THERIOGENOLOGY DETECTION OF OESTRUS IN THE AFRICAN ELEPHANT (Loxodonta africana) P.F. Watson* and F. D'Souza Wellcome Institute of Comparative Physiology, The Zoological Society of London, Regent's Park, London NW1 4RY (Received for publication:

November 19, 1975)

SUMMARY Swabs of mucus and cells from the reproductive tract of a 15 year old female African elephant in captivity were examined. Daily samples were obtained over a l-year period by means of a probe designed to penetrate the urogenital sinus to a depth of 90cm. Dried smears of mucous material showed ferning patterns at intervals of approximately 16 days. Dried spots of supernatant from washings of the swabs also showed intense ferning at 16-day intervals, but with greater regularity. Smears were stained and examined for the presence of squamous cells over a 4-month period. Results indicate a regular occurrence of Keratinisation at approximately 15-day intervals. These observations indicate that the oestrous cycle of this elephant has a duration of approximately 16 days. This is the first detailed study of the oestrous cycle in the African elephant, knowledge of which is essential for artificial breeding. ACKNOWLEDGEMENTS We particularly acknowledge the assistance of Messrs. W.G. Crompton and M.A. Hennessy who obtained all the swabbings. Without their long experience of the elephant this work would not have been possible. We wish to thank Dr. B. Seaton for the use of his computer programne, Dr. R.D. Martin for his encouragement and all the technical staff of the Institute for their able assistance. We were both supported by Ford Foundation Fellowships.

*Present address:

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Department of Physiology, Royal Veterinary College, Royal College Street, London NW1 OTU

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THERIOGENOLOGY INTRODUCTION Captive breeding of African elephants is a rare event and very little is known of the reproductive cycle in the female of the species. Our interest in determining oestrous patterns arose as part of a longterm reject to develop artificial insemination in captive elephants (3, 5p. It is well known that there are considerable hazards involved in maintaining mature males and therefore it is envisaged that future successful breeding of this species will rely heavily on artificial insemination. Short (6) suggested that the oestrous cycle may be approximately 2-3 weeks in duration. Our results confirm this suggestion and were based on the examination of material taken from the elephant's urogenital sinus. MATERIALS AND METHODS A probe bearing a dry gauze swab was designed to sample mucus and cells from the reproductive tract of the elephant at a depth of about 86 cm from the external orifice of the urogenital sinus. During its passage through the lower part of the urogenital sinus, the swab was protected from contamination by the use of an outer polythene tube sealed with a plastic membrane. Swabbings from a mature female African elephant (aged about 15 years) were taken daily. The amount of visible material on the swabs varied but was never very copious, Areas of material on the swab were pressed on to two clean microscope slides. One slide was airdried while the other was flooded with polyethylene 1 co1 fixative and later stained by a modified Papanicolaou method 94; 1 . The swab was then washed in 3 ml distilled water. The washings were centrifuged at 1OOOg for 10 min and drops of the supernatant were dried on a slide at 37°C. The unstained smears and spots were examined for the presence of fern patterns (4); the intensity of ferning was scored subjectively on a O-3 scale. Stained smears were examined for parabasal, intermediate, both mature and degenerating superficial cells, leucocytes and erythrocytes (7). The cells were also scored on a O-3 scale which took into account both the number of cells present from day to day and the ratio between cell types. RESULTS At intervals the stained smear was characterised by a higher proportion of mature superficial cells than any other cell-type. On the few days immediately preceding this picture there were more parabasal and intermediate cells than mature cells, whereas degenerating superficial cells taking a deeper stain predominated on the following few days. The oestrous phase was considered terminated when the squamous cells showed a tendency to clump, fold and disintegrate, though some folded squames were seen at all stages of the cycle.

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Intermediate cells were observed both during and following the time of maximum keratinisation. Large numbers of identifiable leucocytes were never seen but occasional neutrophils were observed 3-4 days after maximum keratinisation and erythrocytes appeared only infrequently and always within 2 days of this stage. There was a marked scarcity of cellular material for three days preceding and three days following the appearance of mature superficial cells. Around the mid-point between consecutive periods of 'oestrus' stained mucus was observed, sometimes in abundance. The results of the ferning and mature and superficial cell scores over the period December-April are presented in Fig. 1. A considerable degree of correlation can be seen between the incidence of fern patterns and the presence of cornified squames. Occasionally the dried spots showed ferning when the smears were negative. The number of consecutive days over which strong ferning occurred never exceeded 2 days. The mean duration of high proportions of mature superficial cells in the smear was 3 days (N = 8, range l-5 days). Periodicity was not immediately apparent from inspection of the data (Fig. 1). All sets of data were therefore analysed for latent periodicity using a computer programne in which a rank was assigned to each observation, taking into account the possibility of missed and multiple observations.* A range of possible cycle lengths (i.e. different possible ranking schemes) were tried and the one giving the best straight line of occurrence versus rank was taken to indicate the actual cycle length. The data for ferning in smears (scores 2 and 3) gave'a periodicity of 16.3 days, while the data for ferning in spots of supernatant (scores 3 only) had a period of 16.4 days. Keratinised cells occurred at intervals of 14.8 days. -*(See Figure 2) DISCUSSION The ferning data had a periodicity of slightly longer duration than that of the cell data. However, there was a greater scatter of points about the line for the latter set of data which indicates that the precision of this method for determining oestrus is less reliable than the method based on mucus ferning. Nevertheless, the period found for this set of data is sufficiently close to that of the ferning data for the difference to be accounted for by day-to-day variations in the swabbing method. Such variation is expected when dealing with an animal of this nature under these conditions. Our data from a captive African elephant indicate an oestrous cycle length of approximately 16 days which supports Short's (6)

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calculations on data from wild African elephants. There are obvious cyclical changes in both the mucus and cells of the upper part of the reproductive tract and these changes correspond well to those seen in other mammal species in periods of pro-oestrus, oestrus and post-oestrus. The 'spotting' technique appeared to be more sensitive when material was limited. In other mammal species (e.g. rat, guinea pig, domestic cat) keratinised cells are indicative of late pro-oestrus and oestrus; ferning of cervical mucus has been used as an indication of ovulation in the human female. Both phenomena result from the action of oestrogens. Using receptivity to the male as an indication of,oestrus, Jainudeen -et al. (2) reported that the Asiatic elephant has a cycle length of approximately 22 days (range 18-27 days). However, no regular pronounced cell or mucus changes were noted from urogenital smears in this species; this was probably due to the fact that smears were taken from the lower portion of the reproductive tract, only 45 cm from the external orifice. It is very likely that the smears were grossly contaminated with urine and other debris. Furthermore, steroid hormones, at least in the human female, appear to affect different areas of the reproductive tract selectively (7). The duration of receptivity in the Asiatic elephant is 3-4 days (2); our data for the African elephant based on fluctuations in vaginal cells and mucus suggest an oestrus duration of 2 days, which again corresponds well with Short's observations of a receptive period of 24-48 hr in a single wild African elephant (6).

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3’ 2’

:’

111#11111 I II IIII.,11,111,111111l IIIII11111.,1,111..11I I

Fig.1 Patterns of change in cells and mucus from the reproductive tract of the African elephant. O-3 represents subjective scores of (A) degree of Keratinisation of superficial cells; extent and intensity of ferning in (B) dried spots of supernatant, and (C) dried mucous smears.

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REFERENCES

208

1.

Disbrey, B.D. and Rack, J.H. Histological Laboratory Methods. E. and S. Livingstone, Edinburgh and London (1970) pp. 327331.

2.

Jainudeen, M.R., Eisenberg, J.F. and Tilakeratne, N. Oestrous Cycle of the Asiatic Elephant, Ele has maximus, in Captivity. J. Reprod. Fert. -27: 321-328 ( TdT-

3.

Jones, R.C. Collection, Motility and Storage of Spermatozoa from the African Elephant Loxodonta africana. Nature, Lond. 243: 38-39 (1973).

4.

Rowland, M. A Simple Test for the Determination of Ovulation, Estrogen Activity, and Early Pregnancy Usin the Cervical Mucus Secretion. Am. J. Obst. Gynec. -63: 81-89 9 1959).

5.

Rowlands, I.W. Artificial Insemination of Mammals in Captivity. Int. Zoo Yb. -14: 230-233 (1974).

6.

Short, R.V. Oestrous Behaviour, Ovulation and the Formation of the Corpus Luteum in the African Elephant. East Afr. Wildlife J. 4: 56-68 (1966).

7.

Wied, G.L. and Bibbo, M. Hormonal Cytology of the Female Genied. A.I. Sherman. tal Tract. In: ‘Pathways to Conception’ Charles C. Thomas, Springfield, Illinois (1971) pp. 188-213.

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Detection of oestrus in the african elephant (Loxodonta africana).

THERIOGENOLOGY DETECTION OF OESTRUS IN THE AFRICAN ELEPHANT (Loxodonta africana) P.F. Watson* and F. D'Souza Wellcome Institute of Comparative Physiol...
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