the course of puberty in the bull seem to occur in a biphasic pattern were first obtained by MACMILLAN and HAFS (1969), who described a similar phenomenon for fructose concentrations in seminal plasma. Testosterone concentrations in blood plasma and testicular tissue obtained from the same bulls (RAWLINGS et al., 1972) also indicated a biphasic testosterone secretion but this observation was not further discussed at that time. The occurrence of a biphasic increase of testosterone in peripheral plasma seems not to be unique for puberty in the bull, since a similar phenomenon has also been observed in the roe deer (Capreolus capreolus) during sexual maturation (puberty) as well as during the reproductive cycle in the mature animal (GIMENEZ et al., 1975; BARTH et al., 1976), similarly in the noctule bat (Nyctalus noctula) (RACEY, 1974). Contrary to observations in the ram (COTTA et al., 1975) and in the bull (MACMILLAN and HAFS, 1968; (RAWLINGS et al., 1972; GOMBE et al., 1973) where an increase of LH in plasma with increasing age was observed, no changes (2 bulls) or a decrease (2 bulls) in LH concentrations was obserrved in the 4 animals examined from birth until the 18th month of age as has been already partially reported elsewhere (SCHAMS and BUTZ, 1972). This is in agreement with earlier observations in cattle (ODELL et al., 1970) and the pig (ELSAESSER et al., 1973). Since it was concluded from castration experiments in sheep and rats (CRIM and GESCHWIND, 1972; GALLOWAY and PELLETIER, 1975; GAY and MIDGLEY, 1969) and from experiments substituting LH and FSH after hypophysectomy (COUROT, 1971) that LH and not FSH is the major hormone responsible for Leydig cell function, the observation described is rather intriguing with respect to the onset of testosterone increase at the beginning of puberty. As far as can be deduced from peripheral plasma levels, the rather constant LH concentrations would suggest that the testosterone increase before puberty cannot be explained by the common desensitization theory (MCCANN and RAMIKEZ, 1964), but rather by a changed sensitivity of the receptor sites in the testes to LH. This theory is also supported by our findings in experiment TI, where episodic variations were investigated. In the 4, 8 and one of the 20 week old animals, plasma testosterone showed no response to the observed changes in plasma LH, while in the other animals examined plasma testosterone responded with an increase to similar rises in LH (see fig. 3). Even though there seems to be a statistically valid relationship during sexual maturation between testosterone and FSH in 3 of the 4 bulls examined in Expt. I, a more direct biologic role of FSH may occur in other processes, like the synthesis of androgen-binding protein which is produced in the Sertoli cells under the specific stimulation of FSH (HANSSON et al., 1973 a; HANSSON et al., 1973 b; HANSSON et al., 1974). However, another interesting specula-

7 Testosterone, Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) 799 tion concerning the role of FSH at the time of puberty was presented by ODELL et a]. (1973) from experiments in rats where they concluded that FSH induces the sensitivity to LH in the testes which would explain the occurrence of puberty without a necessary increase of LH or an alteration in feedback sensitivity. Our data in the longitudinal study, as well as in experiment 11, where significantly higher FSH-levels were found in the 20 and 40 week old animals, would support this theory. WUTTKE and DOHLER (1975) have presented evidence for a functional role of prolactin in the onset of puberty in the rat. Similar conclusions cannot be derived for the bovine species since substantial variations of plasma prolaction levels occur in relation to the season of year (KARG and SCHAMS, 1970; SCHAMS and REINHARDT, 1974) and would tend to mask any such relationship. The prolactin data evaluated in the identical plasma samples of the present experiment were published earlier (SCHAMS and REINHARDT, 1974). The existence of short term fluctuations in testosterone is well known and has also been shown to exist in the bovine species (KATONGOLE et al., 1971; MONGKONPUNYA et al., 1973; SANWAL, 1974; HAYNES et al., 1975). Our investigations have confirmed these observations and further demonstrated that these fluctuations, which seem to occur rather regularly (with the exception of bull Gilch at the age of 14 months), can be measured as early as at the onset of puberty. Similarly, but less distinctly expressed than was reported by KATONGOLE et al. (1971), also in these experiments a positive action of LH with respect to the stimulation of short term testosterone fluctuations was observed. No correlations of biological significance could be established for the short term variations in FSH concentrations. Acknowledgements We are most grateful to the following for their kind gifts: National Institutes of Health, Bethesda, USA for NIH preparation of bovine GH, prolactin, LH, FSH and TSH, and sheep FSH. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn Bad Godesberg. Summary Peripheral plasma levels of testosterone, LH and FSH were determined three times weekly by RIA in 4 bulls from birth through maturation and at 15 min. intervals in 6 males during short periods (8-48 hrs) at different ages (4 weeks-34 months). After an initial period of values below 1 ng./ml. plasma, the subsequent rise in plasma testosterone was characterized by a biphasic pattern. In 3 animals a first increase occurred after the 2Oth, 22nd or 28th week of age (mean values ng./ml.), followed by a plateau or decrease. A second increase, occurred after the 39th, 51st and 54th week of age, respectively (mean values: ng./ml.). In the 4th bull testosterone values exhibited a similar but delayed pattern and were generally lower during the whole trial. LH-levels remained constant in all animals between ng./ml. during the experiment without any tendency to increase with age. FSH increased coincidentally with testosterone in 3 of the bulls, but showed a wave-like trend in the other bull. No changes in LH- and FSHshort term variations were found in relation to age. Testosterone values remained constantly below 1 ng./ml. in the 4 and 8 week old animals and started to exhibit short term variations after 20 weeks of age with a rather constant

8 800 KARG, GIM~NEZ, HARTL, HOFFMANN, SCHALLENBERGER and SCHAMS tendency of high values around 09.00, and h. An LH-peak preceded the observed increases in testosterone in 16 out of 21 observations. Zusammenfassung Testosteron, Luteinisierungshormon (LH) und Follikelstimulierendes Hormon (FSH) im peripheren Plasma von Bullen: Werte von der Geburt bis nach der Pubertat und Kurzzeitvariationen Testosteron, LH und FSH wurden mit Hilfe radioimmunologischer Methoden im peripheren Plasma von 4 Bullen vom Zeitpunkt der Geburt bis zur vollendeten Geschlechtsreife gemessen (Experiment I) und in 15minutigem Abstand bei weiteren 6 Tieren im Alter von 4 Wochen bis 34 Monaten uber einen Zeitraum von 8-48 Stunden (Experiment 11). Experiment I: Nach einer ersten Periode von Testosteronkonzentrationen unter 1 ng./ml. Plasma (Periode I) verlief der darauf folgende Anstieg in 2 Phasen. Bei 3 Tieren wurde ein erster Anstieg (Periode 11) nach der 20., 22., bzw. 28. Lebenswoche (Durchschnittswerte 2,2-8,4 ng/ml) und danach ein Plateau oder Abfall (Periode 111) beobachtet. Dem folgte ein 2. Anstieg (Periode IV) nach der 39., 51. bzw. 54. Lebetiswoche (Durchschnittswerte 1,9-11,9 ng/ml). Beim 4. Bullen zeigte sich ein ahnliches jedoch verzogertes Sekretionsmuster mit durchschnittlich niedrigeren Testosteronkonzentrationen. Die LH-Werte lagen bei allen Tieren konstant zwischen 0,6-2,7 ng/ml und zeigten keine Tendenzen, mit zunehmendem Alter anzusteigen. FSH stieg parallel zum Testosteronanstieg bei 3 der untersuchten Tiere an und zeigte einen nicht korrelierbaren wellenformigen Verlauf bei dem 4. Bullen. Experiment 11: Die episodischen LH- und FSH-Schwankungen wiesen keine Beziehungen zum Alter der Tiere auf. Testosteron lag konstant unter 1 ng/ml Plasma bei den 4 und 8 Wochen alten Tieren; mit dem Alter von 20 Wochen begannen die Konzentrationen im Verlauf eines Tages im peripheren Plasma zu schwanken, wobei - mit der Ausnahme einer Untersuchungsperiode - die Tendenz zu hohen Werten um 0900, 1600 und 2200 erkennbar war. Ein LH-Peak ging in 16 aus 21 Beobachtungen dem Testosteronanstieg voraus. Resume Testostirone, hormone lutiinisante (LH) et hormone folliculostimulante FSH dans le plasma piriphbrique chez des taureaux: taux de la naissance A la puberte et variations pulsatiles On a mesurk la testostkrone, LH et FSH avec des mkthodes radioimmunologiques dans le plasma pkriphkrique de 4 taureaux depuis la naissance B la maturitk sexuelle (experience I) et chez 6 animaux hgks de 4 semaines A 34 mois B des intervalles de 15 minutes durant une pkriode de 8-48 heures (expkrience 11). Expkrience I: Aprks une pkriode de concentration en testostkrone infkrieure A 1 ng/ml plasma (pkriode I), on a constatk une augmentation en 2 phases. Une premiere augmentation a ktk constatke chez 3 animaux aprks 20, 22 et 28 semaines (valeurs moyennes 2,2-8,4 ng/ml) (pkriode 11). Un plateau ou une chute s en suivit (pkriode 111). Une seconde augmentation se manifesta apr& 39, 51 et 54 semaines (valeurs moyennes 1,9-11,9 ng/ml) (pkriode IV). On a obtenu un kchantillonnage semblable mais plus rapide avec des valeurs moyennes de testostkrone plus basses chez le quatrieme taureau. Les valeurs de LH resterent constantes entre 0,6-2,7 ng/ml chez tous les

9 Testosterone, Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) 801 animaux et n'ont pas augment6 avec 1'9ge. FSH augmenta parallklement 2i la testostkrone chez 3 animaux alors qu'il n'y a pas eu de corrklation chez le quatrikme taureau. Expkrience 11: Les variations kpisodiques de LH et FSH n'ont pas eu de rapport avec 1'9ge des animaux. La testostkrone demeura constamment endessous de 1 ng/ml plasma chez les animaux de 4-8 semaines. Les concentrations commenckrent A varier dks la vingtikme semaine au cours d'un jour dans le plasma pkriphkrique et, A l'exception d'une pkriode d'analyse, la tendance 2i des valeurs klevkes fut remarquke 0900, 1600 et Une pointe LH prkckda l'augmentation de la testostkrone dans 16 des 21 observations. Resumen Testosterona, hormona luteinizante (HL) y hormona estimulante del foliculo (FSH) en plama sanguineo perifbrico de toros: niveles desde el nacimiento hasta el final de la pubertad y variaciones a plazo breve Se midieron la testosterona, HL y FSH con ayuda de mktodos radioinmunol6gicos en el plasma sanguineo perifkrico de 4 toros desde el instante del nacimiento hasta consumada la pubertad (experimento I) y en distancias de 15 minutos en otros 6 animales, de edadas comprendidas entre 4 semanas y 34 meses, por un espacio de tiempo de 8-48 horas (experimento 11). Experimento I: Tras un primer periodo de concentraciones de testosterona inferiores a 1 ng/ml plasma (periodo I), el aumento subsiguiente discurri6 en 2 fases. En 3 animales se observ6 un aumento primer0 (periodo 11) tras la semana 20", 22" resp. 28" (valores medios 2,2-8,4 ng/ml) y despuks una meseta o descenso (periodo 111). Siguib a esto un aumento 2" (periodo IV) tras la semana 39", 51" resp. 54" (valores medios 1,9-11,9 ng/ml). Se apreci6 en el tor0 4" un modelo secretor semejante, aunque retardado con concentraciones medias inferiores de testosterona. Los valores de HL se hallaban constantes en todos 10s animals entre 0,6-2,7 ng/ml, no mostrando tendencia alguna a aumentar con la edad. FSH aument6 paralela a1 increment0 de testosterona en 3 de 10s animales examinados, evidenciando un curso ondulante no correlacionable en el tor0 4". Experimento 11: Las fluctuaciones epis6dicas de HL y FSH no evidenciaron ninguna relaci6n para con la edad de 10s animales. La testosterona se hallaba constante, siempre inferior a 1 ng/ml de plasma en 10s animales de 4 y 8 semanas de edad; con la edad de 20 semanas comenzaron a oscilar 10s valores en el curso de un dia en el plasma perifkrico, reconocikndose - con la excepcibn de un periodo experimental - la tendencia a elevados alrededor de 0900, 1600 y Una chspide de HL precedi6 en 16 de 21 observaciones a1 aumento de testosterona. References BARTH, D., T. GIMENFZ, B. HOFFMANN und H. KARG, 1976: Saisonale und rhythmische Veranderungen der Testosteronkonzentrationen im peripheren Blur beim Rehbock (Capreolus capreolus); Beziehungen zu einigen Verhaltensweisen und anderen saisonabhangigen Parametern. Jagdwissenschaft 22, 65. COTTA, Y., M. TERQUI, J. PELLETIER et M. COUROT, 1975: Testosterone et LH plasmatiques chez l'agneau de la naissance la pubertk. C. R. Acad. Sc. Paris, SCrie D 280, COUROT, M., 1971: An experimental study of the endocrine control of the establishment of spermatogenesis in the lamb Ovis aries. Importance of cells of the Sertoli line. D. Sc. Thesis, Paris. CUM, L. W., and I. I. GESCHWIND, 1972: Patterns of FSH and LH secretion in the developing ram; the influence of castration and replacement therapy with testosteronepropionate. Biol. Reprod. 7,

10 802 KARG, GIMENEZ, HARTL, HOFFMANN, SCHALLENBERGER and SCHAMS ~~SAESSER, F., D. K. POMERANTZ, F. ELLENDORFF, K. KREIKENBAUM and A. KONIG, 1973: Plasma LH, testosterone and DHT in the pig from birth to sexual maturity. Acta endocr. (Kbh.) Suppl. 173, 148. GALLOWAY, D. B., and J. PELLETIER, 1975: Luteinizing hormone release in entire and castrated rams following injection of synthetic luteinizing hormone releasing hormone, and effect of testosterone propionate pre-treatment. J. Endocr. 64, GAY, V. L., and A. R. MIDGLEY, 1969: Response of the adult rat to orchidectomy and ovariectomy as determined by LH radioimmunoassay. Endocrinology 84, GIMINEZ, T., B. HOFFMANN, D. SCHAMS, M. HARTL, E. SCHALLENBERGER and H. I<AI<G, 1974: Plasma levels of testosterone in relation to LH and FSH in bulls from birth through puberty. 4th Int. Congr. Hormonal Steroids Mexico City, September 2-7 J. Steroid Uiochem. 5, GIMENEZ, T., D. BARTH, B. HOFFMANN and H. KARG, 1975: Blood levels of testosterone in the roe deer Capreolus capreolus in relationship to the season. Acta endocr. (Kbh.) Suppl. 193, 59. GOMBE, S., W. C. HALL, K. MCENTEE, W. HANSEL and B. W. PICKETT, 1973: Regulation of blood levels of LH in bulls: influence of age, breed, sexual stimulation and temporal fluctuations. J. Reprod. Fert. 35, thnsson, V., F. S. FRENCH, S. C. WEDDINGTON, W. S. MCLEAN, D. J. TINDALL, A. A. SMITH and S. N. NAYFEH, 1973 a: Androgen transport mechanisms in the testis and epipdidymis. International Research Communications System (73-1 1) HANSON, V., E. KEUSCH, 0. TRYGSTAD and 0. TORGERSEN, 1973 b: FSH stimulation of testicular androgen binding protein. Nature, New Biology 246, HANNSSON, V., 0. TRYGSTAD, F. S. FRENCH, W. S. MCLEAN, A. A. SMITH, D. J. 'rindall, S. C. WLDIIINCTON, P. PETRUSZ, S. N. NAYFEH and E. M. RITZEN, 1974: Androgen transport and receptor mechanisms in testis and epidiilymis. Nature, Lond. 250, HAYNIS, N. B., H. D. HAFS, R. J. WATERS, J. G. MANNS and A. RILEY, 1975: Stimulatory effect of prostaglandin F,a on the plasma concentration of testosterone in bulls. J. Endocr. 66, IJOPFMANN, B., und R. HAMIIURGER, 1973: Progesteron in der Milch: radioimmunologische Uestimniung, Bcziehungen zur Gelbkorperfunktion und Milchfettkonzentration. Zuchthyg. 8, KARG, H., and D. SCHAMS, 1970: Prolactin levels in bovine blood under different physiological conditions. In: Lactation, Ed. J. R. Falconer, Butterworths London, KATONGOLE, C. B., F. NAFTOLIN and R. V. SHORT, 1971 : Relationship between blood levels of luteinizing hormone and testosterone in bulls, and the effects of scxual stimulation. 1. Endocr. 50, LINDNER, H. R., 1959: Androgens in the bovine testis and spermatic vein blood. Nature Lond. 283, MACMILLAN, K. L., and H. D. HAFS, 1968: Pituitary and hypothalamic endocrine changes associated with reproductive development of Holstein bulls. J. Anim. Sci. 27, MACMILLAN, K. L., and H. D. HAFS, 1969: Reproductive tract of Holstein bulls from birth through puberty. J. Anim. Sci. 28, MCCANN, S. M., and V. D. RAMIREZ, 1964: The neuroendocrine regulation of hypophyseal luteinizing hormone secretion. Rcc. Prog. Horm. Res. 20, MONCKONPUNYA, K., H. D. HAFS, E. M. CONVEY and W. D. OXENDER, 1973: Blood serum testosterone after sexual preparation or ejaculation, or after injections of LH or prolactin in bulls. J. Anim. Sci. 37, MONCKONPUNYA, K., H. D. HAFS, E. M. CONVEY, H. A. TUCKER and W. D. OXENDER, 1975: Serum luteinizing hormone, testosterone and androstenedione in pubertal and prepubertal bulls after gonadotropin releasing hormone. J. Anim. Sci. 40, OI~ELL, W. D., M. A. HESCOX and C. A. KIDDY, 1970: Studies of hypothalamic-pituitarygonadal interrelations in prepubertal cattle. In: Gonadotrophins and Ovarian Ilevelopment, Ed. Butt, W. R., Crooke, A. C. and Ryle, M. E. & S. Livingstone, Edinburgh & London, pp ODELL, W. D., R. S. SWERDLOFF, H. S. JACOBS and M. A. HESCOX, 1973: FSH induction of sensitivity to LH. One cause of sexual maturation in the male rat. Endocrinology 92, I~ACEY, P. A., 1974: The reproductive cycle in the male noctulc bat, Nyctalus noctulu. J. Reprod. Fert. 41, RAWLINGS, N. C., H. D. HAFS and L. V. SWANSON, 1972: Testicular and blood plasma androgens in Holstein bulls from birth through puberty. J. Anim. Sci. 34, SANWAL, P. C., A. SUNNUBY and L.-E. EDQUIST, 1974: Diurnal variation of peripheral plasma levels of testosterone in bulls measured by a rapid radioimmunoassay procedure. Acta Vet. Scand. 15,

11 Testosterone, Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) 803 SCIIAMS, D., and H. D. BUTZ, 1972: Plasma levels of luteinizing hormone in female and male calves from birth until puberty. 7th Int. Congr. Anim. Reprod. & A. I. Munich, vol. 3, SCHAMS, D., und H. KARG, 1969: Radioimmunologische LH-Bestimmung im Blutserum vom Rind unter besonderer Beriidtsichtigung des Brunstzyklus. Acta Endocr. (Kbh.) 61, SCHAMS, D., und H. KARG, 1970: Studien iiber die SpezifitBt des Radioimmunotests zur Bestimmung des Luteinisierungshormons im Rinderblut. Hoppe-Seyler's Z. Physiol. Chem. 311, SCHAMS. D.. and V. REINHARDT. 1974: Influence of season on plasma prolactin level in cattle from birth to maturity. Horm. Res. 1, SCHAMS, D., and E. SCHALLENBERGER, 1976: Heterologous radioimmunoassay for bovine follicle-stimulating hormone and its application during the estrous cycle in cattle. Acta endocr. (Kbh.) 81, SWANSON, L. V., R. P. WETTEMANN, N. C. RAWLINGS, H. D. HAFS and W. T. MAGEE, 1971: Pubertal relationships of some endocrine and reproductive criteria in Hereford bulls. J. Anim. Sci. 33, THIBIER. M : Peripheral plasma testosterone concentrations in bulls around puberty. J.'Reprod. Fert. 42; 567-i69. WUTTKE, W., K. D. DOHLER and M. GELATO, 1976: Oestrogens and prolactin as possible regulators of puberty. J. Endocr. 68, Address of authors: Siidd. Versuchs- u. Forschungsanstalt fur Milchwirtschaft, Institut fur Physiologie, 8050 Freising-Weihenstephan.