Ondmtepoort ]. vet. Res. 38 (1) 1-62 (1971) REPRODUCTIVE PHYSIOLOGY AND ENDOCRINOLOGY OF NORMAL AND HABITUALLY ABORTING ANGORA GOATS* S. J. VAN RENSBURG(l), Veterinary Research Institute, Onderstepoort SuMMARY VAN RENSBURG, S. J. Reproductive physiology and endocrinology of normal and habitualfy aborting Angora goats. Onderstepoort ]. vet. Res. 38 (1), 1-62 (1971). Recurrent abortion of non-infectious or non-organic origin is exceedingly prevalent in many species including man, yet virtually no information on possible metabolic and endocrine causes was available. In order to study the pathogenesis of this form of gestational failure, an experimental flock was consti tuted which consisted of normal and habitually aborting Angora goats, a species in which the high incidence of abortion constitutes a significant economic problem. The investigation was initially compli cated by the fact that at the time of its inception there were no acceptable theories regarding the cause of the initiation of normal parturition. For this reason experimental work on relevant fundamental aspects was included in the study. Comparative studies on normal and aborting goats entailed: (i) Investigations of sexual behavioural patterns and of breeding performance in mature animals, followed by physiological, clinical and pathological observations on mature does, foetuses and kids. (ii) Development of suitable methods for the precise chemical assay of steroid hormone metabolism in goats. The methods used facilitated detailed studies on luteal function, cortisol metabolism and the excretion of oestrogens. (iii) Investigation of the mohair growth rate as well as its fibre characteristics in relation to repro ductive capabilities. (iv) Experimental reproduction in normal animals of the aberrations found and the investigation of the significance of such aberrations in causing gestation termination. Gestational failure was more prevalent in the heavier, older type of goats which were found to have enlarged pituitaries and which occasionally exhibited clinical signs of disturbed adrenal function. Animals that had aborted exhibited abnormally short oestrous cycles which appeared to be responsible for a lowered conception rate. Their ovaries contained cystic corpora lutea and displayed excessive follicular growth; experimental studies indicated that these changes were secondary to adrenal hyperplasia. Abortions were most frequent during the early part of the fourth month of gestation, a time which coincided with the most rapid increase in the rate of foetal growth and also with the cessation of placental growth. The condition of the foetus destined to be aborted suggested placental insufficiency; growth was retarded, anaemia was usually present and the concentration of some elements in the liver was abnormally low. Changes noted prior to abortion included excessive or deficient urinary oestrogen excretion, exces sive ovarian follicular growth, the sudden onset of maternal adrenal atrophy, accumulation of excessive foetal fluids and degeneration of the placentomes. Control of gestation maintenance by the corpus luteum was confirmed; removal of the corpus luteum from Angora goats at any stage of pregnancy resulted in abortion 40 to 60 hours later. Variations in the level of luteal function during gestation are postulated to be due to a placental lact.ogen-type hormone secreted by the growing placenta. Peripheral plasma levels of progesterone tended to be higher than usual in pregnant aborters, but were depressed shortly prior to abortion in only some individuals. Studies on ovarian secretion rates suggested that this reduction was partly due to a lowered adrenal contribution, which may be expected in view of an observed concurrent adrenal atrophy. Signs of impending abortion were, however, evident while luteal function was still quite normal. The markedly aberrant oestrogen excretion rate of aborters could only be ascribed to an abnormal supply of steroid precursors resulting from altered metabolism in the maternal adrenal glands. Newborn kids destined to perpetuate the abortion defect tended to be heavier than normal and had finer birthcoats. The quantity of mohair produced by the young animal born from aborter stock was ex ceptional and the young males produced 30 per cent more than usual. Adrenal function in young high producing aborter stock was lower than usual. However, established regular aborters had enlarged adrenal cortices and produced smaller quantities of finer mohair. Such findings are consistent with experi mental results obtained with other species, the results demonstrating that corticosteroids inhibit the rate of hair growth and the fibre diameter. Evidence is presented which suggests that the adrenal enlargement found in aborters is an adaptive response favouring the foetus at the expense of hair production characteristics; aborter does which could maintain a higher level of adrenal function throughout gestation carried their foetuses successfully to term. Experimental administration of small doses of corticosteroids to goats during pregnancy prolonged gestation by several days, a situation which resembled successfully adapted aborter does. Dose-related prolongation of gestation was also obtained when small amounts of corticosteroid were administered to the sheep or goat foetus, but slightly higher dosages lead to rapid expulsion of the foetus. When adminis tered maternally to sheep, these steroids caused a moderate reduction of placental progesterone synthesis. However, the same dosage rate given to the foetus caused a more drastic progesterone block, rapidly fol lowed by expulsion of the foetus. Adrenalectomy of the foetus caused indefinite prolongation of gestation in sheep, but not in goats. The investigations have contributed to the concept that normal birth is initiated by the foetal hypothalamus-pituitary-adrenal axis; when the hypothalamus is adequately sensitive to ensure viability *Thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Veterinary Science, in the Department of Physio logy, Faculty of Veterinary Science, University of Pretoria, May 1970, Promotor: Prof.]. M. M. Brown, Department of Physiology, Faculty of Veterinary Science. (1) Present address: Medical Research Council, Private Bag 380, Pretoria. Received 27 January 1971-Editor 1 REPRODUCTIVE PHYSIOLOGY AND ENDOCRINOLOGY OF ANGORA GOATS of the foetus, it responds to the usual prenatal deterioration of the foetal nutritional environment hy stimulating the foetal adrenal glands and the elevated steroid secretions have effects on the foetoplacental unit, resulting in the initiation of parturition, possibly by means of blocking both the production and the action of progesterone. The cause of abortion in Angora goats appears to be intimately related to a high metabolic priority for harr growth, artificially induced by intensive selection and inbreeding. An abnormally low level of adrenal function, coupled with some qualitative changes in adrenal steroid biosynthesis seems to be the responsible mechanism. Physiological adaptation involves adrenal hyperplasia in order to assist the transfer of maternal nutrients to the foetus. Abortion is a consequence of the failure of this mechanism. INTRODUCTION of pregnancy by luteotrophic hormone secretion. This Angora goats are maintained primarily for the pro work and that of others (Short, 1960, 1969) suggest duction of the quality luxury fibre, mohair, a.s opposed that corpus luteum regression is invariably necessary to the usual basic use of the ruminant animal for meat, prior to normal or abnormal expulsion of the foetus milk and wool production. It is also a very old breed, and its role seems essentially permissive. Many early since Angora goats appeared to have existed several changes, indicating abortion at a later stage, were thousand years B.C. This distinctive breed hybridizes found in this work whilst luteal function was still readily with any type of goat, yet it has retained its assessed to be normal or even above the norm. Never essential fleece characteristics. Historical literature theless as a result of Van Heerden's work the problem attributes this survival to careful selection in order to has been contained by eliminating affected animals maintain the highly cherished qualities of the fleece. from the breeding flock, both in South Africa (Van Vogt & Specht (1889) state: "The Angora goat has Heerden, 1964) and in Texas (personal communications). been brought, in certain mountainous countries with a This work was primarily an attempt to define the raw. climate, by careful in-and-in breeding to produce nature of the defect more accurately in order to facili an mvaluable kind of long wool, which envelopes tate future control. The research value of statistical almost the entire body of the animal, and is unsurpassed groups of habitually aborting experimental animals is br delicacy and softness." also unique since even the nature of factors initiating Fashion dictates during the first half of the century the normal termination of gestation was unknown at necessitated ~rastic reductions of the goat population the commencement of this project. As an experimental m South Afnca, consequently only those animals with animal the goat has many distinct advantages (Fletcher, the best production characteristics were retained, and Rogers & Donaldson, 1964) which make it suitable for this nucleus was inbred for many generations (Van physiological studies. Heerden, 1963). The resurgence of the demand for An association with the individual mohair production mohair since 1949 was accompanied by spectacular potential of a goat and the occurrence of abortion has rises in the price of mohair, and attempts to increase been established in this study. It is a variable syndrome the Angora goat population met with considerable and the evidence suggests gestational failure to be a ~ifficulties because of the emerging problem of abor quantitative tendency in certain Angora blood lines, tion; only at this time was the incidence ascertained to rather than a specific defect of certain individuals. The exceed 50 per cent frequently. Such losses barely leave exceedingly constant involvement of the adrenal gland sufficient offspring for normal flock replacement, and and glucocorticosteroid biosynthesis found in afflicted therefore further selection for improvement is pre animals may represent a mechanism of hormonal cluded. The greatest demand is for hair from young control of gene expression; there is evidence that animals, and it is therefore economically advantageous cortisol may act in this way as an effector molecule in to keep the flock as young as possible. This combination induction and repression. As far as the actual cause of of factors has been a serious handicap to the mohair abortions is concerned the results of this study conform industry in South Africa. most acceptably to the theory of Spiegelberg (1891), There is no evidence of a similar problem in other who suggested that parturition was initiated through mohair producing countries such as Turkey, Mada the action of substances secreted by the foetus and gascar, India, Albania and Russia; but in Texas, U.S.A., passed into the maternal blood and that the exciting reliable _information indicates that in flocks consisting substances were elaborated as a result of insufficiency predommantly of the smaller South African type of of nutrition and were an indication that the foetus re goat which has been selected for quality hair, the quired other substances than those supplied to it abortwn rate may be as high as 40 per cent. The majori through the placenta. ty ~f Texan goats are, however, of the large robust type which produce relatively smaller quantities of fairly CHAPTER c?arse hair, and which rarely abort. All types are con siderably more sensitive to stress factors in comparison MATERIALS AND METHODS to sheep, and the sudden onset of cold wet weather Animals results in serious mortality if artificial shelter is not Small groups of Angora goats were purchased from provided. several farmers during the earlier part of this work and The cause of abortions in Angora goats has been during the latter stages the flock consisted predominant the subject of an exceedingly thorough field study by ly of the offspring of these does. Some of the purchased Van Heerden (1963). His data eliminate the possibility groups were alleged by farmers to be does which had that infection plays any role, and show that many ad aborted while others were stated to have bred normally. vers~ ~nv_iron~ental influences seem to participate by In all instances, however, the farmers' breeding history preop1tating Impending abortion. The occurrence of was disregarded. In this work the term "aborter" is abortion was independent of the genotype of the applied to a doe which had been observed to have foetus, and was associated with an inherent maternal aborted at least once while in our flock, regardless of factor. Van Heerden also postulated that the abortions how many normal kids she may have produced. A were due to a hereditary defect of the anterior hypo "normal" doe was an individual which had produced physis as regards the maintenance of the corpus luteum at least two live term kids, and which had never been 2 S. J. VAN RENSBURG observed to have aborted. These definitions differ from collected in bottles containing EDTA and the methods the human "habitual aborter" as abortion is exceedingly used were those described by Morgenthal (1966). common in women (15 to 20 per cent) when compared Urine samples were collected routinely continuously to the rare occurrence of abortion of non-infectious for 48 hours while the doe was confined in a metabo aetiology in small ruminant animals. lism cage. The animals were trained to the cages by The goats were kept in pens equipped with adequate confining them for at least four 48 hour t'eriods, after shelter throughout the year and were maintained under which time they usually entered the cages voluntarily intensive laboratory supervision. Under these circum and appeared quite contented. Collecting vessels were stances relatively few goats (25 to 65 adult does) could surrounded by solid C0 and embedded in an insulated 2 be kept efficiently, and since Angora goats breed only container; the urine was therefore instantly frozen and once a year, the resulting limited supply of experimental no chemical preservatives were used. At the end of 48 animals with known breeding capabilities necessitated hours the sample was fully thawed, the volume recorded the extension of these studies over eight years. and a filtered aliquot was stored at - 15°C for assay. Dried lucerne hay and a concentrate meal containing All foetuses and dead kids were weighed and dissected fish meal, salt, bone meal, ground maize, teff and rapidly after their discovery. Smaller organs were dis lucerne were fed throughout and were supplemented sected free of connective tissue on moistened filter with a small quantity of green lucerne, barley, or oats paper and weighed on an analytical balance. Stillbirths daily. This very constant ration in no way resembles the were not diagnosed unless both lungs failed to float natural browsing diet, yet we found no indication that in water. Goats to be slaughtered were never fasted but reproductive performance was altered in any way. were herded 91,4 m to the Institute abattoir immediately Internal parasites were controlled by biannual prior to being killed by captive-bolt stunning followed drenching and daily cleaning of pen floors; periodic by exsanguination. Corpora lutea removed after faeces examinations invariably yielded negative results. slaughter or surgery were dissected free of connective All goats were shorn and dipped to control external tissue, weighed, incised for inspection of structure; parasites every six months and the date of shearing and one half was then frozen on solid C0 and the other 2 accurate weight of each fleece were recorded. We found half preserved in 10 per cent formalin. Corpora lutea it most convenient to shear at the commencement of collected at a field station for the nutritional stress the breeding season and again six months later when experiment were preserved quite successfully in 5 per kidding commenced. In this way copulation and suck cent formalin. ling by the resulting kid were facilitated, and the stress of shearing during gestation was avoided. In addition, Surgical procedures animals with excessive hair covering of the face were Operations were performed during the course of this trimmed at three-monthly intervals as this condition is work with the purpose of inspecting the genital tract, deleterious to the productive performance of Angora cannulating the ovarian vein, removing the corpus goats (Shelton, 1960). luteum or ovaries, injecting hormones into the foetuses Bucks were housed adjacent to the breeding does and performing foetal adrenalectomies. Animals were throughout the year and at the beginning of February not subjected to any form of presurgical fasting if the a buck was allowed into the doe pen twice daily at 8 a.m. surgery was to be terminal but when recovery was and 4 p.m. The males were observed constantly while necessary, access to water was denied from the evening with tl-te does and were removed after a brief period. before the surgery. Copulation with a fertile buck was usually permitted No sedative premedications were used. Anaesthesia at the first oestrus. A single buck was used to cover all was induced to effect with pentobarbitone and 5 per the does when experiments such as studies on steroid cent dextrose-saline was administered continuously as metabolism during gestation were performed. Teaser an intravenous drip during the operation. Tracheal males were used to assist with oestrus detection and intubation was routine but since no gaseous anaesthetic their sterility as well as the fertility of intact males was apparatus was available the subject was maintained on evaluated as previously described (Van Rensburg, small periodic doses of barbiturate conveniently McFarlane & van Rensburg, 1963). Daily teasing was administered through the dextrose-saline infusion tube. discontinued after July each year but the pen floors The frequent occurrence of apnoea in goats with this and does were inspected daily throughout the year for system was particularly troublesome but good results signs of gestation termination. Clinical examination were obtained by using thiopentone induction and at of the doe, including the use of a vaginal speculum, the same time injecting one mg acetyl-promazine and even laparotomy, was resorted to if there was any intramuscularly. Atropine was only used when pro doubt about the diagnosis of gestation termination. longed surgery and recovery were necessary. Local Kids were weaned individually when 18 weeks old. anaesthetics were not used as their effectiveness in animals which are incapable of expressing pain clearly Sampling procedures is difficult to assess. Usual aseptic precautions of theatre standard were always observed, but since the Animals from which periodic blood samples were atmosphere was inclined to be dusty and wounds were taken were housed immediately adjacent to the labora not dressed penicillin was administered intramuscularly tory and were quite accustomed to being handled and as a routine before the start of each operation. bled prior to the commencement of the experiment. Samples were always drawn between 9 and 10 a.m.; the doe was immobilized where she happened to be stand General laboratory procedures ing in the pen and immediately bled with a 16 gauge Glassware was subjected to acid treatment after use; needle directly into a 75 ml centrifuge tube containing a sulphuric acid containing 5 per cent of a saturated few drops of heparin solution. The blood was centri solution of sodium dichromate was used initially but fuged rapidly for 20 minutes and the plasma frozen; it the bulk of this work was done with glassware washed was always stored at - 15°C for at least two days prior with methanol and hydrochloric acid as described by to steroid assay. For haematology, 5 ml samples were France, Rivera, McNiven & Dorfman (1965). 3 REPRODUCTIVE PHYSIOLOGY AND ENDOCRINOLOGY OF ANGORA GOATS "Analytical Reagent" quality reagents* were usually three successive rinses of 1 ml methanol. Each rinse used, volatile chemicals were fractionally distilled and was evaporated under a stream of nitrogen. The residue residues of aliquots of some of the purified fractions was redissolved in 0,1 ml of methanol which was then were examined by gas chromatography before use. If transferred to the origin of the paper, followed by two necessary, the solvents were purified further, usually as further rinses of 0,1 ml. The descending chromato described by Bush (1961). Particular care is necessary graphic system used consisted of 90 per cent aqueous with solvents used for cortisol assay as volatile im methanol as the stationary phase and petroleum ether purities were present in some batches of methylene (60 to 80°C fraction) as mobile phase; the sheets were chloride which caused severe losses; such batches were allowed to equilibrate for at least one hour and then discarded. Reference steroids were obtained from run for 3 hours. Progesterone spots were located by Steraloids Ltd., and various authentic steroids were also ultraviolet reflex contact photography and eluted in a donated by the British M.R.C. steroid reference col simplified all-glass Zander-Simmer type apparatus with lection. 4 ml methanol. The dried residue was acetylated with Paper chromatography was performed at 32°C in a 0,05 ml acetic anhydride and pyridine for 1,5 hours at thermostatically controlled constant temperature room 43°C, in an attempt to alter the Rf of the impurities. equipped with an air turbulence fan. Whatman No. 20 After evaporating under nitrogen the extract was chromatography paper sheets were laned into three 1,5 rechromatographed and eluted as before. Cholestane em wide strips for the blank, sample and authentic (0,3 J.Lg) was added as an internal standard before the reference steroid and henceforth the "no touch" eluate was evaporated. Freshly distilled tetrahydrofuran technique was used. The sheets were washed in a (100 J.Ll) was used to dissolve the residue for gas-liquid Soxhlet extractor with methanol for 12 hours and chromatography; when it had evaporated to approxi immediately before use by descending chromatography mately 10 to 25 J.Ll a 5 J.Ll aliquot was chromatographed. in an all-glas tank. Procedural losses were assessed by adding duplicates A Beckman model GC 4 gas chromatograph fitted of 11 goat plasma samples to flasks containing 0,112 J.Lg with 1,83 m glass column packed with 3 per cent S.E. 30 of dry progesterone and assaying the samples as above. on Gaschrom Q t was found highly satisfactory for A mean of 87 ± 0,007 per cent (mean ± standard routine steroid gas-liquid chromatography. This error) of the added progesterone was recovered, con instrument was used to quantitate all the urinary sequently the results were multiplied by a factor of 1,15 oestrogen and peripheral blood progesterone extracts to correct for losses. and also in many of the pregnanediol and corpora lutea Corpora lutea: The majority of samples were assayed analyses. Steroid peak areas were quantitated by plani by the method of Short as slightly modified by Van metry; though a somewhat laborious technique, it is Rensburg & van Niekerk (1968) and the same recovery most accurate and this parameter remains constant rate of 74,4 per cent was assumed, therefore the results with slight variations in retention time. Operating were corrected for losses by multiplying by a factor of conditions were usually as follows: on-column inlet, 1,33. Latterly, samples were assayed by the plasma 300°C; column, 210°C; flame ionization detector, method above using an alkaline homogenate of a very 270°C; nitrogen carrier flow, 50 to 60 mlfmin. small aliquot of luteal tissue with very similar results. Mohair samples were clipped from the shoulder over The isolated fractions from both methods from goat the distal part of the scapula. The diameter of the fibre plasma and corpora lutea were characterized as pre was determined as described by Malan, Carter & van viously described by Van Rensburg & Van Niekerk Wyk (1938) from the proximal portion of the removed (1968) and no evidence was found to indicate that the locks and should therefore represent growth during the isolated substance was anything other than progest preceding month or two. Two technicians each made erone. During the course of this work no evidence was 125 lanometer diameter readings independently on found which suggested the presence of considerable each coded sample and each result represents therefore amounts of 201X-hydroxypregn-4-en-one in goats; a the mean of 250 diameter readings. steroid known to occur in the corpora lutea of certain Various elements were determined on samples of species. formalin-fixed liver by means of atomic absorption spectrophotometry. Wet digestion using a combination of perchloric-sulphuric and nitric acids was used to pre Cortisol assqy pare the samples for analyses using the Beckman atomic In ruminant animals the very low plasma concentra absorption system and DB-G grating spectrophoto tion of cortisol and the many interfering compounds meter. present in the plasma considerably complicate this determination. Cortisol is also exceedingly labile in Progesterone assqy comparison to the sex steroids and therefore the utmost Plasma: An aliquot of thawed plasma (5 to 25 ml) care must be paid to procedural details. During the was extracted twice with five times its volume of early part of the project, a paper chromatographic freshly distilled ether and the pooled ether extracts method was used (Van Rensburg, 1965). For the main washed twice with a 15 per cent volume IN NaOH. experiments, a rapid thin layer chromatographic method Two further washes with an approximately 7 per cent was evolved, which has been used extensively on samp volume of deionized, redistilled water followed. A small les from goats, sheep and cattle. The method entails quantity of anhydrous sodium sulphate was added and ethyl acetate extraction, partition between benzene and each flask was allowed to stand for 15 minutes. water, re-extraction with methylene chloride, TLC, and The extract was dried in a vacuum rotary evaporator§ finally performance of the Porter-Silber reaction directly at 43°C, and transferred to small 2,5 ml test tubes with on the silica gel containing the isolated cortisol. *Riedel-De-Haen, A.G., Seelze-Hannover and E. Merck, A.G., Darmstadt t Applied Sciences Laboratory, Inglewood, California §W. Buchi Scientific Apparatus, Flawil, Switzerland 4 S. ]. VAN RENSBURG Method: A frozen sample is fully thawed by placing the is required the use of the newer competitive protein polythene storage bottle in a waterbath at 43°C and binding radioassays is indicated. then an aliquot (25 to 100 ml, 75 ml was usually used) is Eight recovery experiments were performed by placed in a separating funnel for assay. Two volumes of adding a duplicate plasma sample to a flask containing ethyl acetate are added and the mixture is shaken for 5 fLg of dry cortisol and assaying the sample. The approximately 30 seconds and then allowed to stand for amount recovered averaged 72,0 ± 3,65 per cent 5 minutes while the phases separate. The ethyl acetate is (mean ± SE) and therefore all values were multiplied removed and the plasma is extracted with a further two by a factor of 1,4. The duplicates of 10 samples assaying volumes of ethyl acetate. The combined ethyl acetate between 1 to 7 fLg and averaging 3,4 fLg all yielded very extracts are washed twice with 5 per cent of their similar results; the standard deviation (SD) and stand volume of water, again allowing the emulsion to ard error (SE) of the difference of the results from their separate for 5 minutes before discarding the water. The means were 0,071 and 0,022 respectively. Losses are extract is then dried on a rotary evaporator with a good mainly sustained during the chromatographic step and vacuum, taking care not to exceed a waterbath tempera it is important to keep the time of contact of cortisol ture of 43°C. with silica gel to a minimum; leaving the hormone on Five ml benzene is added to the residue, which re the plate overnight will result in loss of most of the dissolves more readily if the flask is rotated briefly in sample. Heating the extract above 45°C and the use of the waterbath and then transferred to a 100 ml separat impure reagents, particularly methylene chloride, also ing funnel. A 5 ml benzene rinse is also added to the resulted in severe losses; no advantage could be found separating funnel and the benzene is shaken gently in rendering extracts slightly acidic before drying and three times with 10 ml portions of water, avoiding the hence this step was omitted. formation of emulsions. The benzene is discarded and The Porter-Silber reaction was extensively checked the pooled water extracts are extracted twice with 75 ml and optimal conditions for the proportions of reagents, methylene chloride. The pooled methylene chloride is time and temperature factors previously found (see washed with 15 ml cold 0,1N NaOH, followed by a Peron, 1962) were confirmed. Heating the extract to 15 ml water wash. A small quantity of anhydrous 60°C for one hour to hasten the formation of the sodium sulphate is added to the methylene chloride and chromogen, resulted in high blank values and a re after 10 minutes the extract is dried in a rotary evapora duced optical density when compared to samples left tor. Two volumes of 0,1 ml methanol, followed by overnight; there was no change in the corrected optical 0,05 ml, are used to transfer the residue to the origin of density in the latter samples between 17 and 23 hours. a silica gel plate, evaporation being assisted by a stream Considerable difficulty was experienced in eluting the of nitrogen. steroid completely from the silica gel and this problem Authentic reference cortisol is applied to each lateral was overcome by adding the reagents directly to the lane of the plate, and a blank lane left for each sample. powder. Premixing the ethanol and Porter-Silber Immediately the transfers are completed, the plate is reagent gave somewhat erratic results but first shaking run in the system ethanol-chloroform, 23:77 at a the silica gel for 1 to 30 minutes with ethanol before constant temperature; 32°C beibg usually used and the adding the reagent yielded satisfactory results. run was generally completed within 50 minutes. The An excellent correlation between the results obtained plates are briefly scanned without delay under ultra and the physiological state of the animal has been noted violet light to locate the cortisol sports and these are in several species, and in this work fluctuations in the removed with a square-tipped spatula while the plate is size of the goat adrenal cortex were consistently de inclined over weighing paper at the edge of a bench. A tected by the above method of cortisol chemical assay. blank of the same weight and Rf value as that of the Adrenocorticotrophin markedly elevated the plasma sample is also taken and each is transferred to small test concentration; two Angora goats injected intravenously tubes with ground-glass stoppers. To each tube 0,8 ml and assayed every half hour up to two hours after ethanol is added and the tube is agitated for 1 minute injection showed maximal values at one-half and one in a mechanical shaker. After adding 1,2 ml freshly hour after injection. The response to ACTH was prepared Porter-Silber reagent (1 mg recrystallized therefore evaluated routinely one hour after injection. phenylhydrazine hydrochloride per ml added to a Adrenal suppression was also detected in a doe treated mixture of 190 ml water and 310 ml concentrated with exogenous steroids for two days. At 8 a.m. on sulphuric acid*), the mixture is again agitated for 1 each day she received 30 mg prednisolone and at 4 p.m. minute and left overnight in the dark at room tempera 10 mg betamethasone was injected intramuscularly. ture. The following morning the tubes are again shaken Assay of cortisol levels in samples taken at 8 a.m. each and then centrifuged. A Hitachi-Perkin Elmer spectro day was as follows: photometer was used for quantitation. It was necessary Day 1 -before treatment: 0,91 fLg/100 ml in this case to raise the standard 1 em cuvettes slightly Day 2- treated : 0,78 fLg/100 ml in their basket in order to obtain full traverse of the Day 3 -no treatment : 0,24 fLg/100 ml incident light. This operation is simple to perform in Day 4- no treatment : 1,46 fLg/100 ml the apparatus used. Absorption was measured at 370, 410 and 450 mfL and the corrected optical density at The isolated steroid from goats plasma was character 410 mfL was obtained by using the formula of Allen ized further by eluting it from the silica gel and sub (1950). jecting it to gas chromatography. Characteristic dis sociation changes found with increasing increments of Comment: The main disadvantage of the method is a column temperature were identical to that observed lack of sensitivity, necessitating large volumes of with authentic cortisol. The ultraviolet absorption of an plasma, although in species such as the horse and dog ethanolic solution as well as the sulphuric acid chromo 5 to 10 ml was found to be adequate. Fluorimetry was gen also provided identical spectra to that obtained not attempted on the final extract but where sensitivity with authentic cortisol. *BDH Micro-analytical grade 5 REPRODUCTIVE PHYSIOLOGY AND ENDOCRINOLOGY OF ANGORA GOATS Urinary oestradiol-17a and oestrone assqy erature 225°C; flame ionization detector block 240°C, In the goat, this assay is complicated by the fact that and nitrogen carrier flow 60 mljmin. oestradiol is in the unstable 17a form (Klyne & Wright, Comment: Exceedingly clean chromatograms were ob 1957). By using a combination of enzymic and acidic tained with the method and the very minor impurities hydrolysis, we were able to show in our preliminary present were also obtained from acetylated blank studies that oestradiol-17a was the major steroid present paper eluate residue. Recovery experiments were not during gestation, together with fair amounts of oestrone, performed, as steroid conjugates were not available. A traces of oestriol, and no detectable oestradiol-17 ~· simpler method used in this laboratory for the assay of For routine purposes, therefore, only oestradiol-17a oestradiol in follicular fluid (Van Rensburg & Van and oestrone were assayed. The method below is based Niekerk, 1968) incorporated several similar steps and a on principles which have been well established by mean of 80 per cent of added hormone was recovered. numerous workers. Twelve urine specimens were assayed in duplicate and the oestrone value were found to range from 9 to Method: The pH of a 10 ml aliquot of thawed urine is 239 ~-tg/24 hours, with a mean of 76; the SD and SE adjusted to 4,8 with cone. HCl and one ml of 0,1 M of the difference of the results from their means were acetate buffer containing 11 mg EDTA is added. The 5,72 and 1,65 respectively. The oestradiol fraction varied enzyme ~-glucuronidase (mollusc-BDH) is added at from 2 to 709 ~-tg/24 hours with a mean of 177; the SD the rate of 300 Fishman units per ml urine and the and SE of the difference of these results from their sample is incubated at 37°C for at least 4 hours before means were 15,9 and 4,5 respectively. being extracted three times with 30 ml ether. Acid The procedures previously used (Van Rensburg & hydrolysis is then performed on the remaining aqueous Van Niekerk, 1968) were all applied to characterize the p~ase by adding 1,5 ml cone. HCl and refluxing for 18 oestrone and oestradiol-17a isolated from goats urine. mmutes. The sample is rapidly cooled under running In addition, the Kober chromogens were characteristic, water and again extracted three times with 30 ml of and identical retention times to the authentic com ether. pounds were obtained on GLC of the free steroid, the The pooled ether extracts are washed with one-sixth acetates, and trimethylsilyl ethers. volume of 1N NaOH saturated with NaCI, followed by a similar' "1ume of water. The ether is evaporated on a rotating Su.J and the residue redissolved in 114 ml of a CHAPTER 2 ~ixture of ether-chloroform 1 :18. This organic solvent SEXUAL BEHAVIOUR AND BREEDING PERFORMANCE 1s extracted three times with 50 ml 1N KOH and once with 10 ml water. The pooled aqueous phase is acidified Angora goats are strictly seasonal breeders; the de to pH 3,0 and extracted once with 100 and twice with creasing length of daylight in autumn stimulates the 80 ml portions of ether. Washing of the pooled ether appearance of oestrous behaviour in the female and the extract is performed once .vith 50 ml of 1N NaOH male also exhibits increased libido in conjunction with saturated with NaCL, once with 25 ml 0,1N HCl and the characteristic buck odour. The breeding season finally with 50 ml water. The ether is dried with a small lasts about four months (March to July in the southern quantity of anhydrous sodium sulphate, evaporated on a hemisphere), during which time the average doe will rotary evaporator and the residue transferred to small exhibit six or seven oestrous cycles if not bred. The tubes by dissolving it in one ml methanol and then length of the oestrous cycle exhibited by goats in Texas evaporating it under nitrogen at 55°C and repeating has been reported to average 19,5 days with 80 per cent with two further rinses of one mi. in the range of 19, 20 or 21 days (Shelton, 1961) and Sheets of chromatography paper are laned into two was found to be of similar duration in this country 1,5 em. wide strips; on the origin of one strip some (19,4 days, Marincowitz, 1962). This is considerably authentlc oestrone and oestradiol-17a are spotted and shorter than the duration of oestrous cycles in milk the sample is transferred to the origin of the other strip. producing goats which average 23 days (Phillips, The descending chromatographic system used was Simmons & Schott, 1943). This study shows that toluene: petroleum ether: methanol: water 5:5:8:2. abnormally short cycles are frequent amongst aborters After one hour equilibration, the chromatograms were and are associated with infertility. run for 2 hours, air dried and the reference strips dipped Duration of gestation in goats is virtually identical to thr~ugh a f~eshly mixed aqueous solution of 2 per cent the five month period of sheep and our mean of 149,4 ferne chlonde and 2 per cent potassium ferricyanide. days resembles the Texas value of 149,2 days. An in The oestradiol and oestrone zones in the sample strip teresting finding reported below is slight prolongation were eluted separately with 2,5 ml methanol, which of the occasional successful pregnancy experienced by was evaporated under nitrogen. The remaining portions the aborter ewe; as demonstrated in Chapter 9 this may of the sample strips were dipped through the detecting well be due to a protective action of hypercortisolism. reagents to establish that the hormone spots had been Much of the characteristics of the gestational failure fully removed. syndrome resembled the field observations of Van Acetates for GLC were formed by adding 0,1 ml of Heerden (1963), but under our experimental conditions both pyridine and acetic anhydride to the residue and more detailed observations were possible. leavinfS the mixture for one hour at 65°C or alternatively overmght at room temperature. After evaporation, the Results residue is redissolved in an accurate volume of tetra Oestrous behaviour hydrofuran according to the expected amounts of The time of commencement of the breeding season: The mean hormone present (50 to 200 ~-tl) and 5 ~-tl is chromato dates_w hen the first oestrus of the year was exhibited by graphed; standard curves are constructed with authentic normal and aborter does were calculated separately for steroids in the same manner. The most convenient seven years. "Aborter" does were those in which the co_nditions found for the Beckman GC 4 used during breeding season under study was destined to end in th1s work were: on-column inlet 290°C; column temp- abortion. This value may reveal any possible differences 6 S. J. VAN RENSBURG in the activity of the hypothalamic-pituitary gona~o­ phenomenon was, however, not a~sociat~d. with abor trophic axis following stimulation, chiefly by decreasmg tion as some potential aborters which exhibited oestrus daylight length and association with males. during pregnancy, carried thei~ kids to term. The Management of the flock was standardized as far as occurrence of this oestrous behaviOur was evenly distn possible, yet the seasonal variation was from the 1st buted between 12 to 60 days after conception. of March to the 25th of April for various groups. For Oestrus after abortion: More than 60 per cent of the ~oes any particular year, however, the dates for normal and observed exhibited oestrus within a month of abortmg. aborter groups were in close agreement. ~borters The majority of these were receptive to the ram within a tended to exhibit oestrus slightly later, but no differenc week, but the intervals were quite irregular and ranged es were significant in any of the seven years studied from the day of abortion up to 29 days later. Those (Table 1). does showing oestrus shortly after ~bortion _usually repeated oestrous behaviour about twice, Invanably at TABLE 1 Average time 1vhen first oestrus of each breeding abnormally short intervals. Only about 25 per cent of season 1vas exhibited those bred were later proved to have conceived, the earliest conception after abortion being the 15th da~. Normals Aborters Surprisingly, three out of four such conceptl?ns ulti mately resulted in the productiOn of a viable kid, whrle Year No. of Average SE No. of Average SE the fourth conception again terminated in a late abor goats day (days) goats day (days) tion. 1962 14 25 April 4,3 6 12 May 9,8 1963 21 28 March 3,0 6 3 April 7,2 Conception rate and intervals of return to oestrus after un- 1964 23 21 April 5,6 7 21 April 13,9 successful breeding 1965 30 23 March 3,9 12 27 March 4,5 1966 34 28 March 1,6 20 2 April 4,2 Conception rates: Anoestrus during an entire breeding 1967 30 3 April 5,7 19 25 March 6,2 season was not encountered in reasonably healthy does. 1968 33 7 March 3,6 22 1 March 6,1 When it did occur, it was due to immaturity, extreme senility or severe organic disease. Differences for each season not significant (P<0,05) A total of 205 breedings with bucks known to be SE= Standard Error fertile was analyzed. Breedings with aborter ammals totalled 98 and the remaining 107 does bred were Oestrus duration: The goats were observed twice daily classed as normal. Does were served twice daily as long for signs of oestrus and the results of four seasons w~re as they remained receptive. pooled for analysis, allowing 12 hours per poslt!ve When bred at a single oestrus, 77 per cent of the observation. normal does and 60 per cent of the aborters were A mean duration of 22,3 hours was found for 157 proved to have conceived. After those that returned oestrous periods exhibited by normal does; 117 periods had been bred at two further periods, a total of 91 per of aborter does lasted a mean of 21,4 hours. Of the cent normal and 80 per cent aborter does had conceived. total of 274 oestrous periods recorded, only six were as In the normal flock, 9 per cent were barren, as compared long as 48 hours. Our goats have therefore a con with 20 per cent in the aborter group. sistently short oestrous period of a day or less and no differences between normal and aborters were apparent. Interval of return to oestrus after unsuccessjul breeding: This analysis was made to obtam some mdrcauon of the Duration of the oestrous rycle: The occurrence of shorter importance of abnormally short cycles and the possible oestrous cycles in aborter does has been previously occurrence of foetal resorption, usually manifested by reported (Van Rensburg, 1964). Analysis of a total of prolonged cycles. Incidental failure to conceive should 58 cycles showed that the average duration for normal be reflected by returns at normal cycle lengths. does was 20,6 days, whereas for aborters it was 16,2 Figure 2 shows that the majority of returns (58 per days (P<0,01). Further studies have confirmed this cent) exhibited by normal does occurred at 19 to 22 or difference. The total of 124 cycles now studied com 38 to 44 days after fertile service. These returns at prises 76 normal doe cycles and 48 exhibited by aborter normal cycle lengths are assumed to be lar!Sely due to does and the distribution of their duration is presented fertilization or nidation failure from miscellaneous in Fig. 1. . causes. The existence of considerable foetal loss within Peak cycling frequency in normals was 20 days and In the normal group is suggested by the 27 per cent aborters 19 days. However, only nine per cent of returning to oestrus between 25 and 32 days. Follow normals' cycles were 19 days or less, whereas 69 per ing sterile service, only 5 per cent were found to have cent of aborter cycles were in this category. Short such prolonged cycles. cycles of 5 to 10 days were common and laparotomy of Returns after short intervals of 18 days or less ac two ewes exhibiting oestrus after such short cycles counted for 58 per cent of the failures on the aborter revealed only very small regressed corpora lutea in the group. A striking peak oc~urred at seven days . a~ter ovaries, together with pronounced follicular growth. service, suggesting early farlure of adequate lutermza Unlike normal does, unusually prolonged cycles were tion. Short cycles were also observed m the group whic_h virtually absent in aborter animals. The four cycles of was subjected to sterile service and this phenomenon Is 37 and 38 days duration were probably double cycles clear evidence of a basic defect in the aborter group. resulting from "silent" or missed oestrous periods. Evidence of resorption of foetuses was ~lsc;> present These basic differences between the two groups suggest as in the normal group, but the data on the rnc~de?ce of that luteal regression is premature in the aborter group resorption are not comparable, as. the maJon~y of and that follicular growth and ovarian oestrogen secre aborters returned without the endocnne opportumty of tion is normal or excessive. conceiving. It must therefore be assumed that the _i~ci­ Oestrus duringpregnanry: Typical oestrus was encountered dence of resorption in aborters capable of mamtammg in approximately 6 per cent of all gestations and w~s one normal oestrous cycle length after fertilization is twice as frequent in aborters as in normal ewes. This very much higher than in the normal group. 7 REPRODUCTIVE PHYSIOLOGY AND ENDOCRINOLOGY OF ANGORA GOATS 40 30 20 ., ..! u> . 10 u 0 .~... 0 5 10 15 20 25 30 35 40 Q) 0 .E c: uQ ) 30 Q; ABORTERS - 48 CYCLES Q. 20 10 5 10 15 20 25 30 35 40 45 Duration of cycles (days) FrG. 1 The length of oestrous cycles in Angora goats NORMAL-33 RETURNS 0 -~ 0 5 10 15 20 25 30 35 40 45 Q) 01 0 ~ 15 ABORTERS-43 RETURNS ~ ,f 10 5 5 10 15 20 25 30 35 40 45 Duration of return interval (days) FrG. 2 Intervals of return to oestrus after unsuccessful breeding 8 S. J. VAN RENSBURG Miscellaneous causes of conception failure, deduced TABLE 2 The incidence of kidding, abortion and barren from returns at normal cycle lengths, accounted for 16 seasons in successive gestations per cent failures in the aborter group, as compared with 58 per cent in the normal group. Percentage of total Gestation Total Kidded Barren Aborted Duration ofg estation Only does which terminated their pregnancies after 1st 97 66 21 13 the 140th day were considered to be within the normal 2nd 77 65 16 20 3rd 56 54 21 25 range, as viability was not recorded before this time and 4th 33 55 15 30 shorter gestations were therefore classed as abortions. 5th 14 21 7 71 A total of 106 single gestations within the normal 6th 5 20 20 60 ± ± range lasted 149,4 0,21 (mean SE) days. The range was from 143 to 153 days. Contrary to many pre Stage of pregnancy at which abortion occurs: The earliest vious reports, not a single gestation of the total of 245 abortions confirmed by recovery of some of the con studied was longer than 153 days. These observations ceptus tissues were two on the 34th day. Abortions were were made over many years in small groups and under frequent from this time up to the 46th day, but there strict laboratory control. With less intensive super after only rarely encountered up to the 90th day. vision, the high frequency of cycles lasting a week or Figure 4 illustrates the high incidence of abortion be less may well result in "recorded" gestation period of up tween 90 and 139 days, with a sharp peak shortly after to 160 days if these short cycles are overlooked. the 100th day of gestation. As will be seen later, the The frequency distribution of these gestation lengths peak incidence of abortion coincides exactly with a will be seen to be skewed in Figure 3. The maximum rapid increase in the growth rate of the foetus. number of births does not occur on the arithmetical Consecutive breeding patterns of individual goats: The out mean of 149 days. Furthermore, it was found that an come of gestation and duration of pregnancy in all 20 additional 15 does which concluded a successful single goats, on which a complete breeding record of at least gestation after having previously aborted, had gestations four seasons was available are presented in Table 3. significantly longer (151,0, SE = 0,41, P< O,OOS) than These does largely represent the troublesome inter the normal does. These differences suggested that there mediate type of goat, as those which had only aborted is an unusual factor which slightly prolongs gestation in or kidded up to three times in succession were usually potential aborters and also in some does which were used for experiments and not retained longer. considered to be normal. Twin pregnancies averaged a day less (148,4, SE = TABLE 3 Consecutive breeding patterns of individual does 0,81, 16 observations) than single gestations, but the difference was not significant. 1 2 3 4 5 Occurrence ofg estational failure K -153 KT-151 KT-146 KT-144 K -148 KD-151 K -150 K -151 K -150 A -100 Incidence of gestational failure: The constitution of the AT-105 K -151 KT-148 A -101 A -105 experimental flock cannot strictly be considered repre K -152 B B K -153 KT-153 sentative of the breed, as aborter does were at times K -152 A -104 A - 98 A - 96 A - 91 K -146 KS -148 A -119 A -117 A -109 specifically selected for purchase. On the other hand, A -108 B A - 46 K -150 A -104 concerted efforts to obtain normally breeding groups KS -149 KD-149 K -152 A-91; K-148 A - 91 were made, but were never fully successful. K -148 KD-145 A - 44 KD-151 A - 38 Of a total of 245 gestations studied, no viable kids K -147 KT-147 KT-147 KT-147 K -150 K -152 KS -150 A -133 were produced in 101 instances (41,2 per cent). Twenty KS -148 A -105 A -118 A -123 one kids (8,6 per cent) died within three days of birth K -151 A - 45 KD-148 K -151 and nine (3, 7 per cent) were stillborn. Gestations ending K -151 A -119 K -149 B in abortion before 140 days amounted to 71 (29,0 per K -150 B K -152 K -148 KD-150 K -149 A -123 B cent). B KD-149 K -151 K -150 This incidence of 29 per cent aborters is quite usual B B K -150 A - 93 for commercial flocks (Van Heerden, 1963, 1964). K -149 A - 92 K -152 K -151 However, the unusually high 12 per cent non-viable B A-34; A-118 A -100 KS -151 kids born at term was possibly due to the optimal K = Kidded; D = Kid died within 3 days; S = Stillborn; management and nutrition of the flock which allowed A = Aborted; T = Twins some pregnancies, that would otherwise have been Numerals adjacent to letters indicate duration of gestation aborted to proceed to term. The incidence of abortion, kidding and barren seasons The data in Table 3 emphasiLe the variable breeding with each successive gestation was then examined. The performance of our type of animal; it is not unusual for does in this study consisted of both purchased mature an "aborter" to produce a normal kid. Nevertheless it animals that had bred previously and maiden does bred does seem that non-viable kids and stillbirths are related in the experimental flock. Only gestations recorded in to the problem of abortion and generally the breeding the experimental flock were analysed. Table 2 clearly performance is inclined to become less productive with shows that age did not influence the incidence of barren each successive gestation, with a strong repetitive seasons. Abortions, however, increased dramatically tendency for the majority of individuals. from 13 per cent in the first gestation studied, to some 70 per cent in the fifth successive pregnancy. It must be Sex and birth weights of offspring emphasized that those does exhibiting a 70 per cent inci Sex ratios of foetuses and kids: In sheep selection for dence of abortion during the fifth gestation were in fineness of birth coat can lead to increased loss of excellent condition, and not showing signs of senility particularly female embryos when the sheep attain such as emaciation. adulthood and are bred (Schinckel, 1955). 9 REPRODUCTIVE PHYSIOLOGY AND ENDOCRINOLOGY OF ANGORA GOATS 25 .. .. ., . c: 20 c0 : -"' ~ Q. .. 15 0 ~ .0 E z::> 10 5 143 145 147 149 151 153 Duration of gestation (days) FIG. 3 The duration of gestation in normal Angora goats 25 20 "' .c.. 0 ~ 0 -.a 15 c 0 ~ .Qa) 10 E z~ 5 30 40 50 60 70 80 90 100 110 120 130 39 49 59 69 79 89 99 109 119 129 139 Range within gestation (days) FIG. 4 The stage of pregnancy at which abortion occurs 10