Endometrial cytology of the normal bitch throughout the reproductive cycle

Samples for endometrial cytology were collected both from live bitches using transcervical uterine cannulation (n=48) and postmortem (n=10). The cells identified were endometrial epithelial cells, leucocytes, erythrocytes, spermatozoa, bacteria and cervical or vaginal cells. The endometrial epithelial cells varied morphologically throughout the reproductive cycle and had signs of degeneration during late dioestrus and during early and mid-anoestrus following dioestrus and postpartum. Neutrophils were the most common leucocytes observed during pro-oestrus, oestrus, dioestrus and early pregnancy, and lymphocytes during anoestrus. Macrophages were frequently seen during anoestrus. Erythrocytes were found in variable numbers at all stages of the reproductive cycle. Spermatozoa were detected in samples collected during oestrus and early pregnancy in bitches which had their last mating one to three days previously. Bacteria were commonly observed during pro-oestrus and oestrus. Cornified cervical or vaginal cells were present during pro-oestrus and oestrus. This study demonstrated that the numbers, types, proportions and morphology of ceils in endometrial cytological samples from normal bitches varied throughout the reproductive cycle.     

Investigation of Cervical Patency and Uterine Appearance in Domestic Cats by Fluoroscopy and Scintigraphy

The cervical patency of six domestic female cats was monitored under sedation by infusion of contrast medium (Omnipaque) into the cranial vagina during early oestrus, mid‐oestrus, late oestrus and interoestrus or a radiopharmaceutical (^99mTc‐HSA) during mid‐ and interoestrus in a non‐ovulatory oestrous cycle. The transport of the contrast medium or the radiopharmaceutical through the cervix and within the uterine horns was observed under fluoroscopy and with the aid of scintigraphy. In three of the queens, transcervical transport of contrast medium was demonstrated in all stages of oestrus, in one queen during mid‐oestrus, late oestrus and 1 day after oestrus, and in two queens only during late oestrus. The relations between the cervical patency to the contrast medium and the oestrous behaviour, cornification of the vaginal cells and the serum oestradiol‐17β concentration were evaluated, and a relationship was found between the cervical patency and the degree of vaginal cornification. Transcervical transport of the radiopharmaceutical was observed in three queens during mid‐oestrus. When the cervix was open, hysterography under a fluoroscope and hysteroscintigraphy were performed. The fluoroscopic and scintigraphic recordings revealed the patterns of the uterine contractions during oestrus in both ascending and descending directions, and the movement of the uterine contents back and forth between the uterine horns. The hysterograms were classified according to the shape of the uterine horns and the appearance of the endometrial lining. Spiral‐shaped uterine horns with a smooth inner contour were observed in two queens, and a corkscrew appearance with irregular filling defects in the uterine lumen was shown in two queens that had developed subclinical cystic endometrial hyperplasia. These findings demonstrated that fluids or particles deposited in the cranial vagina of the cat can be transported into the uterus during some stages of the oestrous cycle. The fluoroscopic and scintigraphic techniques developed in this study may be further modified to permit more detailed studies of uterine contractile patterns and sperm transport in the feline female reproductive tract. Hysterography proved useful to diagnose uterine disease. The information on cervical patency is of value also for the development of techniques for artificial insemination in this species, and should be studied also in the ovulatory cycle.     

A contrast radiographic study of the vagina and uterus of the normal bitch

A method is described for investigating radiographically the shape of the bitch's vagina. The intra-pelvic vagina is readily dilated whereas the intra-abdominal vagina is narrow and tortuous, and cranially is typically ‘spoon-shaped’. Contrast medium entered the uterus during pro-oestrus, oestrus and the post-partum period, indicating patency of the cervix at these times. No cervical relaxation was evident at the end of the luteal phase, i.e. during anoestrus. The possible relevance of these findings to the aetiology of pyometra is discussed.     

Epithelial surface changes and spermatozoa storage in the reproductive tract of the bitch

Spermatozoa are known to bind to the epithelial cells lining the uterine tube in various species, but information in canids is conflicting and sparse. The first aim of this study was to measure the epithelial surface outline (ESO) of different regions of the canine uterine tube in the four stages of the oestrous cycle as an indicator of a changing potential reservoir for spermatozoa. The second aim was to identify the site of sperm storage in the bitch after natural mating. Reproductive tracts were collected from bitches undergoing routine ovariohysterectomy.Histological analysis showed that, when corrected for uterine tube size, the ESO of pro-oestrus (P < 0.005) and oestrus (P < 0.05) tubes were larger than anoestrus, but not metoestrus, tubes. The second study examined reproductive tracts from 12 Beagle bitches at 6, 12, 24 and 48 h after mating. Light and electron microscopy revealed large numbers of spermatozoa in the proximal regions of the uterus and particularly the distal utero-tubal junction (UTJ), with few present in the proximal UTJ or uterine tubes. Spermatozoa were bound by their heads to microvilli on the epithelial surface of the uterine lumen and to ciliated cells in the distal UTJ. This is the first report to measure and document differences in potential epithelial attachment sites of the uterine tubes at different stages of the oestrous cycle and to provide compelling evidence that the main spermatozoal storage site in the reproductive tract of the bitch is the distal UTJ.     

Some observations on the aerobic bacterial flora of the genital tract of the dog and bitch

The aerobic bacterial flora from the genital tracts of 143 bitches and 51 dogs was investigated. Beta haemolytic streptococci (group G), Staphylococcus aureus and Escherichia coli were among the organisms isolated from normal bitches in heat, abnormal bitches and normal dogs. Repeated sampling of two bitches during oestrus, and the dog to which they were mated, showed that bacteria were transmitted from bitch to dog, but did not become established. Guarded swab samples taken from the vagina collected less bacteria than samples from the vestibule. Numerous bacteria were seen in vaginal smears taken from bitches during oestrus, but numbers fell rapidly with the appearance of leucocytes in the smear at the onset of metoestrus.     

Recovery of Mycoplasma spp. from the Reproductive Tract of the Mare during the Estrous Cycle

The sites in the genital tract from which mycoplasmas could be recovered at various stages of the estrous cycle were studied in five Standardbred mares naturally infected with Mycoplasma. Mycoplasma equigenitalium and Mycoplasma subdolum were most frequently isolated from the clitoral fossa as compared to the vagina, cervix, and uterus. The lowest isolation prevalence was observed in the uterus. The recovery of Mycoplasma spp. from the clitoral fossa did not differ at any stage of the estrous cycle; however, recovery from the vagina, cervix, and uterus was variable during the cycle and more organisms were recovered on the day of ovulation than at any other time. From these results it was concluded that the clitoral fossa is the most likely “ecological niche” for Mycoplasma spp. in the mare. Ureaplasmas were not isolated.     

Changes in oestrogen,progesterone and epidermal growth factor receptor concentrations and affinities during the oestrous cycle in the normal mammary gland and uterus of dogs

Changes in the concentrations and affinities of receptors for oestrogen (ER), progesterone (PR) and epidermal growth factor (EGF-R) were studied in mammary glands of healthy bitches with regard to age, the location in the mammary chain and the stage of the oestrous cycle. Uterus was used as the reference tissue for the evaluation of steroid receptors. Mammary and uterine samples from 7 healthy bitches were taken at five stages of the oestrous cycle in such a way that all the locations in the mammary chain were represented at each stage of the cycle (10 samples/dog). ER, PR and EGF-R were detected by biochemical assays using increasing concentrations of tritiated (steroids) or iodinated (EGF) ligands. A significant direct correlation was found between the ER and PR concentrations for mammary and uterine samples. No significant correlation was found between the steroid receptors and EGF-R concentrations. Mammary ER concentrations were significantly higher in bitches of 5 years of age or older than in younger ones; in posterior glands (4th and 5th pairs) than in anterior glands; and in the mid-luteal phase. Mammary PR did not vary significantly with age or location but was significantly lower in the early luteal phase than in other phases. A similar decrease in PR concentrations was observed in the uterus during the early luteal phase and uterine ER and PR concentrations were very low in the mid-luteal phase. Mammary EGF-R were not significantly higher in the early or mid-luteal phase than in pro-oestrus or anoestrus.The differences observed between the uterine and mammary steroid receptor concentrations during the oestrous cycle could be due to different mechanisms for regulating steroid receptor expression in the two tissues. Mammary EGF-R concentrations may be linked, as in other species, to cellular proliferation and/or to the serum progesterone concentrations.Abbreviations BSA bovine serum albumin - EGF epidermal growth factor - EGF-R receptor for epidermal growth factor - ER oestrogen receptor - K _d dissociation constant - LH luteinizing hormone - p probability of error - PR progesterone receptor     

Application of vaginal cytology and plasma Srogesterone eterminations to the management of reproduction in the bitch

The use of vaginal cytology and plasma progesterone determinations in the management of 11 bitches presented to our small animal fertility clinic are described. Nine bitches were mated or artificially inseminated and seven became pregnant. Reproductive patterns detected included failure of ovulation, prolonged pro-oestrus with late ovulation, short pro-oestrus/oestrus with early ovulation, short pro-oestrus/oestrus with late ovulation and normal pro-oestrus/oestrus with late ovulation.     

Tissue concentrations of eosinophilia in the bovine oviduct and uterus of different stages of the oestrous cycle

Numbers of eosinophils in the bovine oviduct and uterus were determined during the oestrous cycle. The eosinophil numbers in the oviduct (ampulla and isthmus) and horn of the uterus during oestrus were significantly higher than during dioestrus. The number of eosinophils in the uterine cervix was lower than in the uterine horn for all stages of the oestrous cycle. In the oviduct, eosinophils accumulated in the lamina propria of the tunica mucosa, in the tunica muscularis and in the connective tissue of the tunica serosa. In the uterus, they were concentrated mainly in the upper parts of the stroma in the endometrium. Degranulation of eosinophils was observed during oestrus when they increased in number in the oviduct and uterus.     

Saliva crystallisation as a means of determining optimal mating time in bitches

Objectives : To chart saliva crystallisation patterns throughout pro-oestrus and oestrus in bitches, and to assess their reliability as a tool for optimising the timing of breeding. Methods : Six beagle bitches with normal reproductive activity were used. Saliva ferning patterns were established during pro-oestrus and oestrus. Vaginal cytology, progesterone (P4) levels and behavioural signs were used to determine the optimum mating time. Receiver operating characteristic (ROC) analysis was performed. Results : Different ferning patterns were scored from 0 to 2. Although variations in saliva crystallisation were noted during the follicular phase of the oestrous cycle in bitches, the test did not prove accurate; test sensitivity was 40·6%, i.e. too low to reliably discriminate the fertile period, while specificity was 86·1%. Clinical Significance : This test could be used to complement other methods of determining the fertile period in bitches, but its potential capacity remains to be demonstrated.     

Vaginal and uterine microflora of adult dogs

Aerobic and anaerobic microflora were identified and quantitated in 82 vaginal and 78 uterine samples obtained from mature bitches during different stages of the estrous cycle. The mean +/- SD of total bacterial counts/100 mg of vaginal contents of the 82 bitches was log 5.0 +/- 1.5, ranging from log 2.4 to log 8.8. The count at the estrous stage (log 7.8 +/- 0.7) was significantly higher (P less than 0.05) than that at the anestrus (log 4.4 +/- 1.0), pregnancy (log 5.9 +/- 1.3), and postpartum (log 5.1 +/- 1.5) stages. The common organisms isolated from the vaginas were Bacteroidaceae, streptococci, Pasteurella spp, and mycoplasmas. Organisms were isolated from 48 (68%) of 78 uterine samples. The range of total counts/100 mg of uterine contents was from log 1.6 to log 8.3. Staphylococci and mycoplasmas were frequently isolated from the uterine contents. Although many uterine microfloras were similar to vaginal microfloras, some uterine culture had a single isolate identified. There were no pathologic findings in most of the uteri. Seemingly, vaginal bacteria frequently flow into the uterus, yet they rarely cause uterine infection.     

Changes in the weight, collagen concentration and content of the uterus and cervix of the ewe during pregnancy

The dry and wet weights of the uterus (caruncular and intercaruncular areas) and cervix were measured in non-pregnant (n = 5) and pregnant (n = 25) ewes post mortem; for the latter, five were obtained for each of the 5 months of gestation. The total collagen tissue content was measured in both areas of the uterus and cervix by hydroxyproline analysis and image analysis of Haematoxylin-Van Gieson stained tissue sections. Both wet and dry uterine weights increased significantly with gestational age (P < 0.001). The water content of uterine and cervical tissue remained constant, at between 83 per cent to 85 per cent and 76 per cent to 80 per cent, respectively. There was a close correlation between the two methods used to determine the collagen content (r = 0.96, P < 0.001), and between the increasing weight of the uterus during pregnancy and the total collagen content of tissues (r = 0.97, P < 0.001). At all stages, the total collagen content of the cervix [mean (SEM) 96.2 (5.4) mg g(-1)] was significantly greater (P < 0.001) than that of the caruncular mean [mean (SEM) 24.3 (1.4) mg g(-1)], and the intercaruncular areas [mean (SEM) 29.0 (1.0) mg g(-1)]. The changes in uterine and cervical weights and collagen content of the tissues were similar to those reported in other related species.     

The regional differences of the pH-value in the vagina,cervix, and uterus of cows during interestrus

This study aimed to ascertain the pH value of the vagina, cervix, and uterus in cows during interoestrus. Measurements were made with an ambulant, digital pH meter with a flexible probe and a measuring frequency of one value per second. The genital tract (vagina, cervix, uterus) of 50 cows were analyzed with this instrument immediately post mortem. The sexual health of the animals was evaluated via clinical, histological, and microbiological tests. Statistically significant regional differences in the pH value were documented (p < 0.01). Whereas a decrease was measured between the values for the vagina (6.92 +/- 0.51) and the cervix (6.22 +/- 0.31), increased values were measured in the uterus (6.62 +/- 0.32). The physiological pH value in the genital tract of the cow should be taken into consideration for the development of new therapeutic measures in terms of the local application of probiotically active bacterial strains.     

Immunohistochemical Studies on Oestrogen Receptor Alpha (ERα) and the Proliferative Marker Ki-67 in the Sow Uterus at Different Stages of the Oestrous Cycle

In order to better understand physiological changes during the different stages of the oestrous cycle, immunohistochemistry was used in the present study to investigate the distribution of oestrogen receptor alpha (ERα) as well as the proliferative marker Ki‐67, in the sow uterus during the oestrous cycle. Uterine samples were collected from multiparous sows with normal reproductive performance at selected stages of the oestrous cycle: at late dioestrus (d 17), prooestrus (d 19), oestrous (d 1), early dioestrus (d 4) and dioestrus (d 11–12), respectively. The tissue samples were fixed in 10% formaldehyde, embedded in paraffin and subjected to immunohistochemistry using monoclonal antibodies against ERα (C‐311) and Ki‐67 (MM‐1). In general, the immunostaining of both ERα and Ki‐67 was confined to nuclei of the target cells. Variations were seen, not only at the different stages of the oestrous cycle, but also in the different tissue compartments of the uterus. In the epithelia, the strongest ERα staining and highest amount of positive Ki‐67 cells were found at early dioestrus. In the myometrium, the highest levels of staining of both ERα and Ki‐67 positive cells were found at pro‐oestrus and oestrus. For the proliferative marker, Ki‐67, no positive cells were found at dioestrus and late dioestrus in the epithelium and myometrium. In the connective tissue stroma (subepithelial layer), the highest number of ERα positive cells were found at oestrus, which was significantly different compared with other stages (p≤0.05), whereas the levels of Ki‐67 positive cells were relatively low and did not differ between the stages examined. Significant correlations between the number of ERα positive cells in the stroma and Ki‐67 positive cells in the epithelia were observed. This suggests indirect regulatory mechanisms on epithelial proliferation via ERα in the stroma. In conclusion, these findings in the sow uterus show that the presence of ERα as well as Ki‐67 protein varies not only between different stages of the oestrous cycle but also between different tissue compartments of the uterus. These findings indicate various regulatory mechanisms and stress the importance of localising ERα and proliferating cells in different uterine tissues.     

The development of cystic endometrial hyperplasia in the bitch following serial uterine biopsies

Serial uterine biopsies were performed on two bitches during three oestrous cycles. The uterus was biopsied once in one other bitch and twice in another case following exteriorization and palpation of the uterine horn on four occasions earlier in the cycle. Following the serial biopsies both bitches developed a vulval discharge for a duration of 8-21 days. This first appeared between metoestrus days 14 and 30. By the 5th week after oestrus the uteri of both bitches developed cystic endometrial hyperplasia with inflammatory cell infiltration which regressed spontaneously. This lesion did not develop in the two other bitches, and it may have been caused by the surgical interference with the endometrium during the period of luteal development.     

Immunohistochemical and qPCR determination of the expression and serum level of anti‐Müllerian hormone in pre‐pubertal,intact and ovarian remnant syndrome detected bitches

The aim of this study was to determine the serum concentrations, ovarian presence and expression of anti‐Müllerian hormone (AMH) in pre‐pubertal, bitches with signs of ovarian remnant syndrome (ORS) and intact bitches. In addition, we aimed to verify the suitability of serum AMH concentrations for diagnostic purposes in sterilized bitches and/or in suspected cases of ORS in the field of veterinary medicine. For this purpose, 36 healthy female dogs divided into six groups: proestrus, oestrus, dioestrus, anoestrus, pre‐pubertal and ORS. Serum AMH concentrations were determined by electrochemiluminescence immunoassay, and ovarian presence and distribution of AMH was confirmed by immunohistochemical and qPCR techniques. According to the results of qPCR, while the expression values of AMH were at the highest concentrations in the proestrus and oestrus, there was a statistically significant decrease in these values at the later stages of the cycle (p < 0.05). According to hormone analysis, the serum AMH values of the ORS group had decreased significantly compared with the proestrus and oestrus (p < 0.05). Although serum AMH levels of ORS group were increased compared with anestrus and pre‐pubertal groups, this increase was statistically non‐significant (p > 0.05). Immunohistochemically, AMH expression was first observed in the granulosa cells of primordial follicles in folliculogenesis. Expression values were the highest in the proestrous and oestrus groups, but values from bitches in later stages of the cycle were statistically significant decrease in comparison with these groups (p < 0.05). As a result, AMH concentration and expression were found to be higher in proestrus and oestrus than in other periods (p < 0.05). In addition, the measurable level of AMH concentration in bitches with ORS is an indication that it can be used in the diagnosis of ORS.     

Distribution of mast cells in vaginal, cervical and uterine tissue of non-pregnant mares: investigations on correlations with ovarian steroids

Mast cells constitute part of the cellular immune system of the genital tract. They play a potential role in cervical remodelling during parturition and are subject to the influence of ovarian steroids. In this study, the influence of oestradiol‐17β and progesterone on the distribution of mast cells in equine vaginal, cervical and uterine tissue was evaluated. Genital tracts were retrieved from healthy mares at a local slaughterhouse. The cervix was divided along the longitudinal axis into five equivalent regions from cranial to caudal (R1 to R5). Toluidine blue staining was used for the detection of mast cells. Hormone values were determined via radioimmunoassay. In cervical tissue, mast cells were present at higher frequency and higher density than in vaginal and uterine tissue. Statistically significant differences were obtained between cervical R1 (cranial) and uterus as well as cervical R2 and uterus (p < 0.05). In cervical tissue, an association between the presence of mast cells and peripheral oestrogen concentrations was observed, whereas such correlation could not be established for vaginal and uterine tissue. With increasing oestradiol‐17β concentrations, the density of mast cells in cervical tissue increased to a statistically significant degree in the cranial (r = 0.46; p = 0.05) and caudal (r = 0.5; p = 0.03) regions. We propose that mast cells play a role in the cyclic remodelling of the equine cervix during the course of the oestrous cycle. Elucidation of mechanisms controlling cervical remodelling could contribute to development of therapies for mares showing a lack in cervical opening during oestrus.     

Permanent Lateral Deviation and Stenosis of the Cervix in an Infertile Jennet

Congenital abnormalities of the uterus and the cervix are rare in horses, often associated with infertility, despite normal ovarian activity and sexual behaviour. An abnormality of the cervix in a 5‐year‐old jennet with a history of infertility was described. Clinical and ultrasound examination of the genital tract showed a normal development of the uterus with regular cyclic activity in both the ovaries. Vaginoscopy revealed a normal development of the vagina and a cervix deviated on the left side of the vagina by a thick dorsoventral fold. The lateral deviation was permanent in any stage of oestrus cycle. Furthermore, the cervical os was narrow and digital penetration was not realizable. Using a 5‐mm catheter, the full penetration was not possible even during oestrus; moreover, the inoculation of fluid could be revealed in uterus by ultrasound (fluid was not present before inoculation), demonstrating a minimal patency. An abnormal and permanent lateral displacement of a stenotic cervix was suspected as the primary cause of infertility in the jennet.     

The relationship between the prevalence of uterine lesions and the use of medroxyprogesterone acetate for canine population control

SUMMARY The prevalence of uterine disease was established during desexing of 175 bitches in the Torres Strait and Cape York, 42 of which had been treated with injectable medroxyprogesterone acetate (MPA) for oestrus postponement. The prevalence of uterine lesions was 45% for treated bitches, 5% for untreated bitches, and 14.9% for the sample population. A highly significant relationship (P<0.01) between MPA treatment and uterine lesions was established. A significant association (P<0.05) between age (>2 years old) and uterine lesions was found, most likely attributable to a significantly higher proportion (P<0.01) of MPA-treated bitches in the older population. There was no significant difference in the effect of MPA on the prevalence of uterine lesions between older and younger bitches. There was no effect of parity on the prevalence of uterine lesions.