The effects of 5-bromo-2-thienyl-ethyl-ketone thiosenicarbazone on ovarian cyclicity and ovulation in the rat.

Pseudopregnant and cyclic rats were injected for 5 to 26 days with daily doses of 5 and/or 3 mg of 5-bromo-2-thienyl-ethyl-ketone thiosemicarbazone (70026) starting on Day 0 (the day of oestrus). The vaginal smear cytology, record of ovulation and ability to breed and conceive were compared with the results for corn oil-injected controls. Both doses of 70026 were found to cause a reappearance of pro-oestrous and/or oestrous vaginal smears within 4 to 6 days in the pseudopregnant rats, but ovulations did not occur. The 5-mg dose of 70026 inhibited ovulation and interrupted the oestrous cycle in cyclic rats, even though the daily 3-mg dose seemed to have little effect on ovulation, ovarian cyclicity, breeding or conception. In spite of the absence of an ovulation accompanying the induced pro-oestrous and/or oestrous vaginal smears in the pseudopregnant rats, the pattern of the vaginal smears suggested the occurrence of a 'delayed pseudopregnancy' in most of the pseudopregnant rats treated daily with 3 mg, but in few of those treated with 5 mg, 70026.     

Oestrous cycle of captive southern hairy-nosed wombats (Lasiorhinus latifrons) in South Australia, Australia

There is limited information available on the oestrous cycle of female southern hairy-nosed wombats (Lasiorhinus latifrons). This is mainly due to an extremely poor breeding success in captivity and the difficulty in routine recapturing of these cryptic, semi-fossorial animals in the wild. The aim of this study was to characterise the oestrous cycle of this species by monitoring peripheral plasma concentrations of progesterone and oestradiol, assessing changes in vaginal cytology, pouch condition and the urogenital sinus. Eight adult female wombats were monitored during the breeding season (July-December) over 2 years (2002-2003). Samples were collected up to three times a week. Vaginal smears contained several cell types, categorised by morphology, as either superficial epithelial cells or parabasal-intermediate cells. Leucocytes were also counted. Plasma progesterone profiles showed a mean oestrous cycle length of 36.33+/-0.67 days with a peak progesterone concentration of 139.53+/-10.62nmol/L. Levels of oestradiol peaked at a mean level of 467.33+/-44.32pmol/L on average 5 days before a rise in plasma progesterone values. The proportion of epithelial cells in vaginal smears varied throughout the cycle, with a high percentage of superficial epithelial cells observed during the follicular phase. During periods when progesterone concentrations were high, a greater percentage of parabasal-intermediate cells was observed. In conclusion, this study has characterised the oestrous cycle of the southern hairy-nosed wombat and confirmed that changes in vaginal smears together with pouch and urogenital sinus details could be used to determine signs of oestrus in this species.     

Reproductive functional anatomy and oestrous cycle pattern of the female brush-tailed porcupine (Atherurus africanus,Gray 1842) from Gabon

In the present study, we examined certain features of the functional anatomy of the female genital tract of the wild brush-tailed porcupine (Atherurus africanus) to obtain data on the reproductive biology of this African forest rodent. Two consecutive experiments were performed. The aim of the first was to establish macroscopic and microscopic features of the genital organs, and to explore correlations between predominant ovarian structures and vaginal contents in 20 wild, mature females. In the second experiment, we inspected the external genitalia and vaginal smears of a further 10 females in captivity on a daily basis for 90 days. The uterus of the brush-tailed porcupine is bicornuate and composed of two separated uterine horns, a uterine body and cervix. The genital tract does not present a vaginal vestibule. Thus, there is no portion common to genital and urinary tracts. Females in the follicular phase of the oestrous cycle showed increased cornification of the vaginal epithelium and a high density of eosinophilic cells in vaginal smears. The vulva and vaginal opening were open, reddish and tumefacted. In luteal phase or in pregnancy, epithelial cornification and eosinophilic features were notably reduced and the vagina presented a pale, non-tumefacted vulva and a vaginal closure membrane. Females in captivity showed spontaneous cycles, a polyoestrous reproduction pattern and, based on features of the external genitalia and vaginal smears, their oestrous cycle length was 27.1+/-6.4 days (n=12).     

Endometrial cytology and computerized morphometric analysis of epithelial nuclei: A useful tool for reproductive diagnosis in the bitch

New diagnostic approaches are required to recognize early canine hypofertility or infertility. We suggest that the identification of different cytologic types, cellular aspects, and nuclear features of the endometrial epithelial cells may be suitable for this purpose. This study was performed on the bitch (Canis familiaris) during the physiologic reproductive cycle and in uterine diseases. We also applied computerized cytomorphometry to evaluate nuclear area, perimeter, diameter, density, aspect, and roundness of endometrial epithelial cells in healthy dogs (N = 35) at different stages of the reproductive cycle (before puberty, during proestrus, estrus, diestrus, and anestrus) and in bitches affected by uterine disorders (N = 10). The stage of the estrous cycle was determined by vaginal cytology and progesterone evaluation and also confirmed by clinical and histologic observations. Samples for endometrial cytology were collected in vivo by uterine flushing with transcervical uterine cannulation. After uterine sampling, each dog underwent OHE or uterine stump revision. Cytologic analyses were compared with histologic examinations to verify the uterine condition. The uterine cellular population was represented by endometrial epithelial cells, erythrocytes, neutrophils, lymphocytes, eosinophils, macrophages, plasma cells, and cervical or incidental vaginal cells. Bacteria and amorphous material were observed. The proportion of different cells and nuclear features in the cytologic samples varied throughout the stages of the reproductive cycle and between normal and pathologic uterine conditions. The computer-assisted nuclear morphometry, performed in cytologic specimens by means of the six nuclear parameters chosen to evaluate the endometrial epithelial cell population, proved to be useful for determining the stage of the reproductive cycle. Furthermore, this system was demonstrated to be a valid support to diagnose and distinguish uterine disorders.     

小鼠发情周期卵泡发育动态及其对超数排卵的影响

该文探讨了小鼠发情周期中阴门状态、阴道脱落细胞类型变化规律、卵泡发育规律及其相互关系,并比较了发情周期不同阶段的超排效果。结果表明,采用阴门状态观察法和阴道脱落细胞涂片法,能有效判断小鼠发情周期阶段。卵巢组织切片观察结果表明,在发情周期不同阶段,小鼠的卵泡发育和黄体的生成与消退存在明显的规律性变化;小鼠发情周期中,其阴门状态、阴道脱落细胞种类及卵泡发育动态之间存在相关关系;发情周期不同阶段开始超排的小鼠,其配种见栓率和回收胚胎平均数均存在明显差异,发情前期显著优于发情后期与间情期(P<0.05),并高于发情期,但差异不显著(P>0.05),即阴门状态观察法与阴道脱落细胞涂片法均可用于小鼠发情周期阶段的判断,发情前期为最适宜的小鼠超排时期。     

Pouch appearance is a reliable indicator of the reproductive status in the Tasmanian devil and the spotted-tailed quoll

Female Tasmanian devils (TDs) Sarcophilus harrisii and spotted-tailed quolls (STQs) Dasyurus maculatus were monitored to assess changes in plasma progesterone and faecal oestrogens/progestagens, vaginal smears and qualitative changes in pouch appearance during the oestrous cycle. Pouch condition was characterized based on size, colour and secretions, and was found to accurately reflect reproductive status, being significantly correlated with changes in both sex steroids and vaginal cytology. During the follicular phase, pouch redness and secretions were maximal, and associated with increased sex steroid concentrations, a karyopyknotic index of >90% and the onset of copulation. Post-ovulation, pouches became wet and deep and developed a glandular appearance; plasma progesterone/faecal progestagen concentrations remained high and sustained throughout the luteal phase. These features were identical during the pregnant and non-pregnant oestrous cycle. This study demonstrated that pouch appearance is a reliable physical indicator of the stage of oestrous in the TD and STQ, and provides an alternative non-invasive method for evaluating the ovarian cycle of these threatened species. This technique can be readily applied to monitor individuals in free-ranging or captive populations, and will aid as a practical tool for improved breeding management.     

Vaginal oestrus during the reproductive and non-reproductive period in European ground squirrels

Most hibernating animals are thought to be monoestrous because reproductive activity is subject to strong time constraints. In previous studies, female European ground squirrels (Spermophilus citellus) turned out to have elevated oestradiol levels during late lactation and after weaning, indicating ovarian activity during summer. Therefore, we monitored vaginal cytology, endocrine changes, and vulval development in semi-free-living female European ground squirrels throughout one active season. Vaginal oestrus, defined by the predominance of cornified cells in smear samples, was found during the mating period shortly after vernal emergence. This phase was followed by metoestrus, characterised by the appearance of leukocytes, and a subsequent anoestrous phase. During weaning or postlactation, a second vaginal oestrus was documented in all experimental females, again followed by a metoestrous and an anoestrous phase lasting until hibernation. In line with the second vaginal oestrus, plasma oestradiol concentrations peaked during postlactation. Progesterone levels were elevated from gestation to postlactation, and titres were marginally higher during vaginal oestrus in summer than in spring. Vulval swelling was more pronounced during the first than the second vaginal oestrus. The second oestrous cycle was non-reproductive, as males were sexually inactive with regressed testes during summer. We assume that the second oestrous cycle and the accompanying endocrine changes have beneficial effects on prehibernatory fattening and reproductive performance in the subsequent season. This might allow females to become oestrous immediately after emergence from hibernation in spring.     

Ultrasonographic and laparoscopic evaluation of the reproductive tract of the captive female African lion (Panthera leo)

The use of transabdominal ultrasonography to assess the oestrous cycle has not been previously described in the African lion (Panthera leo). Twelve sexually mature lionesses and five female cubs had their reproductive organs assessed by transabdominal ultrasound. Ovarian findings were compared to laparoscopic findings while performing laparoscopic ovariectomy or salpingectomy. Vaginal cytology was performed and serum progesterone levels were determined. By combining all data the oestrous cycle stage of each lion was determined. One lion was far pregnant and was not operated on. In adults a uterine body could be seen ultrasonographically in 67% of lions while mural structures could be distinguished in 44% of lions. Five uterine horns could be seen in 3 lions. In 12 adults 10 ovaries were found of which eight had discernable follicles or luteal structures. During laparoscopy 12 active ovaries were seen with luteal structures seen in 11 ovaries and follicles in 2 ovaries. Using laparoscopy as the gold standard, ultrasonography had a sensitivity of 66% and specificity of 83% to detect ovarian reproductive activity. Two uterine cysts and a cluster of periovarian cysts were seen in three different lions. Three lions were pregnant, two were in oestrus, three in a luteal phase (dioestrus), and four were in anoestrus. Transabdominal ultrasound in combination with serum progesterone levels and vaginal cytology can be used to assess ovarian cyclical activity with reasonable accuracy in captive bred lions.     

Determination of ovulation time in bitches based on teasing, vaginal cytology, and elisa for progesterone

The estrous cycle of 16 mature mongrel female dogs was monitored to evaluate the accuracy of teasing, vaginal cytology and quantitative ELISA progesterone assay to determine ovulation. The dogs were presented to male, and blood samples and vaginal swabs were taken daily during proestrus and estrus. Selected serum samples collected during estrus were assayed for endogenous LH by radioimmunoassay (RIA). Plasma samples collected during proestrus and estrus were assayed for progesterone with a commercially avialable ELISA kit. Ovulation was considered to take place 48 h after the preovulatory LH peak. Vaginal cytology smears were stained with Wright's stain and evaluated for the percentage of superficial squamous cells. Day 1 of diestrus (Day 1) was defined as a drop of 20% or more in the total number of superficial cells. Two standard curves (linear and best fitted curves) commonly used with ELISA were compared together and with the RIA progesterone assay. Ovulation was estimated to occur when progesterone concentration was 4.9 +/- 1.0ng/ml (mean +/- SD, n = 15), with a range of 3.4 to 6.6 ng/ml. Based on vaginal cytology, ovulation took place 6.9 +/- 1.6 d (n = 15) after 80% of the squamous cells were superficial and 6.8 +/- 1.4 d (n = 16) before Day 1. Ovulation took place 2.1 +/- 3.9 d (n=11) after the first day of standing estrus and 8.8 +/- 1.5 d (n = 10) before the last day of receptivity. The two standard curves were found parallel to each other and to the RIA progesterone assay. Based on the results of the present study, ELISA progesterone assay and determination of the first day of estrus by vaginal cytology are reliable methods for predicting ovulation, whereas the last day of receptivity as determined by teasing and Day 1 as determined by vaginal cytology are reliable methods to retrospectively estimate ovulation time.     

Plasma progesterone concentrations and length of the first spontaneous oestrous cycle in pubertal rats

The length of the first spontaneous oestrous cycle in pubertal Wistar-Imamichi strain rats determined by vaginal smears varied from 5 to 18 days. The variation was ascribed to the period (3-16 days) of the stage of vaginal smears consisting of leucocytic cells (L stage). Plasma progesterone concentration and the decidual reaction in the uterus were used as indicators of the function of the corpus luteum and the L stage period was categorized as short, lasting for 3-6 days (average 4 days) with non-functional corpora lutea, or long, lasting 9-16 days (average 12 days) and with functional corpora lutea. Rats with the long L stage showed nocturnal and diurnal prolactin surges, but no daily changes in prolactin values were observed in rats with a short L stage. Daily changes in prolactin concentrations were maintained by the administration of progesterone in rats ovariectomized on Day 6 of the L stage. Plasma progesterone values on Day 6 of the L stage decreased with ergocornine treatment on Days 4 and 5 of the L stage and administration of bovine prolactin restored the level. These results indicate that the L stage observed in the first oestrous cycle is maintained by a positive feedback relation between progesterone and prolactin secretions.     

Vaginal bacterial flora and cytology in proestrous bitches: role on fertility

The study of canine vaginal cytology underwent limited evolution over the years. Presence and significance of inflammatory cells in vaginal smears are little considered aspects in the bitch. Moreover, occurrence of vaginal bacteria in breeding bitches during follicular phase of the reproductive cycle, in absence of clinical signs of infection, involves the difficult question of antibiotics administration. The aim of this study was to relate findings in vaginal cytology (presence of neutrophils, lymphocytes, eosinophils, erytrocytes and bacteria) and microbial environment during proestrus with fertility outcomes (development of pregnancy, uterine infection, resorption, abortion and neonatal mortality). Bacteria sensitivity to antibiotics normally used in small animal practice was also evaluated. Bacteria isolated from vagina, in order of frequency, were Enterococcus faecalis, Streptococcus β-haemolyticus, Pasteurella multocida, E. coli, Klebsiella pneumoniae, Proteus mirabilis, E. coli haemolyticus, Arcanobacterium pyogenes, Streptococcus spp., Staphylococcus spp. and Acinetobacter spp. No mycoplasmas were observed. The present study showed that proestrous cytological aspects do not affect fertility. Eosinophils were never detected, while erythrocytes were always detected. During diestrus, E. coli was found in all pregnant bitches that developed clinical symptoms of uterine disorders (n = 3), resulting in uterine infection, resorption or abortion, but without statistical significance. Vaginal presence of Streptococcus spp. in proestrus was instead negatively associated with development of uterine infections (P = 0.005). Therefore, Streptococcus spp. could have a protective competitive role against more dangerous pathogens affecting fertility of the bitch. Among the 12 antibiotics tested, Gram-negative bacteria showed a significant sensitivity towards the amoxicillin and clavulanic acid association (P = 0.038). However, antibiotic treatment before mating, on the basis of positive culture, yet in the absence of clinical signs, seems to be unnecessary besides harmful leading to imbalance in vaginal commensal flora with adverse effects on fertility. In conclusion, vaginal bacteria, neutrophils, lymphocytes and erytrocytes should be considered as physiological aspect in the bitch during proestrus that does not require antibiotic therapy when asymptomatic.     

Characteristics of the oestrous cycle and influence of social factors in grey short-tailed opossums (Monodelphis domestica)

Characteristics of the oestrous cycle of grey short-tailed opossums were studied by vaginal smears. The period of oestrus was identified by a sudden proliferation of epithelial cells which lasted about 6 days (range 3-12 days), followed by a leucocytic infiltration. Oestrous cycle length showed a bimodal distribution of 14.4 days (5 cycles, range 11-17 days) and 32.3 days (10 cycles, range 28-39 days). Females housed with males showed more days of epithelial cell proliferation than did females housed alone, and oestrous periods tended to occur in synchrony, suggesting that social factors may influence the oestrous cycle in this species.     

Performing Vaginal Lavage,Crystal Violet Staining,and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-β-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.     

The oestrous cycle of the brown lemur, Lemur fulvus.

Examination of vaginal smears and observations of the colour and tumescence of the genitalia and discharge of mucus of 9 brown lemurs indicated that the oestrous cycle lasted for 30 days and that cycles occurred between September and July. The timing of cycles of females housed in visual isolation differed from that of females able to see other lemurs, indicating that the oestrous cycle in this species can be influenced by different housing conditions.     

Estrus induction in the bitch using a combination diethylstilbestrol and FSH-P

The purpose of the study was to induce estrus and ovulation in normal bitches using a combination of diethylstilbestrol (DES) and follicle stimulating hormone of porcine pituitary origin (FSH-P). Thirteen mature mongrel female dogs were divided into two groups, the first group was treated for estrus induction during late anestrus and the second group during mid-anestrus. The dogs were monitored by teasing, vaginal cytology, and hormonal assay during the induced (n = 13) and the previous spontaneous estrous cycle (n = 9). Six of eight and three of five bitches came into standing estrus in the first and second group, respectively. Of the bitches that came into estrus, three conceived in the first group and one in the second. The average induced litter size was 7.0 versus 7.5 for the colony. Based on vaginal cytology the induced proestrus and estrus lasted 1.7 (0 to 3) and 12.9 (4 to 24) d, respectively, while the spontaneous proestrus and estrus lasted 5.8 (0-17) and 12.8 (9-15) d, respectively. Progesterone profiles were similar between the induced and spontaneous estrous cycles, although the progesterone peak was higher during the spontaneous cycle. The preovulatory luteinizing hormone (LH) surge was observed in only one induced estrous cycle. Modest results were obtained with this therapy. However, the litter sizes were normal and the induced cycles were very similar to the physiologic ones. No side effects were seen with the oral form of DES.     

长爪沙鼠发情周期规律与判定方法

目的研究长爪沙鼠发情周期,揭示发情规律,优化判定方法。方法连续18 d采集50只长爪沙鼠阴道上皮脱落细胞涂片,采用角化细胞计数法研究长爪沙鼠发情周期规律。比较瑞氏染色、HE染色和直接镜检判定发情周期4个时相的优缺点。结果长爪沙鼠的发情周期有稳定型、不稳定型、假孕三种类型。其中稳定型占68.6%,发情周期为(106.3±35.0)h,可分为4个时相。4个时相角化细胞的比例分别为发情前期(13.5±7.8)%、发情期(86.7±9.9)%、发情后期(27.9±12.8)%和发情间期(3.3±2.8)%。结论角化细胞计数能准确地判定长爪沙鼠的发情周期及各个时相。直接镜检法能快速反映阴道脱落细胞的形态。     

高原鼢鼠发情周期阴道细胞变化特征及血清促性腺激素含量动态

In order to determine the change characteristics of vaginal cells in the estrus cycle and the duration of the estrus cycle, vaginal smears and HE staining methods were used to observe and count the types, morphological changes, and proportions of vaginal cells in plateau zokor (Eospalax baileyi). The hormone secretion dynamics of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the serum of plateau zokor in the estrus cycle were determined by ELISA. The results showed that the main vaginal cells of plateau zokor were leukocytes, nuclear epithelial cells, incompletely cornified epithelial cells, and complete cornified epithelial cells. The proportion of nuclear epithelial cells in the proestrus was significantly higher than that during the other three periods (48. 4 ±3. 09)% (n=12, P < 0. 05). The proportion of complete cornified epithelial cells in the oestrus was the highest and significantly higher than that in the other three periods (59. 73 ±7. 59)% (n=15, P < 0. 05). The proportion of leukocytes in the metestrus and dioestrum was significantly higher than that in the proestrus and oestrus (P < 0. 05). The expression level of LH was the highest in the metestrus (4. 709 5 ±1. 094 0) ng/mL (n=6, P < 0. 05), which showed an increasing trend in proestrus and oestrus, and a decreasing trend in the metestrus and dioestrum. There was no significant difference in the expression level of FSH among the four periods (P > 0. 05); the estrus cycle duration of plateau zokor was 16 to 19 d, the characteristics of vaginal cell types in different stages of the estrus cycle were obvious, and the relative number was statistically significant. The study provided a basis for vaginal smear identification in the estrous cycle and reproductive physiology study of plateau zokor.     

Reproductive cycles in Tarsius bancanus

The reproductive condition of three pairs of Bornean tarsiers (Tarsius bancanus) was documented for 16 months. Each pair was housed separately under a constant photoperiod (L:D = 11.5:12.5) similar to that in their native habitat. Reproductive cycles of females were monitored visually for 6 months and were then monitored with vaginal smears and measurements of external genitalia for an additional 10 months. Progressive proliferation and cornification of vaginal epithelial cells during proestrus and estrus was accompanied by an enlargement of the external genitalia. Cycle lengths averaged 24.0 ± 3.2 days. The external genitalia were swollen each cycle for a period of 6–9 days. Copulations occurred on the 1st day of estrus, which lasted 1–3 days. No menstruation was observed. There was no evidence of seasonality of estrous cycles with this photoperiod. Testicular measurements of males showed no overt seasonal changes. There was variation in testicular size, and one male copulated at a range of testicular sizes. The data indicate that T. bancanus has estrous cycles similar to those of prosimian primates and some New World monkeys and that these cycles occur throughout the year under constant photoperiodic conditions.