Pheromaxein, the pheromonal steroid-binding protein, is a major protein synthesized in porcine submaxillary salivary glands.

Submaxillary salivary gland tissue from large White, Göttingen miniature and Meishan (Chinese) breeds of pig, and European wild boars, was incubated with [35S]methionine. The radiolabelled amino acid was incorporated into protein in all incubations as demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Specifically [35S]methionine was predominantly incorporated into the alpha- and beta-charge isomers of pheromaxein, a 16-androstene steroid-binding protein, as shown by SDS-PAGE in combination with vertical isoelectric focusing on polyacrylamide slab gels. The synthesis of pheromaxein occurred in submaxillary gland tissue from both sexes, including tissues stored frozen at -70 degrees C for long periods. There was little evidence for pheromaxein synthesis in parotid gland tissue or skeletal muscle. Total protein, pheromaxein and total 16-androstenes were determined in the submaxillary gland cytosols of six mature Göttingen miniature boars and a positive correlation was found between these glandular constituents. The amounts of endogenous pheromaxein relative to total protein in the submaxillary gland cytosols (range 10.3-18.0%), together with the predominant synthesis of this protein in vitro, indicate that pheromaxein is a major protein produced in porcine submaxillary glands, particularly in those of the male.

An enzyme-linked immunosorbent assay for cortisol in the saliva of man and domestic farm animals.

An enzyme-linked immunosorbent assay has been developed for the direct assay of cortisol in the saliva of man, sheep, calf and pig. The assay is sensitive (integral of 1pg), highly specific and reproducible, and has the advantage over radioimmunoassay in being cheaper and quicker to carry out. The relatively-non invasive method of collecting saliva on cotton buds coupled with simple assay equipment provide a good potential for assessing cortisol status in studies on stress and welfare in farm animals as well as the possibility of monitoring cortisol status in mammals generally.

The isolation, purification and some properties of pheromaxein, the pheromonal steroid-binding protein, in porcine submaxillary glands and saliva.

Pheromaxein, the 16-androstene steroid-binding protein with a relative molecular mass of 15,000 was isolated in sub-milligram quantities from the submaxillary gland and saliva of the Gottingen miniature boar, after a fourfold purification involving the following methods: ultrafiltration for submaxillary gland cytosols and ethanol precipitation for saliva, Concanavalin-A-Sepharose affinity chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis, 'Extractigel-D' affinity chromatography (to remove sodium dodecyl sulphate) and fast protein-liquid chromatography. Yields of purified pheromaxein obtained after fast protein-liquid chromatography represented 10-20% of total protein present in an ultrafiltrate of a submaxillary gland cytosol. Fast protein-liquid chromatography separated the alpha- and beta-charge isomers of pheromaxein which were shown to have isoelectric points of 4.78 and 5.35 respectively on flat-bed isoelectric focusing. Some data are provided for the variable occurrence of the isomeric forms of pheromaxein in relation to different breeds of pig. Five 16-unsaturated steroids showed the highest binding to pheromaxein. Other steroids of the 5 alpha- and 5 beta-androstane series also showed some binding to pheromaxein, i.e. 17 beta-hydroxy-5 alpha-androstan-3-one (19.2%), with 5 alpha-androstan-3-one, which has a similar urinous odour to 5 alpha-androst-16-en-3-one, showing the greatest binding (42.6%) relative to 5 alpha-androst-16-en-3-one (100%).

Factors affecting the pheromone composition of voided boar saliva.

The pheromone binding protein 'pheromaxein' which binds the pheromonal 16-androstene steroids in the saliva of the male pig (boar), was degraded and lost its binding activity in saliva incubated in air for 72 h at 21 degrees C and 37 degrees C. However, pheromaxein and its binding activity were retained in saliva incubated for 168 h at 4 degrees C. When the 3H-labelled pheromones 5 alpha-androst-16-en-3 alpha-ol (3 alpha-androstenol), 5 alpha-androst-16-en-3-one (5 alpha-androstenone) and 5 alpha-androst-16-en-3 beta-ol (3 beta-androstenol) were incubated with boar saliva for 168 h at 21 degrees C, 3 alpha-androstenol was primarily converted to 5 alpha-androstenone and 5 alpha-androstenone to 3 beta-androstenol; 3 beta-androstenol was unchanged. Evidence was obtained for microorganisms being responsible for these steroid transformations.

Plasma prolactin in relation to changes in oestrous cyclicity following olfactory bulbectomy in post-pubertal pigs.

Prolactin was determined by radioimmunoassay in the peripheral plasma of five post-pubertal Large White gilts before and after olfactory bulbectomy. In the oestrous cycle before bulbectomy and in cycles after bulbectomy, the highest concentrations of prolactin were found during the period from the late luteal phase to oestrus. Throughout the prolonged summer anoestrus which occurred after bulbectomy, the amounts of prolactin were similar to base-line quantities found during the oestrous cycle between Days 3 and 12. These results differ from those found for the seasonally breeding European wild sow where high prolactin levels are associated with a summer anoestrus.

Behavioural and morphological effects of 5 alpha-dihydrotestosterone and oestradiol-17 beta in the prepubertally castrated boar.

Three groups of 5 prepubertally castrated boars received twice weekly subcutaneous injections from 16 to 28 weeks of age of 5 alpha-dihydrotestosterone propionate (1 mg/kg), oestradiol-17 beta dipropionate (0.1 mg/kg), or a combination of the two steroids. The pigs were tested each week for sexual behaviour with an oestrogen-treated ovariectomized gilt. 5 alpha-Dihydrotestosterone did not induce copulatory behaviour whereas oestradiol initiated mounting, although this behaviour was not maintained. However, the combined hormonal treatment induced the full pattern of copulation, including ejaculation, in 4/5 pigs. Oestrogen-treated animals also showed champing and salivation. None of the pigs treated with 5 alpha-dihydrotestosterone showed lordosis when exposed to a mature boar. Increases in the size of the penis, the ischio- and bulbocavernosus muscles, seminal vesicles and bulbourethral glands were produced by 5 alpha-dihydrotestosterone, and, with the exception of the penis, this hypertrophy was further enhanced by additional treatment with oestradiol. Increases in the weight of the prostate and muscular urethra were due mainly to the effects of oestrogen; this steroid caused extensive development of fibromuscular tissue and also increased mammary teat length. In the submaxillary salivary glands, synergism between 5 alpha-dihydrotestosterone and oestradiol resulted in hypertrophy of the serous cells.

Sexual dimorphism involving steroidal pheromones and their binding protein in the submaxillary salivary gland of the Göttingen miniature pig.

Submaxillary glands of mature Göttingen miniature pigs were examined for the presence of a sexual dimorphism. Gland weights, serous cell hypertrophy and total protein in the glands were much greater in male than female pigs. High concentrations of the pheromonal 16-androstene steroids were present in the glands of males and exceeded 2 mmol/g in some animals; this was primarily due to 5 alpha-androst-16-en-3 alpha-ol. The high concentration of 16-androstene steroids in boar glands was correlated with the presence of large amounts of binding protein for these steroids in the glands; smaller amounts of the binding protein were detected in female glands. These findings are similar to those found in domestic pigs, but the degree of sexual dimorphism assessed from these findings is more extreme in the miniature pig.

Development of some male characteristics supported by oestrone but not dehydroepiandrosterone in the boar.

Prepubertally castrated boars received subcutaneous injections twice weekly, from 13 to 35 weeks of age, of dehydroepiandrosterone (2 mg/5 kg) or oestrone (1 mg/5 kg). Dehydroepiandrosterone did not support the growth and secretory activity of the accessory organs, or induce copulatory behaviour. However, oestrone caused hypertrophy of the prostate, seminal vesicles and bulbourethral glands which was due to an increase in fibrous stromal tissue and not to the secretory epithelium. Oestrone also induced some male mating behaviour patterns in the presence of an oestrous gilt, although penile extrusion and ejaculation did not occur. The morphological and behavioural effects of the steroid treatments were supported by steroid profiles in blood plasma as seen in comparison with androgen and oestrogen values of intact and untreated castrated boars. It is concluded that oestrogen in the intact boar might enhance the secretion of the accessory organs by affecting the neural control of the secretory processes rather than by increasing the amount of secretory epithelium in the glands.

In-vitro metabolism of unconjugated androgens, oestrogens and the sulphate conjugates of androgens and oestrone by accessory sex organs of the mature domestic boar.

Minces of the prostate and seminal vesicles of the mature boar were incubated with the following major testicular steroids of the pig: [3H]dehydroepiandrosterone, [3H]5-androstene-3 beta, 17 beta-diol, [3H]oestrone, [3H]oestradiol-17 beta and their respective sulphate conjugates (excluding oestradiol-17 beta). Incubations were also carried out with [3H]testosterone, [3H]5 alpha-dihydrotestosterone and [3H]5 alpha-androstanediols. Minces of the epididymides were incubated with [3H]dehydroepiandrosterone and [3H]oestrone sulphates. The prostate and seminal vesicles converted dehydroepiandrosterone predominantly to weak androgens, whereas 5-androstene-3 beta, 17 beta-diol was primarily converted to testosterone; testosterone and its 5 alpha-reduced metabolites were metabolized in a manner typical of androgen end target organs. Unconjugated oestrone and oestradiol-17 beta were interconverted by the prostate and seminal vesicles. The metabolism of C19 steroid sulphates was less than 1% in all incubations; some oestrone sulphate, however, was converted to unconjugated oestrone and oestradiol, particularly by the caput epididymidis. The significance of these results is discussed in relation to recent studies in vivo.

Changes in oestrous cyclicity following olfactory bulbectomy in post-pubertal pigs.

Bilateral olfactory bulbectomy was carried out on 16 post-pubertal gilts during the winter and spring of 2 consecutive years. Eight bulbectomized gilts in Exp. 1 had 2-7 regular periods of oestrus before becoming anoestrous for greater than 100 days in the summer and autumn. During November, 3 of these gilts were placed with b boar and became oestrous within 9 days, were mated and established normal pregnancies; the remaining pigs also resumed normal oestrous cycles when transferred to other pens. The following year anoestrus again occurred during the summer in gilts and in sows after weaning. In Exp. 2, 8 bulbectomized gilts remained in the same pens throughout the experiment, 4 were housed with a vasectomized boar and 4 away from boars. As in Exp. 1, the same periods of anoestrus occurred throughout the year, whereas 4 unoperated pigs housed nearby had continuous oestrous cycles. Ovulation without oestrus occurred in some bulbectomized pigs even after gonadotrophin treatment. Prolonged anoestrus in Exp. 1 was associated with non-ovulatory levels of plasma oestrogen and low plasma progesterone and LH values. Behavioural, morphological and endocrine data indicate that the effects of bulbectomy on oestrous cycles in post-pubertal gilts are mediated at the hypothalamus. The seasonality of the effects seems likely to be due to bulbectomy unmasking a photoperiodic influence.

Metabolism of C19 steroids in vitro by the submaxillary salivary gland of mature and immature domestic pigs.

Homogenates of the submaxillary glands of immature and mature pigs were incubated with 3H-labelled C19 steroids which have been shown previously to be metabolized in vitro by the submaxillary gland of mature boars. Dehydroepiandrosterone was metabolized largely to androstenedione, 5 alpha-androstane-3,17-dione and androsterone, and to small amounts of testosterone, 5 alpha-dihydrotestosterone and 5 alpha-androstanediols. Testosterone yielded predominantly 5 alpha-androstane-3 alpha, 17 beta-diol with smaller amounts of other 5 alpha-reduced products, i.e. 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 beta, 17 beta-diol, 5 alpha-androstane-3,17-dione and androsterone; 5 alpha-dihydrotestosterone and the two epimeric 5 alpha-androstane-3 alpha/3 beta, 17 beta-diols were interconverted. These and earlier results show that the porcine submaxillary gland has the capacity in vitro to metabolize selected C19 steroids in a way which is not related to either sexual maturity or sex of the animal; in this respect the findings support certain aspects of previous histochemical studies.

Degeneration and regeneration of the olfactory epithelium after olfactory bulb ablation in the pig: a morphological and electrophysiological study.

The olfactory bulbs were removed surgically from Large White male and female pigs, 10--12 weeks of age. At intervals of 3, 7, 14, 42 and 84 d after bulbectomy, the pigs were sacrificed and portions of olfactory mucosa were removed from the ethmoturbinate and septum regions of the nasal cavity; olfactory mucosa was also removed from unoperated pigs. A piece of each tissue sample was processed for light microscopy. The remaining tissue was placed in Ringer-Locke solution, saturated with O2/CO2 at room temperature, and the electrical activity of the olfactory epithelium was investigated in vitro by passing a stimulus of butyl acetate vapour over the epithelium. Slow negative potential changes (electro-olfactogram, e.o.g.) induced by butyl acetate were recorded. During the first two weeks after bulbectomy there was a rapid decrease in the height of the olfactory epithelium associated with the disappearance of the e.o.g. response. However by 42 and 84 d after bulbectomy, partial recovery of the height and some electrical activity of the olfactory mucosa had occurred. In some pigs, the insertion of a stainless steel lining over the cribriform plate to prevent any association of regenerating axons with forebrain tissue had no effect on the regenerative characteristics studied.

The extragonadal origin of 5 alpha-reduced androgens in the peripheral circulation of the adult male rabbit.

The extragonadal source of the high levels of 5 alpha-reduced androgens in the peripheral blood of the male rabbit has been investigated. Adult 3-day castrated rabbits each received a single intravenous injection of 3H-labelled testosterone, and blood plasma was collected at various intervals thereafter. Two h post injection, animals were killed, various accessory sex organs and peripheral tissues removed, and radioactively labelled testosterone and its metabolites quantified by radio-gas-liquid chromatography (radio-g.l.c.). Five min after the injection of 3H-labelled testosterone, the major labelled metabolite in blood plasma was 5 alpha-dihydrotestosterone with small amounts of 3 alpha/3 beta-androstanediols and androstenedione. After 2 h significant quantities of 3H-labelled 5 alpha-dihydrotestosterone had accumulated in the accessory sex organs, whereas only small amounts were found in "non-sexual" peripheral tissues such as skeletal muscle. However, substantial amounts 3-day castrated rabbits, when they were incubated in vitro with 3H-labelled testosterone; the most active were skeletal muscle, liver and abdominal skin. Some formation of 3 alpha/3 beta-androstanediols occurred in all tissues. It is concluded that in the male rabbit skeletal muscle and skin are likely to be a significant extragonadal source of 5 alpha-reduced androgens for the maintenance of accessory sex organs such as the prostate and epididymis, which have low 5 alpha-reductase activity but a high capacity to retain 5 alpha-dihydrotestosterone.

A study of some major testicular steroids in the pig in relation to their effect on the development of male characteristics in the prepubertally castrated boar.

Prepubertally castrated boars received subcutaneous injections twice weekly, from 12 to 38 weeks of age, of testosterone or 5-androstenediol (5-androstene-3 beta,17 beta-eiol) (2 mg/5 kg), or each of these steroids in combination with oestrone (1 mg/5 kg). All steroid treatments induced growth and secretory activity of the accessory organs. The prostate was maintained best by testosterone, and the seminal vesicles by 5-androstenediol. No specific effects of oestrone on these organs were found. Champing of excess saliva was induced in all treated pigs when they were introduced to an oestrous gilt, but only boars receiving testosterone displayed intense mating behaviour including mounting and, in 3 animals, ejaculation; no specific effects of oestrone on behaviour were seen. Low normal levels of unconjugated androgen, and normal to high levels of unconjugated oestrogen were circulating in the treated pigs. There was evidence that peripheral metabolism of exogenous steroids had occurred: 5-androstenediol to testosterone, these two androgens to oestrogen, and sulphation of oestrogen. The sulphate conjugates of dehydroepiandrosterone, 5-androstenediol and oestrone predominated in the peripheral plasma of mature intact boars. Concentrations of 3 alpha-androstenol (5 alpha-androst-16-en-3 alpha-ol) in the submaxillary glands of androgen-treated pigs were similar to those in untreated castrates, thus indicating that androgens are probably not precursors of the pheromonal 16-androstene steroids in vivo.

Lack of effect on sexual behaviour or the development of testicular function after removal of olfactory bulbs in prepubertal boars.

Bilateral olfactory bulb ablation was carried out surgically on 8 prepubertal Large White boars when they were 10-12 weeks of age. Between 26 weeks and slaughter at 47-49 weeks of age, androgen was determined in peripheral blood plasma of bulbectomized and unoperated control animals. The pigs were exposed to oestrous female pigs to observe mating behaviour, and to alien boars to observe aggressive behaviour. Saliva produced during behaviour tests was extracted with diethyl ether and levels of the pheromonal 16-androstene steroids in the extracts were determined by a colorimetric assay. After slaughter the testes, accessory organs and submaxillary glands were weighed, and pices of tissue together with olfactory epithelium were processed for light microscopy; fructose and zinc were determined in the seminal vesicles. The results showed that, contrary to findings in some rodents, prepubertal bilateral bulbectomy in the male pig had no significant effect on mating or aggressive behaviour, or testicular function in so far as complete spermatogenesis was present and normal levels of androgen and pheromone were maintained together with the integrity of the accessory organs. However, in keeping with findings in other species, the height of the olfactory epithelium was generally reduced in the bulbectomized pigs.