Concentrations of progesterone and luteinizing hormone in the serum of diestrous bitches before and after hysterectomy

Blood was collected from 6 adult bitches (3 pregnant and 3 nonpregnant) daily for 59 days after the onset of diestrus, and serum progesterone concentrations were determined. The bitches were hysterectomized at various times during diestrus [diestrus day (DD) 5, 15, or 25] to determine whether the uterus has a role in maintaining luteal function in the pregnant or nonpregnant bitch. Concentrations of serum luteinizing hormone (LH) were also measured to determine whether LH concentrations decrease when luteal function abates (experiment 1). All 6 bitches in experiment 1 had progesterone concentrations greater than 1.0 ng/ml during the 2-month period, indicating that neither the pregnant nor nonpregnant uterus was necessary for maintaining luteal function in the bitch. Concentrations of LH appeared to increase in the serum of 5 of the 6 bitches when progesterone concentrations were declining, indicating that cessation of luteal function does not appear to result from a lack of LH. Blood was collected from the 6 bitches used in experiment 1 twice weekly during the 2nd diestrus after hysterectomy (experiment 2) to determine whether alterations in luteal function that might be observed during experiment 1 were due to some aspect of the surgical procedure, rather than to absence of a uterus. Progesterone concentrations in the serum were lower (P less than 0.05) during the 10 days after surgery in the 2 bitches hysterectomized on DD 5 than in the 4 bitches remaining intact during this period (experiment 1).(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo investigation of luteal function in dogs: Effects of cabergoline,a dopamine agonist,and prolactin on progesterone secretion during mid-pregnancy and -diestrus

The role of prolactin on luteal function in dogs was investigated in vivo. The function of prolactin in mid-luteal phase was compared in pregnant and nonpregnant dogs. A dopamine agonist, cabergoline, known for its prolactin secretion inhibitory effects, was injected subcutaneously at a dose of 5 μg/kg body weight in five pregnant and five nonpregnant Beagle bitches. Mean plasma prolactin and progesterone were dramatically suppressed for 4 to 5 days after injection in both groups when compared with control pregnant and non-pregnant animals, whereas no effect on luteinizing hormone (LH) secretion was observed. The decline in plasma progesterone occurred after that in prolactin, suggesting plasma progesterone was impaired by inhibition of prolactin secretion. These results confirm the luteotropic importance of prolactin in pregnant bitches, and also demonstrate its importance in luteal phase of the nonpregnant dog.Second, to demonstrate that the effects of cabergoline were mediated by prolactin inhibition and not by a direct action on the corpus luteum, concomitant administration on Day 30 of cabergoline and prolactin (375 μg IV twice daily on Days 30 and 31) or cabergoline and LH (750 μg IV twice daily on Days 30 and 31) was affected in two groups of five pregnant animals each. Results showed that only prolactin was able to reverse the negative effects of cabergoline on circulating progesterone. This confirms the indirect mode of action of the dopamine agonist, cabergoline on corpus luteum function.Third, further investigation on the precise luteotropic role of prolactin was made by IV injection of 375 μg pure canine prolactin twice daily in five pregnant bitches on Days 30 and 31, and in five pregnant bitches on Days 40 and 41. No direct stimulatory effect of prolactin on plasma progesterone secretion occurred. Nor was there a noticeable effect on plasma LH secretion. These results suggest that prolactin is unable to directly stimulate progesterone secretion by the corpus luteum of pregnancy.The results of this study suggest that prolactin is an essential luteotropin in the dog from mid-luteal phase in both pregnant and nonpregnant animals. However, it appears to act by sustaining corpus luteum lifespan and function rather than by direct stimulatory effects on progesterone secretion.     

The Use of Epostane in an Attempt to Reproduce Cystic Endometrial Hyperplasia in the Bitch

The aetiology and pathogenesis of spontaneous cystic endometrial hyperplasia (CEH) in the bitch is not yet completely understood. Recent research based on the expression of uterine sex hormone receptors in spontaneous cases of CEH suggested that a temporary progesterone deficiency during late oestrus-early metoestrus may be responsible for the onset of CEH development. In the present study a temporary progesterone deficiency during late oestrus–early metoestrus was experimentally created using an inhibitor of progesterone synthesis, epostane. At day 49 of metoestrus, there was a significant reduction in the size of the uterine wall, mainly due to endometrial atrophy, and there was also a significant increase in the mucus-filled uterine lumen in the bitches that had been treated with epostane compared to the control bitches. No significant differences in the expression of sex hormone receptors was observed between the two groups. As no CEH developed in the epostane-treated bitches, an additional oestrogenic stimulus may be required to stimulate endometrial proliferation. Therefore, it is suggested that deficient luteinization of the corpus luteum may be the trigger in the pathogenesis of CEH, as the secretion of varying amounts of sex steroids depends on the degree of luteinization.     

The intra-uterine position of canine foetuses and their sequence of expulsion at birth

In 14 bitches, laparotomy was performed between the 45th day and 54th day of gestation. In total, 89 foetuses were marked individually by means of transuterine injections of a radiopaque substance. After expulsion, each pup was provided with a colour code. By radiographs one day after birth, all pups of a litter could be identified by the radiopaque markings. In this way the order and presentation at birth of each pup could be compared with the uterine position and presentation at laparotomy. When after expulsion of a pup, one or more were left in each uterine horn, the next pup was produced by the contralateral horn in 43 cases (78·2%) and by the ipsilateral horn in 12 cases (21·8%). One horn was never observed to be completely empty before the other horn started expelling pups. In six of eight bitches in which each two uterine horns did not contain an equal number of foetuses, the first pup was born from the horn containing most pups. The presentation at birth has been observed in 73 pups. For five of these (6·9%), the presentation differed from the one observed at laparotomy; in all five cases the original posterior presentation had changed into an anterior one.     

Matrix Metalloproteinase (MMP)-2 and MMP-9 Activity in the Canine Uterus Before and During Placentation

The aim of the present study was to demonstrate the presence and localization of MMP-2 and -9 by means of RT-PCR and immunohistochemistry (IHC) within the canine uterus from the pre-implantation stage until mid-gestation and to determine MMP-2 and -9 activities by means of zymography. For this purpose, samples of the uterus and salpinx from bitches were obtained after ovariohysterectomy. Pre-implantation stages (5-12 days after mating, n = 11) were determined by verifying embryos after flushing the uterus. Further groups were determined as implantation (15-19 days after mating, n = 9), post-implantation (20-30 days after mating, n = 9) and placental stages (30-45 days after mating, n = 3). A non-pregnant group (17-30 days after mating, n = 4) served as control. MMP-2 and -9 positive cells were detected in all specimens from pregnant and nonpregnant bitches, however, with different distributions. MMP-2 was present in endothelium and smooth muscles of blood vessels and the myometrium of pregnant and nonpregnant bitches, additionally in the surface epithelium of the oviduct. The latter also stained positive for MMP-9. During placentation, MMP-2 was detected mainly in fetal blood vessels and trophoblastic cells. Higher MMP-2 activity was observed in the endometrium and myometrium of all pregnant groups compared with the nonpregnant group (p < 0.05). The pregnant groups did not differ significantly from each other (p > 0.05). MMP-9 was present in blood vessels, smooth muscle cells and epithelia, such as maternal surface epithelial cells, uterine crypts and glands. During placentation, the deep uterine glands and the epithelium of the glandular chambers were immunoreactive to MMP-9. Highest MMP-9 activities were reached in the endometrium of the pre-implantation group (23.2% of total MMP-9) and placental parts (33.3%).     

Follicular cystic ovaries and cystic endometrial hyperplasia in a bitch.

Treatment of cystic ovaries in a bitch was unsuccessful with human chorionic gonadotropin, but aspiration of the follicles at laparotomy was successful. Follicular aspiration is described, with special emphasis on preventing bursal adhesions. Aspiration of persistent follicles is a simple treatment for cystic ovarian disease that is nonresponsive to parenterally administered hormones. The persistent estrus associated with cystic follicles is a primary cause of cystic endometrial hyperplasia (CEH) and severe cases of CEH are associated with infertility. Rapid intervention (surgical or hormonal) will limit the severity of CEH and, therefore, decrease infertility in breeding bitches.     

Is Mechanically Induced Cystic Endometrial Hyperplasia (CEH) a Suitable Model for Study of Spontaneously Occurring CEH in the Uterus of the Bitch?

Cystic endometrial hyperplasia (CEH) was experimentally reproduced by the intraluminal insertion of a silk wire in the uterus of metestral bitches to obtain a model for the study of spontaneously occurring CEH in the bitch. This mechanically reproduced CEH corresponded histologically very well to spontaneously occurring CEH. With respect to sex hormone receptor expressions, however, there was no similarity. In the mechanically induced CEH, progesterone receptor expression was reduced in the epithelial cells (surface epithelium and endometrial glands) and slightly increased in the stromal fibroblasts and myometrium as compared with the normal metestrus uterus. The oestrogen receptor expression in mechanically induced CEH was reduced in the epithelial cells (surface epithelium and endometrial glands) and more or less unchanged in the stromal fibroblast and myometrium as compared with the normal metestrus uterus. This is in contrast to the increased sex hormone receptor expressions in all uterine cell types, observed in spontaneously occurring CEH. The mechanically induced CEH model corresponds immunohistochemically more to early placentation and to pyometra in the bitch. Thus this experimental CEH model is not suitable for pathogenic studies of spontaneously occurring CEH in the bitch.     

山羊子宫内胆碱能神经分布及妊娠时的变化

采用乙酰胆碱酯酶组化法，研究了山羊子宫内胆碱能神经的分布，结果，山羊子宫颈是经较丰富，在浆膜和肌层内有神经束伴血管而行并分支分布于血管壁，在粘膜及其皱褶上皮下，粘液腺上皮有神经丛分布，妊娠时子宫颈部的神经分布与未妊娠时相比无明显变化，子宫角部神经密度均低于子宫颈部，其内环行肌层中及其与内膜交界处神经密度略高，神经束伴血管而行并分支分布于血管壁，在子宫腺上皮下及内膜上皮下无神经分布，妊夺时作胎盘内无神经分布外，仍有神经束伴血管而行交分支分布于血管壁，在分布于血管壁的神经纤维减少，结果提示，胆碱能神经主要支配山羊子宫内血管壁及颈部粘液腺上皮和粘膜上皮，妊 时子宫颈部胆碱能神经无明显变化，而子宫角内支配血管壁的胆碱能神经纤维减少。     

Oxytocin receptors in dioestrous and anoestrous canine uteri

The aim of the study was to localize oxytocin receptors (OTR) and measure mRNA expression of OTR in the canine uterus with and without the influence of progesterone. Uterine samples were taken from nine anoestrous and eight dioestrous bitches during ovariohysterectomy. Histological changes were evaluated in haematoxylin and eosin (HE)‐stained samples. Purified polyclonal antibody for OTR was used in immunohistochemistry to localize receptors in uterine layers. Relative mRNA concentration of OTR was evaluated with real‐time PCR from full‐thickness uterine samples taken from the middle horn and the body. Myometrial smooth muscle cells, endometrial luminal epithelium (LE) and deep and superficial glandular epithelium were positively stained for oxytocin receptors in non‐pregnant animals. No significant difference in staining intensity was detected between uterine middle horn and body. However, the staining intensity of LE was significantly higher in dioestrous than in anoestrous uteri (p < .05). Leucocytes and endothelium of blood vessels were also positively stained for OTR. Real‐time PCR showed no significant differences in OTR mRNA expression between the middle horn and the body of the uterus, or between anoestrous and dioestrous uterus. No correlation was noted between OTR mRNA expression and blood progesterone concentration. In conclusion, despite the apparent inactivity, the uterus of the non‐pregnant bitch expresses OTR. The distribution or relative expression of OTR does not differ between uterine horn and body in dioestrus or anoestrus except in LE. LE may have more oxytocin‐dependent activity during dioestrus than anoestrus.     

Role of progestin-induced mammary-derived growth hormone in the pathogenesis of cystic endometrial hyperplasia in the bitch

Endogenous progesterone and synthetic progestins may induce hypersecretion of growth hormone (GH) of mammary origin, hyperplastic ductular changes in the mammary gland, and the development of cystic endometrial hyperplasia (CEH) in dogs. It was investigated whether progestin-induced mammary GH plays a role in the pathogenesis of CEH in the bitch. During 1 year, bitches with surgically excised mammary glands and healthy control bitches received medroxyprogesterone acetate (MPA). Before and after MPA treatment, uterine and mammary tissues were collected for histological, immunohistochemical, and RT-PCR examination. After MPA administration, the mammary tissue in the control dogs had differentiated into lobulo-alveolar structures and CEH was present in all uteri of both dog groups. In the MPA-exposed mammary tissue of the control dogs, GH could only be demonstrated immunohistochemically in proliferating epithelium. After treatment with MPA the dogs of both groups had immunohistochemically demonstrable GH in the cytoplasm of hyperplastic glandular uterine epithelial cells. RT-PCR analysis of the mammary gland tissue after MPA administration demonstrated a significant higher GH gene, and lower GHR gene expression than before treatment. In the uterus, the expression of the gene encoding for GH was significantly increased in the mastectomized dogs, whereas in the control dogs the expression of the gene encoding for insulin-like growth factor-I had significantly increased with MPA administration. MPA treatment significantly down regulated PR gene expression in the uterus in both dog groups. These results indicate that progestin-induced GH of mammary origin is not an essential component in the development of CEH in the bitch.     

Prevalence of fetal resorption in stray dogs in Yucatan, Mexico

OBJECTIVES: To determine the prevalence of fetal resorption in 77 pregnant bitches and its association with the age, size and body condition score of the bitch and to determine the frequency of transuterine migration of embryos. METHODS: The number and position of embryos or fetuses and placental zones were registered postmortem. The prevalence of fetal losses was determined by comparing the number of corpora lutea with the number of embryos or fetuses in each bitch. The prevalence of transuterine migration of embryos was determined by correlating the number of fetuses in each horn and the number of corpora lutea in the ipsilateral ovary. RESULTS: Fetal resorption zones were demonstrated in 42.9 per cent of the bitches. However, pregnancy continued in 84.9 per cent of cases. Of the fetal losses, 25.9 per cent were determined by counting the number of corpora lutea and viable embryos or fetuses. Young bitches tended to have a higher probability of fetal resorption than adult bitches (P<0.06). The presence of fetal resorption zones was not associated with size or body condition. Transuterine migration of embryos had occurred in 15.8 per cent of the bitches. CLINICAL SIGNIFICANCE: This study demonstrated that embryo resorption is a normal event in the bitches, with a higher prevalence than previously thought.     

山羊子宫内肽能神经分布及妊娠时的变化

采用免疫组化方法，研究了山羊子宫内含P物质（SP）和含血管活性肠肽（VIP）神经的分布。结果，妊娠及未妊娠山羊子宫颈内有粗细不等的神经束行经于外膜和肌层中，神经分支形成丛状分布于血管壁，子宫颈部未见SP神经元胞体及VIP神经元胞体；未妊娠山羊子宫角内SP神经和VIP神经均呈丛状围绕血管并分布于血管壁；妊娠中期的孕角和非孕角均有胎盘形成，除胎盘内无神经分布外，SP神经及VIP神经同样分支形成丛状分布于血管壁。结果提示，山羊子宫内SP神经和VIP神经主要支配子宫内血管，妊娠时除胎盘内无神经分布外，SP神经及VIP神经的分布无明显变化。     

Postural Influence on Systemic Blood Pressure in Large Full-term Pregnant Bitches during General Anesthesia

Effects of positioning on systemic blood pressure were evaluated in six full-term pregnant golden retriever bitches during general anesthesia. Two positions (dorsal and left lateral recumbency) were examined. Anesthesia was induced with thiamylal sodium and maintained with halothane in oxygen. Direct arterial blood pressures were recorded from the bitches in each position. Effects of positioning for each bitch during general anesthesia were reexamined after the bitch weaned its pups; thus, each bitch served as its own control. Maternal posture had no significant effect (p greater than 0.05) on systemic blood pressure. Pregnancy had no significant effect (p greater than 0.05) on systemic blood pressure. Supine hypotension did not occur in anesthetized large, full-term pregnant bitches. Dorsal recumbency was an acceptable position for cesarean sections performed with the patient under general anesthesia.     

Timing the mating of dogs on the basis of blood progesterone concentration

The optimal time for mating bitches was determined by measuring the progesterone concentration in peripheral blood three times a week after the start of vulval bleeding. Of 104 bitches with reduced fertility 81 (78 per cent) became pregnant and of 112 bitches with normal fertility 105 (94 per cent) became pregnant. Of 121 bitches mated once, 102 (84 per cent) became pregnant, and of 95 bitches mated more than once, 84 (88 per cent) became pregnant. The mean (+/- sd) interval between the start of vulval bleeding and the optimal time for mating in 88 bitches was 11.8 +/- 3.1 days. The blood progesterone concentration appears to be an excellent indicator of the best time for mating, particularly in bitches with reduced fertility.     

Serum relaxin and progesterone concentrations in pregnant, pseudopregnant, and ovariectomized, progestin-treated pregnant bitches: detection of relaxin as a marker of pregnancy

Serum concentrations of relaxin and progesterone were measured by specific radioimmunoassays in pregnant, pseudopregnant, or ovariectomized (between gestation weeks 4 and 5) pregnant Labrador Retriever bitches. Daily administration of 17 alpha-ethyl-19-nortestosterone was performed to maintain gestation in the ovariectomized pregnant bitches. This synthetic gestagen was selected because it did not interfere with the assay for endogenously secreted progesterone concentration in serum. Serum progesterone concentration was high in ovarian-intact pregnant or pseudopregnant bitches, but the mean progesterone concentration in pseudopregnant bitches (evaluated at 4 weeks after mating) was only 56% of the concentration in pregnant bitches. After ovariectomy, serum progesterone concentration decreased to undetectable values. Unlike progesterone, serum relaxin concentration increased during the latter half of pregnancy in the ovarian-intact and in the ovariectomized pregnant bitches, but relaxin was not detectable at any time in the pseudopregnant bitches. The amount of relaxin measured in the ovariectomized pregnant bitches was less (P less than 0.05) than that in ovarian-intact bitches, suggesting that the ovaries may have contributed to the total circulating relaxin concentration in the latter. Placental production of relaxin might have accounted for the serum relaxin concentration after ovariectomy; thus, the ovary and placenta each may secrete relaxin during gestation in bitches. Regardless of its source, measurement of serum relaxin concentration may offer a useful way of distinction between pregnancy and pseudopregnancy in dogs.     

Measurement of C‐reactive protein and Prostaglandin F2α Metabolite Concentrations in Differentiation of Canine Pyometra and Cystic Endometrial Hyperplasia/Mucometra

Canine pyometra is a dioestrus period disease in which systemic inflammatory response syndrome (SIRS) is a common outcome due to the response of the body to the bacterial infection. The purpose of this study was i) to differentiate canine pyometra and cystic endometrial hyperplasia (CEH)/mucometra by measuring serum C‐reactive protein (CRP) and prostaglandin F_2α metabolite (PGFM) concentrations in blood and ii) to compare serum concentrations of CRP and PGFM in bitches with a pathological uterus (pyometra or CEH/mucometra) to concentrations in bitches with a healthy uterus. Mean CRP concentrations were found significantly higher (p < 0.001) in dogs with pyometra compared to those with CEH/mucometra or healthy uterus. However, no statistical difference could be detected between the groups for mean PGFM concentrations. Mean white blood cell count (WBC), alkaline phosphatase (ALP) and total protein concentrations were found significantly higher (p < 0.001) in dogs with pyometra. Escherichia coli was the most frequently isolated microorganism from dogs with pyometra (64.3%). Edwardsiella spp. was detected in a single case of pyometra for the first time. In conclusion, our results demonstrate that serum CRP concentrations were increased in dogs with pyometra and thus we conclude that serum CRP concentration but not PGFM might be useful as a marker to differentiate a case of CEH/mucometra from pyometra in female dogs. To the authors' knowledge, this is the first report in which Edwardsiella spp. has been isolated in the canine uterus.     

Transport of spermatozoa in the reproductive tract of the bitch: observations through uterine fistula

Using unilateral uterine fistulas, the time required for spermatozoa to reach the end of the fistula after natural mating, artificial insemination (AI) in a normal standing posture (NP), and AI standing on the head (SH) was investigated in each of three stages of estrus. Conceptivity in these bitches was also investigated. Five experimental bitches were tested during a total of 8 estrous periods. The results are as follows; the time required for spermatozoa to reach the end of the fistula was almost the same in the early and middle stages, i.e., 30 sec to 1 min after natural mating and SH and less than 2 min for half the bitches in NP, although no intrauterine transport could be observed in the other half. In most cases of mating during the late stage no spermatozoa were found after any of the 3 methods of insemination. Five animals became pregnant in these experiments, but the other three failed to conceive. The implantation of fertilized ova occurred also in the fistulated uterine horn in all cases of pregnancy.     

THE INDUCTION OF OESTRUS AND OVULATION IN THE BITCH USING PREGNANT MARE SERUM GONADOTROPHIN AND HUMAN CHORIONIC GONADOTROPHIN

Seventeen bitches (11 anoestrous, 4 prepubertal, 1 pregnant and 1 postpartum) were treated with pregnant mare serum gonadotrophin (110 iu/kg) at weekly intervals up to the occurrence of oestrus or a maximum of 3 treatments. Oestrus was induced in 8/11 anoestrous bitches only. The between bitch ovarian response was very variable. Ovulation occurred in 7/8 PMSG induced oestrous bitches. The 3 oestrous bitches treated with human chorionic gonadotrophin (500 iu) at the start of oestrus had a better ovulatory response than those treated with PMSG alone.     

Assessment of the optimal age for a preventive ultrasonographic screening of the uterine health in bitches

Studies about prevalence of uterine pathologies in bitches are scarce. Although correlation between age and uterine disorders was documented, the most suitable age for a preventive sonographic screening has not been proposed yet. Present study aimed to estimate the eligible age for an ultrasonographic screening of uterine abnormalities in dogs. Data regarding ultrasound examination and clinical records of non‐pregnant intact females were retrospectively analysed. The age of each bitch was expressed as age ratio (actual/maximum age expected for the respective breed). The cut‐off age ratio was determined by a ROC curve for overall uterine abnormalities. Frequencies of different abnormalities below and over the cut‐off derived from the ROC curve were calculated and statistically analysed by chi‐Square and OR. Prevalence of three categories of ultrasonographic findings was as follows: cystic endometrial hyperplasia (CEH) 18%; uterine collections (UC) 10.5%; masses (M) 1.3%. By the cut‐off age ratio (0.325), derived by ROC curve (AUC = 0.91; SP 84.23%; SE 79.2%; PPV 83.4%; NPV 80%), 228 cases were divided into two subgroups: bitches over (exposed group: n.83) and below cut‐off (control group: n.145). All abnormalities resulted more frequent in exposed group: OR was 24.96 (p < 0.0001: 71.1% over vs. 9% below cut‐off) for overall abnormalities; 13.68 (p < 0.0001: 40.9% vs. 4.8%) for CEH; 6.13 (p < 0.002: 21.7% vs. 4.1%) for UC; 12.65 (p = 0.09: 3.6% vs. 0%) for M. Cystic endometrial hyperplasia represents the most common finding in adult bitches, followed by UC. A preventive sonographic screening for uterine abnormalities should start from 33% of expected longevity to preventively select animals requiring further evaluations.