Summary. Lipoproteins were separated by centrifugation and column chromatography and included in citrate-based semen diluents. One lipoprotein fraction was particularly effective in preventing injury to bovine spermatozoa during dilution and freezing and thawing when assessed using motility and release of hyaluronidase as criteria of cell damage. Radioactive labelling of egg-yolk lipids demonstrated that egg-yolk components remained associated with spermatozoa even after extensive washing to remove diluent. The pattern of labelling of lipids extracted from the washed spermatozoa did not indicate that particular lipids became associated with spermatozoa. Increases in the specific radioactivity of lipid extracts from washed spermatozoa lent support to the contention that lipoproteins become firmly bound to the cells.
J. A. FOULKES and P. A. WATSON
Ministry of Agriculture, Fisheries and Food, Cattle Breeding Centre, Shinfield, Reading RG2 9BZ
(Received 4th November 1974)
One of the earliest visual signs of sperm damage is the loss of acrosomal integrity. Consequently, assay of the release into the seminal plasma of hyaluronidase, which is localized in the acrosome (Mancini, Alonsa, Bacquet, Avarez & Nemirosky, 1964; Gould & Bernstein, 1973) should provide an early and sensitive indication of damage sustained by the spermatozoa during processing of semen for artificial insemination (A.I.). Previous biochemical tests of sperm viability have included the assay of phenylalanine α-ketoglutarate transaminase released into boar seminal plasma (Gemert, Hendrike & Van der Horst, 1972). A correlation was found between high enzyme activity in seminal plasma and poor fertility. The release of glutamic oxaloacetic transaminase has been considered an indication of cell damage (Brown, Crabo, Graham & Pace, 1971) and assay of this non-acrosomal enzyme has been investigated
B. J. MacDonald and J. A. Foulkes
Summary. The dye 1-anilino-naphthalene-8-sulphonate (ANS) bound at 94·5 ± 2·7 pmol per 106 spermatozoa (K D = 1·83 ± 0·13 × 10−5 m) and 7·30 ± 0·17 pmol per mg seminal plasma protein (K D = 2·19 ± 0·06 × 10−5 m). Equilibration of excess ANS with a mixture of washed spermatozoa and seminal plasma resulted in a significant increase in fluorescence over that calculated by summation of the individual levels (P < 0·05). A less pronounced but significant increase was seen when an egg-yolk lipoprotein, previously shown to have cryoprotective properties, was similarly added to washed spermatozoa (P < 0·05). This increase in fluorescence was not reduced by washing spermatozoa–lipoprotein mixtures, suggesting that an interaction between the lipoprotein and the cell membrane had occurred.
J. A. Foulkes and D. L. Stewart
Summary. A lipoprotein separated from egg yolk by centrifugation and column chromatography was used to prepare a diluent containing 2·9% trisodium citrate.2H2O (pH 7·0) and 7% glycerol. Ejaculates from Friesian bulls were split between this and two control diluents containing egg yolk, glycerol and citrate or lactose and used to inseminate once 1782 unselected Friesian cows. By 16 weeks after insemination with semen frozen in the egg yolk-lactose, egg yolk-citrate or lipoprotein-citrate diluents, 66·4,67·1 and 66·1% of cows, respectively, had not returned to service.
A. D. Cookson, A. N. Thomas, and J. A. Foulkes
Summary. Antiserum was raised in rabbits against an egg-yolk lipoprotein fraction previously shown to have cryoprotective properties. It was used in conjunction with alkaline phosphatase-conjugated goat anti-rabbit IgG to demonstrate lipoprotein interaction with bovine spermatozoa. The lipoprotein bound firmly to spermatozoa and was not removed by extensive washing, suggesting that the lipoprotein had become irreversibly associated with the sperm membranes.
D. A. Ross, H. S. Dhadwal, and J. A. Foulkes
Summary. An objective method of assessing bull sperm motility by specifying the mean swimming speed and number of motile spermatozoa in a sample is described. Laser light was conducted into diluted semen samples using a fibre-optic Doppler anemometer (FODA). The signal correlation of the back-scattered laser light was modelled using least squares computer curve fitting of the resulting data. Agreement was found between the mean swimming speeds obtained and those measured using time lapse photography.
J. A. Foulkes, D. Sweasey, and R. G. Goodey
Summary. Three groups of hens were fed a standard egg-production mixture or similar diets enriched with either sunflower oil or beef tallow. The resulting egg yolks were used to prepare bovine semen diluents. Changes in the lipid fatty acid composition of the diluents were demonstrated by gas–liquid chromatography. No significant effects of the treatments were seen on bovine sperm motility after freezing and thawing, or on fertility.
M. J. Sauer, J. A. Foulkes, A. Worsfold, and B. A. Morris
Summary. A simple direct-addition microtitre plate enzymeimmunoassay (EIA) for progesterone in whole milk is described. The assay used antiserum raised against 11α-hydroxyprogesterone 11-hemisuccinate (progesterone 11-hemisuccinate) and a heterologous label prepared by conjugation of 11α-hydroxyprogesterone 11-glucuronide (progesterone 11-glucuronide) with alkaline phosphatase using an active ester procedure. The sensitivity, analytical recovery, linearity of response and precision of the assay compared favourably with radioimmunoassay (RIA). Results from EIA of milk samples were compared with determinations made after isolation of progesterone by HPLC (r = 0·910). Milk samples (200) were assayed by RIA at both the Milk Marketing Board and the Cattle Breeding Centre and the results were correlated with EIA performed at the Cattle Breeding Centre (r = 0·890 and r = 0·833 respectively). Calving data were obtained from a further 110 cows for which the milk progesterone EIA had provided a pregnancy test 24 days after AI; 46 cows were correctly identified as non-pregnant and 58 as pregnant and there were 4 false positive and 2 inconclusive results.
P. D. P. Wood, J. A. Foulkes, R. C. Shaw, and D. R. Melrose
Summary. Nineteen young Hereford bulls were used to study the relationship between semen characteristics and fertility in artificial insemination following 15 320 inseminations. Seven measures of sperm motility, morphological abnormalities, the release of hyaluronidase, ATP content and sperm head measurements were examined as predictors of fertility (49-day fixed-interval non-return rate). Two assessments of motility, three categories of abnormal spermatozoa, acrosomal changes and the release of hyaluronidase had predictive power. Multiple regression analysis showed that a combination of sperm motility after dilution in saline, motility after thawing and the proportion of coiled tails and proximal protoplasmic droplets provided the best prediction of fertility and allowed bulls to be ranked in order of observed non-return rate (%) with a Spearman correlation better than +0·80.