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A. G. HUNTER and H. D. HAFS

Summary.

Bombardment of bovine spermatozoa with glass beads and subsequent extraction in phosphate buffered sodium chloride yielded soluble sperm-specific proteins (approximately 10% yield) sufficient for physicochemical analysis.

Moving boundary electrophoresis revealed three sperm-specific components with mobilities of 2·0, 3·8 and 5·1×10-5 cm2 volt-1 sec-1 at pH 8·6 in barbital (μ = 0·10). The proportions of these components were 20, 50 and 30%, respectively. Sedimentation velocity ultracentrifugal analysis revealed at least three sedimentation gradients. The two major gradients had S20 values of 1·7 and 12·6. The third exhibited polydispersion. Diffusion coefficients of 4·2 and 10·2×10-7 cm2 sec-1 were calculated for two sperm-specific antigens by an agar-gel-diffusion method.

Three protein fractions were isolated by chromatographic elution from diethylaminoethyl (deae) cellulose with an ionic gradient. The first protein fraction eluted was not bound to the deae cellulose. It migrated toward the cathode in agar-gel electrophoresis at pH 8·4, and was firmly bound to carboxymethylcellulose at pH 6·0, 7·6 and 11·0. A second protein fraction from the deae cellulose appeared just after the ionic gradient commenced, and a third protein fraction appeared when the ionic gradient approached 0·2 m-sodium chloride.

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A. G. HUNTER and H. D. HAFS

Summary.

The antigenicity and cross-reactions of bovine seminal constituents were studied using complement fixation, agar-gel-diffusion, and immuno-electrophoresis. Bovine ejaculated spermatozoa, seminal plasma, and blood serum contained common antigens such that antisera produced with one also reacted with the other two. Spermatozoa per se were antigenic when injected intradermally with Freund's adjuvant into rabbits. In addition, ejaculated spermatozoa were coated with seminal plasma proteins. At least some of these coating antigens were found on spermatozoa from the vas deferens and must therefore have been deposited before the contributions of the major accessory sex glands came in contact with the spermatozoa. Antisera prepared against epididymal spermatozoa reacted with epididymal and ejaculated spermatozoa but not with seminal plasma or blood serum.

Ejaculated spermatozoa possessed at least seven antigens. At least five were shared with seminal plasma and at least one with blood serum. Absorption of anti-sperm immune sera with seminal plasma and blood serum revealed that epididymal and ejaculated spermatozoa shared at least three protein antigens which were not found in seminal plasma or blood serum.

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K. R. STEVENS, H. D. HAFS and K. T. KIRTON

Summary.

The pH, protein concentration, and amount of oestrous uterine fluid were determined in mature Dutch type rabbits from 1 to 20 weeks after ligation of the uteri. Uterine fluid accumulated at the rate of 2·10 to 4·48 ml per cornu per week. These fluids were serous, slightly turbid, and colourless or slightly yellow. The protein concentration decreased from 5·13 mg/ml after the first week to 1·82 mg/ml after the 20th week of accumulation. The high initial protein concentration may have been due to contamination of the uterine fluid with blood at the time the uterus was ligated. The in-utero pH of fluids was 7·64. A rapid increase in pH with time and handling after removal from the uterus indicated the importance of carbon dioxide in buffering uterine fluids.

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CLAUDE DESJARDINS, K. T. KIRTON and H. D. HAFS

Summary.

Bovine follicular fluid was compared with blood serum or blood plasma with respect to the concentration of protein and free amino nitrogen, the number of antigens and the number and mobilities of electrophoretic components. The protein concentration of follicular fluid (7·08 g/100 ml) was less than that of blood serum (9·10 g/100 ml), but no significant difference existed between the values of free amino nitrogen in the two fluids (4·31 and 3·97 mg/100 ml, respectively).

Rabbit antisera to follicular fluid, blood serum or blood plasma gave several precipitin lines (at least 7, 7 and 8 respectively) when reacted with their homologous antigens in agar–gel-diffusion studies. After absorption with blood serum, there was one antigen (presumably fibrinogen) in blood plasma and follicular fluid that was not found in blood serum. These results were substantiated by immuno-electrophoresis.

Moving boundary electrophoresis in five different buffers revealed at least eight components in follicular fluid and blood serum and at least nine in blood plasma. Minor differences were observed in the electrophoretic mobilities of some components found in both follicular fluid and blood serum.

The results showed that the major macromolecular components of follicular fluid and of blood were similar, but that some minor macromolecular ones may differ.

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K. R. STEVENS, H. D. HAFS and A. G. HUNTER

Summary.

Fluids were obtained from 185 rabbit uteri 1 to 20 weeks after uterine ligation. The uterine fluid proteins were concentrated with an ultrafilter and characterized by diffusion in agar gel, moving boundary electrophoresis, and immunoelectrophoresis. Eight electrophoretic components were identified by means of moving boundary electrophoresis. One which migrated as a pre-albumin and another which migrated as an alpha-globulin were not found in blood sera. Agar-gel diffusion tests revealed thirteen antigenic components in uterine fluid. Three precipitin lines appeared to be specific to uterine fluid after absorption of guinea-pig antisera to rabbit uterine fluid with rabbit blood sera. However, at least five antigens which could not be found in blood sera were identified in uterine fluid by means of immunoelectrophoresis. The mobilities of two were similar to prealbumins and the mobilities of the remaining three were similar to beta-globulins. The results indicated that at least two classes of proteins, which do not exist in rabbit blood serum, may be found in uterine fluids obtained by ligation.

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C. DESJARDINS, K. T. KIRTON and H. D. HAFS

Summary.

Each of twelve rabbits was ejaculated for 5-week periods at frequencies of: once a week (1 × F) ; four times on Friday (4 × F) ; twice on Monday, Wednesday and Friday (2 × MWF) ; and once daily except Sunday (1×M-S). Average sperm outputs/ejaculate (×106) for the four ejaculation frequencies were: 273 for 1 × F; 114, 207, 141 and 80 for first, second, third and fourth ejaculates, respectively, at 4×F; 78 and 128 for first and second ejaculates, respectively, at 2 × MWF; and 86 for 1 × M-S. Repetitive ejaculation significantly increased weekly sperm output (P<0·01). Component of variance analysis of weekly sperm output indicated that differences among bucks were magnified by the more intensive ejaculation frequencies, but not as much as average sperm output.

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K. T. KIRTON, H. D. HAFS and A. G. HUNTER

Summary.

Four consecutive ejaculates were collected within 1 hr, at weekly intervals for 4 weeks, from each of five mature Holstein bulls to determine the influence of repetitive ejaculation on the levels of some seminal constituents. The pH of the fresh semen and the concentrations of fructose and citric acid in the seminal plasma increased (P<·01) while the concentration and total amount of free amino nitrogen declined from first to fourth ejaculates (P<0·01). Repetitive ejaculation did not significantly influence the concentrations of protein and zinc in the seminal plasma (P ≃ 0·20) or the total citric acid and fructose per ejaculate (P ≃ 0·20). Moving boundary electrophoresis of the proteins and agar-gel-diffusion studies of the antigens in seminal plasma revealed that each protein component observed in first ejaculates was also found in fourth ejaculates. However, the proportion of the protein components in fourth ejaculates differed from that in first ejaculates. The results suggested that the total contribution of the seminal vesicles to the seminal plasma was relatively constant, but their relative contribution increased, from the first to the fourth ejaculate, at least partly at the expense of the contribution of the epididymides.

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R. H. FOOTE, H. D. HAFS, R. E. STAPLES, A. T. GREGOIRE and R. W. BRATTON

Summary.

At any one time a large proportion of individually caged, sexually mature female rabbits failed to copulate. Intravenous administration of purified pituitary luteinizing hormone (plh) (Armour) produced ovulations in sexually receptive and nonreceptive does alike. Does with histories of infertility ovulated after the injection of plh. The routine administration of 2·5 mg of plh to fifty-seven virgin and multiparous Dutch-Belted does averaging 2 to 3 kg, followed by artificial insemination, resulted in 91% of the does kindling and 307 young being born. Administration of either 0·5 mg or 1 mg of plh per kg to random members of fourteen pairs of does, accompanied by artificial insemination, resulted in no difference between the number of ovulations or the number of young per treatment (P>0·05). Fertility and litter size were normal when sixteen does were re-injected with plh and inseminated after weaning their previous litters.

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R. Webb, G. E. Lamming, N. B. Haynes, H. D. Hafs and J. G. Manns

Summary. Doses of 125, 250 or 500 μg LH-RH were injected i.m. into suckled beef cows on approximately Day 11 of an oestrous cycle synchronized by prostaglandin treatment. There was a positive linear relationship between dose of LH-RH and the area under the measured LH peak. Administration of 500 μg LH-RH as a single injection to suckled cows 13–32 days post partum resulted in LH release but failed to induce normal ovarian activity. A small transient rise in plasma progesterone for 6–9 days occurred at the expected time after injection in 50% of animals. Administration of 500 μg LH-RH to suckled beef cows approximately 20–30 days post partum and a second injection approximately 10 days later at the time when the resulting transient rise in plasma progesterone had returned to basal values induced normal cyclic activity (as shown by progesterone concentrations and observed oestrus) at 35 days compared with 70 days for untreated controls. Pituitary responsiveness to LH-RH, as assessed by LH levels, was found to increase up to 20 days post partum.

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N. B. Haynes, H. D. Hafs, K. Purvis and J. G. Manns

Department of Physiology and Environmental Studies, University of Nottingham School of Agriculture, Sutton Bonington, Loughborough LE12 5RD, Leicestershire, U.K.

As part of a study on pituitary-testicular feedback mechanisms, the plasma hormone levels of 9 Friesian bulls, 6-8 months old, were examined at the time of unilateral castration. Blood (10 ml) was taken from indwelling jugular cannulae at 30 min intervals for 48 hr, a testis being removed under local anaesthesia (Lignocaine, Pharmaceutical Man. Co.) from each animal after 24 hr. Testosterone concentrations in plasma were measured by the radioimmunoassay method of Haynes, Hafs, Waters, Manns & Riley (1975) and LH concentrations by the radioimmunological procedure described by Oxender, Hafs & Edgerton (1972). One bull had no detectable testosterone after unilateral castration and was not included in the analysis.

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A summary of the results for 8 animals is given in Table 1. Hormone concentrations before castration were in accord with previous