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.     

Zearalenone applied per os provides adverse effects in structure of chosen parts of bitch reproductive system

Both earlier investigations and complete formulas of pet food for dogs show that professionally prepared pet food is based in 30% on the material of the plant origin. Our own investigations prove that animal feeding stuffs of the plant origin are very often vectors of many undesirable substances (including micotoxins) that are unfavourable for carnivores. Zearalenone is a micotoxin, which frequently occurs among them. It was revealed in pet food in quantities from 5.0-299.5 microg/kg. It is a very dangerous xenobiotic because of its quick and easy absorption in the organism after per os application in monogastric animals. It is also a sterid and despite its plant origin it binds estrogen receptors in the reproductive tract and causes its hypo- and dysfunction. It is clearly visible in young, growing organisms and those during the menopause. In bitches, dysfunctions of the reproduction system, such as: endometritis-pyometra syndrome, prolonged oestrus, ovarian cysts and others are often found. It is supposed that specific for this species hormonal regulation of the reproduction processes based on the long progesterone and prolactin cycles, and on high sensibility to estrogens plays an important role in the etiopathogenesis of these dysfunctions. The application of hormones in bitches in therapeutic and biotechnical (contraception) purposes are the other factors, which should be taken into account in this aspect. There are some suggestions that therapeutic management, unfortunately connected with mistakes in the medical art, is often a cause of pathological condition in a reproductive organ in bitches. However, another reason of these disorders may be that bitches take zearalenone with the commercial pet food. The long-lasting intake of the feed with a low dose of this micotoxin may be the factor, which complicates hormonal regulation of the reproduction processes and is the cause of many disorders. Our own pilotage investigations showed that application of zearalenone at a dose of 200 microg/kg b.w. for 7 days leads to apoptotic-like changes in granule cells in the bitch reproductive system, atresia of follicular cells and oedema and hyperplasia of the oviductal and uterine cells. They could also indicate a follicular phase of the oestrus cycle in bitch. However, it did not take place.     

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.     

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 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.     

Laparoscopic sterilization of the bitch and queen by uterine horn occlusion

Laparoscopic sterilization techniques, originally developed for use in women, were evaluated in the bitch and queen. In the first study (study I), the uterine horns of 6 bitches and 3 queens were occluded by electrocoagulation or plastic clips. The sites of occlusion were midway along the length of 1 cornus and at the uterotubal junction on the contralateral side. Both procedures effectively occluded the uterine horns, as evidenced by a distinctly visible separation of the reproductive tract. Laparoscopic examination 1 year after surgery revealed an enlarged, thin-walled, and fluid-filled uterine segment cranial to the midcornus occlusion sites in all animals. The contralateral horn was normal in appearance, except for the separation from the ovarian bursa. Three of the bitches developed pyometra (confined to the distended uterine segment) at 24 months, at 53 months, and at 72 months after sterilization, respectively. In a subsequent study (study II), 1 adult and 5 prepubertal bitches were sterilized by laparoscopic electrocoagulation of both uterine horns at the uterotubal junction adjacent to the ovarian bursa. Upon reexamination 1, 2, and 4 years later, the uterine horns of these females were normal in appearance, but were separated from the adjacent ovarian bursae. These females continued to be clinically healthy. Laparoscopic sterilization offers a rapid and safe alternative to ovariohysterectomy and, because of its minor invasive nature, can be performed on young, prepubertal animals. Such a procedure may have particular value as a simple, practical means of sterilizing dogs and cats on a mass basis.     

Analysis of integrins and vascular endothelial growth factor isoforms mRNA expression in the canine uterus during perimplantation period

Integrins are the major receptors within the extracellular matrix (ECM) that mediate several functions connected with cell life and metabolism, such as cell adhesion, migration, cytoskeletal organization, proliferation, survival, and differentiation. A vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors. It has been suggested that the expression of this gene may play crucial physiological roles in reproductive organs. All investigated endometrial tissues were isolated on day 10-12 after mating. Control bitches, used in this study, were in metestrus, which was determined according to the vaginal cytology and progesterone level in blood. Early pregnancy was verified by flushing the uterine horns with PBS. Total RNA was isolated from the bitches endometrium by means of the Chomczyński and Sacchi method, treated by DNase I, and reverse-transcribed into cDNA. A quantitative analysis of integrins alpha2b, beta2 and beta3, VEGF 164, 182 and 188 cDNA was performed by RT-PCR. In results we have shown an increased expression of all investigated genes (integrins alpha2b, beta2 and beta3, VEGF 164, 182, and 188) in pregnant bitches uterus as compared to non-pregnant females (P < 0.001). Our results indicated that the expression of genes encoding integrins and vascular endothelial growth factors is different in relation to the time of the embryo implantation and it is increased in the first period of this process. This may be associated with the induction of specific mechanisms responsible for receptivity of uterus following the embryo attachment. In addition, all of investigated genes are up-regulated in a pregnancy-specific manner and the increased expression of these genes may regulate the uterus function during the implantation of canine embryos.     

Lactoferrin in Canine Sera: A Pyometra Study

The concentration of lactoferrin was measured in canine sera from groups of healthy male dogs as well as pregnant and non-pregnant female dogs and was compared with that of bitches with pyometra. Lactoferrin concentrations were higher in bitches with pyometra. The role of elevated lactoferrin concentrations in the suppression of lymphocyte activity was examined in sera from bitches with pyometra in a series of investigations. Although the sera from bitches with pyometra were capable of suppressing lymphocyte activity, lactoferrin was not found to be involved in this action.     

Experimental Bovine Genital Ureaplasmosis II. Granular Vulvitis, Endometritis and Salpingitis Following Uterine Inoculation

Twenty-three virgin Holstein heifers received uterine inoculations with ureaplasma and were necropsied one to thirteen days later. Three heifers inoculated intracervically were necropsied on days 3, 5 and 11.

Granular vulvitis was produced on average by 3.6 days in fourteen of sixteen uterine inoculated heifers monitored for four or more days. Two cervically inoculated heifers monitored for over four days also developed granular vulvitis by the fourth day.

At necropsy, ureaplasma was recovered from 94% of uterine horn cultures for the first four days postinoculation and 50% during days 5 to 7. Thereafter all uterine cultures were negative. The percentage of positive ureaplasma recoveries from uterine tube flushings was lower than for uterine horns but remained positive for a longer period. By day 7, three of four uterine tube flushings were still positive. No bacterial pathogens were isolated from the uterine horns or uterine tube flushings.

On histopathology 50% of uterine inoculated heifers had endometritis up to six days postinoculation and a slightly higher percentage (58%) had salpingitis. Endometritis was not found in any heifers after day 6. Residual salpingitis was present in one heifer on day 7. Endometritis was present in cervically inoculated heifers necropsied on days 3 and 5 but not on day 11. Salpingitis was not found in any of the three cervically inoculated animals.

The study concluded that some strains of ureaplasma are pathogenic for the upper reproductive tract of the cow and should be considered significant when isolated from cases of granular vulvitis, endometritis or salpingitis.

     

Intrauterine and intravaginal insemination with frozen canine semen using an extender consisting of orvus ES paste-supplemented egg yolk tris-fructose citrate

Our previous report indicated that addition of Orvus ES Paste (OEP) to the extender of frozen canine semen protected acrosomes and maintained sperm motility after thawing. In this study, artificial insemination (AI) using the frozen semen was carried out. The frozen semen was prepared using egg yolk Tris-fructose citrate, and the final concentrations of glycerol and OEP were 7% (v/v) and 0.75% (v/v), respectively. AI was performed during the optimal mating period predicted from the peripheral plasma progesterone level. In intrauterine insemination (IUI), the bitches were laparotomized and 1 x 10(8) spermatozoa were infused into one of the uterine horns. In insemination of non-OEP supplemented semen, 3 x 10(8) spermatozoa were inseminated. In intravaginal insemination (IVI), 10-40 x 10(8) spermatozoa were inseminated. Conception was obtained in nine of 10 bitches (90.0%) that underwent IUI. The number of newborns was from 1 to 7 (mean 3.6 +/- 0.9). The mean ratio of the number of puppies to the number of ovulations in the inseminated uterine horn was 71.8%. The number of puppies did not exceed the number of ovulation in the inseminated uterine horn. Conception using non-OEP supplemented frozen semen was unsuccessful in all four bitches. In IVI, conception was not obtained in any of the six bitches that received insemination of 10 x 10(8) or 40 x 10(8) spermatozoa, but two of three bitches that received insemination of 20 x 10(8) spermatozoa were fertilized. It was shown that a high conception rate can be obtained by IUI using OEP-supplemented frozen canine semen. Developmenmt of a non-surgical method of IUI and a method of freezing canine sperm applicable to IVI is necessary.     

Protease-induced hyperactivation of canine spermatozoa associated with disappearance of lectin-binding glycoproteins on their surface

The relationship between the disappearance of glycoproteins from the surface of canine sperm and sperm capacitation was investigated in vitro. The protease (PR) concentration in flush fluids of the uterine horns and oviducts removed from 6 estrous, 5 diestrous, and 5 anestrous bitches was measured with a protease assay kit. Ejaculated sperm collected from 10 dogs were incubated for 4 hr in Eagle's MEM supplemented with 1 or 5 microg/ml PR, or to which no PR had been added (control). The glycoproteins on the surface of the sperm were stained with 4 different FITC-lectins (Con A, PHA-E, PNA, and WGA), and the percentages of hyperactivated (HA-) sperm and acrosome-reacted (AR-) sperm were evaluated. The mean PR concentration (5.95 microg/ml) in the flush fluid from the oviducts of the estrous bitches was significantly higher than in the fluid from their uterine horns (1.00 microg/ml; P<0.01). The PR concentrations of the flush fluids from the uterine horns and oviducts of both the diestrous and anestrous bitches were less than 0.05 microg/ml. Before incubation the acrosomal regions or entire heads of all sperm clearly stained with each FITC-lectin, but the percentages of sperm binding the 4 FITC-lectins decreased after incubation. The percentages of lectin-binding sperm in the MEM containing 5 microg/ml PR were significantly lower than in the control MEM (P<0.05 and 0.01). The mean percentages of motile sperm and HA-sperm after incubation in the MEM with PR were higher than in the control MEM, but there were no differences in the percentages of AR-sperm. The results indicate that HA-movement of sperm is induced by the disappearance of glycoproteins from the surface of canine sperm as a result of the action of PR in the oviductal fluid of estrous bitches.     

Long-term prevention of estrus in the bitch and queen using chlormadinone acetate

Estrus was prevented with weekly oral administration of 2 mg chlormadinone acetate for 2.0 to 9.8 y in bitches and queens. Abnormalities, including mammary or uterine disorders, or both, were noted in 7 out of 14 bitches and 9 out of 24 queens during this long-term treatment.     

Escherichia coli strains isolated from the uterus and urinary bladder of bitches suffering from pyometra: comparison by restriction enzyme digestion and pulsed-field gel electrophoresis.

Pyometra (uterine inflammation with accumulation of pus in the uterus) is regarded as one of the most common illnesses in bitches. The ethiology and pathogenesis are complex with both hormonal and bacterial elements. The bacteria most frequently isolated from the uterine content is Escherichia coli.In this study, 84 E. coli strains from the uteri of 70 bitches suffering from the disease were examined and their DNA-profiles compared by restriction enzyme analysis and pulsed-field gel electrophoresis (PFGE). Through variations in DNA-profiles of the E. coli isolates, this study indicates that pyometra is caused by E. coli originating from the normal flora of the dogs and not by certain clones spread between animals.E. coli strains from the urinary bladder and the uterus of six of the bitches suffering from simultaneous urinary tract infection and pyometra were examined and compared as above. The DNA-profiles of the isolates from each of the six bitches were 100% identical. This study supports the theory suggesting that in cases of simultaneous urinary tract infection and E. coli pyometra, the urinary tract and uterus are infected with the same bacterial strain.To evaluate whether the uterus was infected with a single clone of E. coli or if multiple clones were present, eight to 16 colonies of E. coli isolated from pyometra samples from a further 10 bitches were examined. All bacterial colonies from the culture of the same bitch showed identical DNA-profiles.In 14 of the 70 bitches, two macroscopically different but biochemically identical E. coli colony types were isolated. The two colony types from the same bitch proved to have identical DNA-profiles in 13 cases and almost identical in the remaining bitch.     

Changes in the mast cell distribution in the canine ovary and uterus throughout the oestrous cycle

This is the first study describing the relation between the phase of the oestrous cycle and the number of mast cells (MCs) in the canine ovaries and uterine horns. The total number of MCs was counted in cortex ovarii, medulla ovarii, endometrium and myometrium. The number of MCs was compared in all of these areas among the bitches in the early follicular phase, luteal phase and anoestrus. MCs were the most numerous in the early follicular phase and in the luteal phase; however, they were significantly less numerous during anoestrus. Based on the results, it was concluded that the number of MCs in ovaries and uterine horns fluctuates during the oestrous cycle. The results of this study may contribute to clarifying the role of MCs in the reproductive organs of bitches.     

Genital lesions and distribution of amastigotes in bitches naturally infected with Leishmania chagasi

Recent reports indicate that Leishmania chagasi has tropism to the male canine genital system, which is associated with shedding of the organism in the semen, supporting the hypothesis of venereal transmission. The aim of this study was to describe the lesions and assess parasite load in the genital system of bitches with canine visceral leishmaniasis (CanL). Symptomatic (n=5) and asymptomatic (n=5) bitches seropositive for CanL were randomly selected at the Center for Zoonosis Control (Belo Horizonte, State of Minas Gerais, Brazil). Five serologically negative, healthy, adult bitches also from the CZC were used as controls. Samples from genital organs (vulva, vagina, cervix, uterine body, uterine horns, uterine tubes, and ovaries), liver, and spleen were histologically evaluated and processed for immunodetection of Leishmania sp., and PCR. The most significant histological change was a mild to moderate vulvar dermatitis, characterized by a histio-plasma-lymphocytic infiltrate. This change was detected in all asymptomatic, four symptomatic, and three uninfected control bitches. In one symptomatic and one asymptomatic bitch intracytoplasmic amastigotes were observed within macrophages in the inflammatory infiltrate. Samples from all the segments of the genital tract were positive in at least one infected animal, in the absence of detectable amastigotes in the tissue. These findings support the notion that L. chagasi does not have genital tropism in the bitch, which is in contrast to our previous findings in naturally infected male intact dogs.     

Surgical uterine drainage and lavage as treatment for canine pyometra

Pyometra is a common post-oestral syndrome in bitches. Classical treatment consists of either ovariohystorectomy or medical intervention. Surgical uterine drainage and lavage via direct trans-cervical catheterisation using a 5% povidone-iodine in saline solution was performed successfully in 8 bitches with pyometra. All bitches conceived and whelped without complications subsequent to this treatment. It is concluded that this method offers an effective alternative treatment for canine pyometra with shorter recovery times as well as good clinical recovery and pregnancy rates in bitches destined for further breeding.     

Molecular studies on oestrogen α and progesterone receptors and histomorphometric analysis of canine uteri following aglepristone treatment

Aglepristone, a competitive progesterone antagonist, is successfully used in various progesterone-dependent conditions. This study investigated uterine histomorphometric analysis, and expressions of the oestrogen α receptor (ERα) and progesterone receptor (PR) in uteri of bitches following the single dose of aglepristone treatment. Twelve client-owned healthy diestrous bitches were used in the study. The single dose of aglepristone (Alizine^®, 10 mg/kg) was injected subcutaneously 5 days before ovariohysterectomy in the treatment group (n = 6); bitches without treatment served as a control group (n = 6). Uteri were collected for histomorphometric analysis, ERα and PR gene, and protein expressions studies. The mRNA expressions of ERα and PR were determined by RT-qPCR. Immunohistochemical analysis was used to evaluate the ERα and PR protein expressions using an H-score in five parts of the uterus. The results demonstrated glandular epithelium height significantly decreased (p < .05) and ERα mRNA increased (p < .01) in treated dogs. Of the treated bitches, lower expression levels of ERα were observed in the luminal epithelium, crypt and glandular epithelium, with higher expression in the endometrial stroma and myometrium (p < .05); however, PR expression decreased in the luminal epithelium, crypt and glandular epithelium (p < .01). In conclusion, reduction of the uterine glandular epithelium and ERα mRNA upregulation together with changes in ERα and PR expressions were observed in the treated bitches. However, changes in uterine ERα and PR expressions in the treated bitches depended on tissue layers. The treatment had no effect on serum oestradiol and progesterone levels.     

Induction of dog sperm capacitation by glycosaminoglycans and glycosaminoglycan amounts of oviductal and uterine fluids in bitches

Ejaculated sperm collected from 12 beagle dogs were incubated in canine capacitation medium (CCM), supplemented with 5 microg/ml chondroitin sulfate A (CS), 5 microg/ml hyaluronic acid (HA), or 5 microg/ml heparin (HP) for 7 hr at 38 degrees C in a 5% CO2 in air atmosphere to investigate the effects of glycosaminoglycans (GAGs) on dog sperm capacitation. The percentages of motile sperm, hyperactivated sperm (%HY), and acrosome-reacted sperm (%AR) in all media were examined after 4 hr and 7 hr of incubation. The oviducts and uteri of 9 anestrous and 18 estrous beagle bitches were removed under halothane inhalation anesthesia to measure the total GAG amounts in oviductal and uterine fluids. The lumens of the ampulla of the oviducts, isthmus of the oviducts, and the uterine horns were each flushed with 1 ml HEPES-EDTA fluid. Total GAG amounts in the flush fluids obtained were measured with a spectrophotometer. Sperm motility (51-59%), %HY (79-86%), and %AR (31-36%) in CCM supplemented with CS, HA, or HP were significantly higher after 7 hr of incubation than when incubated in CCM without GAGs (P<0.01 or 0.05). The mean total GAG amounts in the fluids from the ampulla and isthmus of the oviducts and the uterine horns in the estrous bitches were higher than in the anestrous bitches. These results indicate that GAGs in the oviductal and uterine fluids in estrous bitches are associated with in vivo sperm capacitation.     

Recording uterine motility in the nonanesthetized bitch

Spontaneous and drug-induced uterine motility (UM) was recorded in 5 nonanesthetized bitches for 2 to 4 days. Catheter-tip pressure transducers were surgically implanted in 1 uterine horn, tunneled subcutaneously to exit from the skin over the dorsal lumbar area, and protected by a bandage. On the day after implantation, spontaneous UM was recorded in the awake bitch. Effects of IV prostaglandin (PG) F2 alpha (5 micrograms/kg of body weight) and oxytocin (0.05 USP U/kg) and IM PGF2 alpha (25 micrograms/kg) were measured. Estradiol (1 to 25 micrograms/kg) was administered and the study was repeated 24 hours later. In awake bitches, spontaneous UM was 190% greater than UM in anesthetized bitches. Uterine motility was increased by more than 100% after IV PGF2 alpha or oxytocin and by 52% after IM PGF2 alpha. Estradiol abolished spontaneous UM, but did not affect drug-induced responses. Seemingly, spontaneous and drug-induced UM can be documented in the nonanesthetized bitch.