Ovarian function in the captive black mastiff bat, Molossus ater J. J. Rasweiler IV Department of Obstetrics and Gynecology, Cornell University Medical College, 515 East 71st Street, New York, NY 10021, U.S.A. Summary. In nearly all animals from a laboratory breeding colony that were examined (85/86) the right ovary was significantly larger than the left. Although primordial follicles were present in both ovaries, Graafian follicles and CL were noted only in the right ovary. The left ovary usually had a much less prominent intraovarian vascular supply, and it is suggested that this may play a central role in limiting the ability of follicles to grow on that side. Many of the bats examined very soon after the intro- duction of stud males had well-developed CL, sometimes of 2\p=n-\3different ages, and uteri that had probably been subjected to stimulation by luteal hormones. Such observations made on females that had been housed with a stud male only for 24 h indicate that the black mastiff bat is a spontaneous ovulator with a functional luteal phase. It was common to observe an extended period after the introduction of a stud male during which spermatozoa were present in the vaginal smears from a female almost every day. Most of the ovulations that resulted in pregnancies appear to have occurred during this period. Of the 72 bats with CL, 11 possessed 2 or more CL of the same age, indicating that multiple ovulations can sometimes take place. The right ovaries of all females examined during advanced pregnancy had non-atretic, vesicular or Graafian follicles in addition to the CL of pregnancy. Keywords: black mastiff bat; ovarian asymmetry; ovulation Introduction The black mastiff bat (Molossus ater) is an insectivorous, colonial species which is common in many tropical areas of the New World. Recently, efforts have been successfully undertaken to breed this bat in captivity (Rasweiler, 1987), so that it could be utilized as a model in investigations focussing upon the morphogenesis of the chorioallantoic placenta. The present paper deals with some of the observations made on ovarian function in the captive animals, most of which were killed primarily for the placental studies. Materials and Methods Animal procurement. All of the bats utilized in this study were captured with mist nets as they left their roosts in buildings in central Trinidad, West Indies, to forage. Collections were made during early July, mid-August and late November. Animal care and management. The bats were maintained in a laboratory colony established at Cornell University Medical College according to the procedures described previously (Rasweiler, 1987). The colony was kept in a warm room (29-31°C) with the lights on from 04:00-16:00 h. The females were housed in sexually-segregated groups of 8-15 animals per cage from the time of their arrival in New York. Between 2 and 7 months later a single male was introduced into each of these cages. Vaginal smears were then obtained from the females each morning and checked for spermatozoa. Many of the females housed with males for prolonged periods exhibited a period of 10-50 days during which spermatozoa were present in the vaginal smears almost every day. Spermatozoa were also observed sporadically in the smears before and after this interval (Rasweiler, 1987). It was suspected that ovulation occurred at Downloaded from Bioscientifica.com at 10/11/2021 11:21:05AM via free access some time during the period when spermatozoa were present for many days in succession. In an effort to test this hypothesis and to obtain reproductive tracts in different stages of pregnancy, females were killed at various times after the first appearance of spermatozoa in the vaginal smears for 3 days or more in succession. To investigate further the influence of the male upon ovarian function, one group of 8 females was killed after having been sexually isolated for 7 months. During the last 24 h these animals were housed with a stud male to collect information on their sexual receptivity and sperm transport in the female tract. A single vaginal smear was obtained from each of these females immediately before death. All of the subjects of this study were killed between January and June of the year after capture. Hislological procedures. Females were killed between 09:00 and 12:00 h and their reproductive tracts were removed for histological examination. When large enough to be easily removed from the tracts, conceptuses were fixed separately, measured and weighed. The tracts were fixed in Zenker's fluid for 10-12 h, washed overnight in running tap water and processed through graded ethyl alcohols, cedar wood oil and Histosol (National Diagnostics, Inc., Somerville, NJ, U.S.A.) to paraffin wax. Most of the tracts were then serially sectioned at 6 µ . Because of their size the only exceptions were the tracts obtained late in pregnancy. In these cases usually just the ovaries and that portion of the uterus containing the chorioallantoic placenta were serially sectioned. The histological sections were stained with haematoxylin and eosin, Masson's trichrome procedure, or the periodic acid-Schiff (PAS) technique and haematoxylin. Some sections from each tract were incubated for 1 h at 37°C in 01% a-amylase (1,4-a-D-glucan- glucano-hydrolase; Sigma Chemical Company, St Louis, MO, U.S.A.) dissolved in a 0-02 M-phosphate buffer (pH 6) to remove any glycogen before being stained by the PAS procedure. Parallel sections were incubated under similar conditions in the buffer alone and then stained. The glycogen-hydrolysing ability of the enzyme solution was confirmed by tests on sections of liver and skeletal muscle. Reproductive condition of laboratory-maintained bats. Female black mastiff bats in the following reproductive states were examined in this study: non-pregnant with no large follicles or corpora lutea (10 bats); non-pregnant with healthy, large vesicular or Graafian follicles (12 bats); non-pregnant with recently formed CL (17 bats); non-pregnant carrying a newly-released secondary oocyte (1 bat); pregnant with a tubai embryo (1 bat); pregnant with uterine conceptuses varying from unimplanted embryos to near-term fetuses (45 bats). Results Reproductive seasonality in the wild bat population Some of the observations made on black mastiff bats in this study may reflect seasonal breeding patterns in the wild population and the time of year at which the experiments were carried out. It Figs 1 & 2. Sections through the left (Fig. 1) and right (Fig. 2) ovaries from a non-pregnant bat (No. 52B) killed 5 days after the introduction of a stud male. Note the marked size difference between the two ovaries. Two CL of different ages are evident in the right ovary (see also Figs 9 & 10). Both ovaries are surrounded by bursae which have openings (not shown) to the peritoneal cavity near the mouth ofeach oviduct. PAS-haematoxylin, 31. Downloaded from Bioscientifica.com at 10/11/2021 11:21:05AM via free access Figs 3 & 4. Medium-power view of the left (Fig. 3) and right (Fig. 4) ovaries from Bat 59B. Numerous primordial follicles are present in the cortices of both ovaries, but no growing follicles are evident in the section of left ovary shown. Both ovaries also contain abundant interstitial gland tissue. Note that the intraovarian vascular supply of the right ovary is much more prominent than that of the left ovary. PAS-haematoxylin, 80. therefore seems important to present the following preliminary evidence that reproductive synchro¬ nization does occur in the species. Some of the females taken in July (25/215) and August (10/118) were pregnant and aborted or gave birth between capture and mid-September. This may under¬ estimate the proportion of pregnant bats in the wild population at those times because the precise reproductive condition of all the recently captured females could not be determined. In contrast, none of the females (N = 180) captured in November aborted or delivered young after being brought into captivity. Downloaded from Bioscientifica.com at 10/11/2021 11:21:05AM via free access Downloaded from Bioscientifica.com at 10/11/2021 11:21:05AM via free access Fig. 7. Section of right ovary from Bat 54B showing the Graafian follicle. This contained an oocyte at the germinal vesicle stage. This animal was killed 5 days after the introduction of the stud male and 4 days after the first appearance of spermatozoa in her vaginal smears. PAS- haematoxylin, 80. Differences between right and left ovaries In nearly all of the female black mastiff bats differences were noted between the right and left ovaries. With the exception of one animal, the right ovary was always larger (Figs 1^4). Both ovaries contained primordial follicles, but it was not readily apparent whether the total number of these follicles differed consistently between the two sides in all animals, because the primordial follicles tended to be more widely dispersed along the cortical periphery in the larger right ovary than in the left. Follicular development beyond the primordial stage was generally retarded in the left ovary. In most bats (81/86) the largest follicles seen on the left side were only primary follicles with enlarged oocytes or secondary (non-antral) follicles with no more than 5-6 layers of granulosa cells. A few vesicular follicles were noted in the left ovary in 4 animals, but these were all atretic. Healthy Graafian follicles and CL were observed only in the right ovary (Figs 2, 4, 7, 8, 13, 14). In pregnant animals the conceptuses were always in the right oviduct or uterine horn. Both ovaries contained an abundance of interstitial gland tissue (Figs 3-6) in all specimens examined, but the total volume of this component was generally much less in the smaller, left ovary.