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R. P. AMANN, H. H. KOEFOED-JOHNSEN, and H. LEVI

Summary.

Sixteen rabbits were injected with thymidine-3H and tissue sections or spermatozoa smears were evaluated by autoradiography. Two successive ejaculates were collected every second day. Analyses of labelled germ cells in twenty-four testes taken 1 hr to 32 days postinjection showed that one cycle of the seminiferous epithelium required 10·7 ± 0·5 days and that labelled spermatozoa should enter the caput epididymidis 31·4 days post-injection. Labelled spermatozoa were found in the proximal portion of four capita epididymidum 32 days and one caput 33 days post-injection, but not in eleven other capita epididymidum 28 to 31 days post-injection. Labelled spermatozoa first were detected in ejaculated semen from five rabbits on Days 38, 39, 41, 42 and 42. The mean number of grains/spermatozoon in autoradiograms of successive samples agreed closely with the scintillation count activity curve. However, neither curve represented the excretion pattern of spermatozoa labelled as preleptotene spermatocytes. The first samples containing labelled spermatozoa had a low percentage of highly labelled spermatozoa while, through mixing with spermatozoa labelled as spermatogonia, subsequent ejaculates contained up to 55% labelled spermatozoa but individual cell activity was lower. Labelled spermatozoa were excreted over a period of 2 to 3 weeks. Epididymal spermatozoa transit time averaged 9·0 days, but it ranged from 6·6 to 10·6 days even with a uniform semen collection schedule. Combined with autoradio--graphic evaluation of tissue sections or semen smears, intravenous injection of 0·1 to 0·3 mc/kg body weight of thymidine-3H should be adequate for experiments of this type.

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R. P. AMANN, J. F. HOKANSON, and J. O. ALMQUIST

Summary.

A cannula and collection system are described which permit continuous quantitative recovery of the sperm effluent from the proximal ductus deferens without contamination by accessory gland fluids. In addition to concentric polyethylene cannulae, it features a 90° copper elbow soldered at one end to a copper flange which is sutured to the posterior neck of the scrotum. The cannulae are directed through the elbow into an easily removable collection vial. In the first two bulls unilaterally cannulated, daily samples of about 2·8 × 109 spermatozoa were obtained via the cannula for more than 3 and 9 weeks.

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R. P. AMANN, G. J. KILLIAN, and A. W. BENTON

Summary.

Polyacrylamide gel electrophoresis was used to evaluate rete testis fluid (RTF) and plasma from the cauda epididymidis (CEP) obtained from conscious Holstein bulls. The major protein in RTF had an electrophoretic mobility similar to that of blood serum albumin, but the seemingly analogous protein in CEP had a slower relative mobility. Bovine CEP contains at least three proteins, one glycoprotein and possibly three other PAS-positive components not detected in RTF or blood serum. In addition, esterases, acid phosphatases and several β-glucuronidases have been detected in CEP which were absent in RTF. Agarose immunoelectrophoretic analyses confirmed the basic differences among the three fluids. Although RTF and CEP share certain proteins with blood serum, each fluid is biologically unique. Certain proteins detected in CEP but not in RTF or blood serum probably originated from the testicular spermatozoa while others may represent epididymal secretion.

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J. Naden, R. P. Amann, and E. L. Squires

Summary. Puberty was studied using 15 colts of Quarter Horse phenotype. Total scrotal width was measured every 8 weeks from 48 to 96 weeks. Blood samples were taken from 8 colts at 8, 16 and 24 weeks and then every 4 weeks until 100 weeks to measure changes in LH, FSH and testosterone concentrations. Seminal collections were attempted monthly from 48 to 64 weeks and every 2 weeks thereafter until puberty, and resumed every 3rd day from 96 weeks for 15 ejaculates. For all collections, times to erection, mount and ejaculation and seminal characteristics were recorded. Age at puberty was defined as the first ejaculate containing 50 × 106 spermatozoa, with ≥ 10% motile. Colts were castrated at 2 years to enable determination of daily sperm production (DSP), epididymal sperm reserves and normality of spermatogenesis. Total scrotal width increased linearly from 48 to 96 weeks. Age at puberty averaged 83 weeks (56–97 weeks). Changes in serum concentrations of LH and FSH were parallel, rising at 36–40 weeks, declining after 40 weeks and rising again at 68–80 weeks. Testosterone was low until 68 weeks after which concentrations rose slowly to 80 weeks and increased rapidly to a plateau at 92 weeks. Sexual behaviour and seminal characteristics differed (P < 0·05) between puberty and 2 years, except for time to erection, time to mount, and percentage of motile spermatozoa. DSP at 2 years averaged 1·7 × 109 and daily sperm output (DSO) averaged 1·1 × 109. The correlation between DSP and DSO was 0·83 (P < 0·01). There were 9·57 × 109 spermatozoa/epididymis of which 67% were in the cauda.

Keywords: puberty; hormonal changes; stallions; testes; semen; sexual behaviour

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G. W. Robb, R. P. Amann, and G. J. Killian

Summary. Rats were considered to be pubertal at 50 days of age when spermatozoa were first found in the tail of the epididymis. Sperm production/g testis increased up to 75 days of age and testicular weight increased until 100 days of age. Sperm reserves in the tail of the epididymis were not maximal until 100 days of age. Therefore, Wistar rats are not sexually mature until 100 days. Sexually mature rats had testes weighing 3·7 g, produced 86 × 106 spermatozoa or 24 × 106 spermatozoa/g testicular parenchyma daily, and their paired epididymides contained 295 × 106 spermatozoa in the head + body and 440 × 106 spermatozoa in the tail.

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D. P. Froman, R. P. Amann, P. M. Riek, and T. T. Olar

Summary. Eight experiments were performed to validate an extraction technique for canine acrosin and to quantitate the acrosin activity of fresh and frozen–thawed spermatozoa. Acrosin activity from fresh spermatozoa differed amongst dogs and was influenced by the interval since previous ejaculation. Freezing and thawing spermatozoa induced a loss of acrosin activity that differed with the extender in which the spermatozoa were frozen. The assay of acrosin activity, in conjunction with motility estimates, provides a more complete evaluation of the efficacy of seminal extenders in attenuating freezing injury than do motility estimates alone.

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J. K. VOGLMAYR, J. F. KAVANAUGH, L. C. GRIEL Jr, and R. P. AMANN

The development of a technique for cannulating the rete testis permitted continuous collection of spermatozoa and fluid from the testis of conscious rams for up to 5 months (Voglmayr, Scott, Setchell & Waites, 1967; Voglmayr, 1970). When this technique was employed on bulls, the flow of rete testis fluid began to decline 4 to 5 days after surgery and ceased by Day 11 (Voglmayr, Larsen & White, 1970). Histological examination revealed that fibrous tissue had blocked the orifice of the catheter (J. K. Voglmayr, unpublished observations). Early cessation of flow was unusual in rams even if several attempts were required to insert the flexible catheter into the rete testis (J. K. Voglmayr, unpublished observations). This paper describes a modified

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J. Naden, E. L. Squires, T. M. Nett, and R. P. Amann

Summary. The pituitary response to exogenous GnRH was studied in 8 colts of Quarter Horse phenotype from 32 to 96 weeks of age. Colts were from dams treated daily from Day 20 to 325 of gestation with (1) 2 ml neobee oil per 50 kg body weight (controls); or (2) 2 ml altrenogest per 50 kg body weight. GnRH challenges (5 μg/kg body weight) were administered every 8 weeks from 32 to 96 weeks of age to estimate pituitary content of LH. Blood samples were collected every 20 min for 4 h before GnRH and 15, 30, 45, 60, 90, 120, 180, 240 and 360 min after GnRH. Serum concentrations of LH and FSH were determined for the 2 pre-GnRH and all post-GnRH samples. Baseline concentrations (mean of 2 pre-GnRH samples) of LH and FSH were not affected by treatment (P > 0·05). Serum concentrations of LH declined from 40 to 56 weeks and rose again between 72 and 80 weeks. Basal concentrations of FSH declined from 32 to 56 weeks, and varied widely after 56 weeks. The maximum LH response to GnRH (highest concentration after GnRH minus baseline) declined steadily in both groups for 48 to 64 weeks but remained relatively constant in both groups after 64 weeks. The maximum FSH response to GnRH declined from 32 to 64 weeks then remained relatively constant in both groups. The GnRH-induced gonadotrophin release remained low with a transient increase at 72 weeks for both hormones. There was no treatment effect (P > 0·05) on time to maximum gonadotrophin release after GnRH, and the time to maximum release varied widely throughout the study. Changes in basal concentrations of LH were associated with changes in LH release after GnRH; whereas basal concentrations of FSH were associated with changes in FSH release only from 32 to 56 weeks of age. The prepubertal changes in secretion of LH in stallions may therefore be associated with a reduction and restoration of the ability of the pituitary to secrete LH as assessed by discharge following exogenous GnRH. However, this relationship does not hold true for FSH after 56 weeks of age.

Keywords: puberty; stallion; exogenous GnRH; LH; FSH

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D N R Veeramachaneni, J S Palmer, R P Amann, C M Kane, T T Higuchi, and K-Y F Pau

We studied sequelae of prenatal plus infantile exposure of male rabbits to vinclozolin, because it is ingested by women and children. Female Dutch-Belted rabbits (7–10/group) were treated daily per orum from gestation day 15 through post-natal week 4 to provide 0, 7.2, or 72 mg vinclozolin/kg dam’s body weight/day. Vinclozolin had no effect on maintenance of pregnancy, growth of pups, age at testicular descent or weight of organs. Concentrations of serum LH or testosterone at 6, 12, or 24 weeks of age were unaffected. However, FSH was lower (P < 0.05) in both vinclozolin groups at all three ages. Following injection of GnRH at 12 or 24 weeks, the increase in FSH was less (P < 0.05) in both vinclozolin groups, as was testosterone at 12 weeks of age. After full sexual maturity, 2 of 7 low dose rabbits were uninterested in female or male teasers and never achieved erection or ejaculation. Overall, rates of ejaculation failure were: control 0% (0/48), low dose 29% (12/42), and high dose 5% (3/60). Daily sperm production per gram of testis and total number of sperm per ejaculate in both vinclozolin groups were similar (P > 0.1) to controls. However, semen from vinclozolin rabbits contained over two times more (P < 0.05) morphologically abnormal spermatozoa, mostly nuclear and acrosomal defects, than semen from controls. Seminiferous tubules with degenerative changes were more frequent (P < 0.05) in vinclozolin rabbits than in controls. Lesions included syncytia of spherical spermatids and desquamation of germ cells. Hence, developmental exposure to vinclozolin caused presumably permanent changes in copulatory ability, secretion of FSH, and spermiogenesis.