Early embryo development, number, quality, and location and the relationship with plasma progesterone in dogs.

This research assessed the developmental stages and morphological quality of dog embryos collected during different stages of pregnancy as well as the relationship with serum progesterone recorded at insemination and embryo collection. Embryos were collected from 23 young mature bitches, that had been inseminated with fresh semen 3-6 days after the LH surge (day 0). Embryo flushing was performed on pregnancy days 8-11 (Group 1), 12-15 (Group 2), or 16-20 (Group 3). The location, number and morphological characteristics of the embryos were evaluated. A total of 120 embryos and 25 unfertilized oocytes were collected from bitches with a total of 156 corpora lutea (CL). The mean total embryo yield (total of embryos/CL) was 76.7 ± 5.9%, and the mean embryo recovery rate (number of flushed embryos/number of CL) was 70.6 ± 6.6%. The mean ovulation rate was 6.8 ± 0.5 and the mean number of embryos per bitch was 5.2 ± 0.6. Oocyte fertilization occurred following oocyte maturation. Most embryos in Group 1 (70.0 ± 18.6%) were collected at the 2 to 16 cell stage. The morula stage was first observed on day 11. Expanded blastocysts (EBLs) and hatched blastocysts were first flushed from the uterus on days 13 and 14, respectively. The EBL was the most abundant stage in Groups 2 and 3. After day 19, some embryos (n = 8) had already adhered to the endometrium. Although most recovered embryos were classified as very good, a greater number of low quality embryos was collected in the later gestational periods. A significant variation in the embryonic stages and location of embryos in early canine pregnancy was observed, as embryos entered the uterus independently of their developmental stage. Embryo yield and quality were independent of the serum progesterone concentration at insemination and recovery.

Decidualization of the canine uterus: From early until late gestational in vivo morphological observations, and functional characterization of immortalized canine uterine stromal cell lines.

The apparent lack of classical mechanisms for maternal recognition of pregnancy is one of the most intriguing features of canine reproduction. Consequently, similar levels of circulating luteal steroids are observed in pregnant and non-pregnant dogs. However, the early pre-implantation canine embryo locally modulates uterine responses to its presence, facilitating the successful onset of pregnancy. As a part of this interaction, the canine uterus undergoes a species-specific decidualization. Maternal stroma-derived decidual cells develop, the only cells of the canine placenta expressing progesterone receptor (PGR). There exists an acute need for an in vitro stable cell line model for canine decidualization. Therefore, herein our goal was to establish, immortalize and characterize such a cell line. We immortalized three monolayer dog uterine stromal (DUS) cell lines by stably transfecting them with SV40Tag oncogene. Cells retained their mesenchymal character for over 30 passages, as evidenced by VIMENTIN staining. Genomic incorporation of the SV40Tag protein was confirmed by immunofluorescence and Western blot analyses. Cells submitted to a classical in vitro decidualization protocol (N6,2'-O-dibutyryladenosine-3',5'-cyclic monophosphate) revealed upregulated gene levels of selected major decidualization markers (e.g. PRLR, PGR, IGF1, PTGES). Additionally, the basic decidualization capability of PGE2 was demonstrated, revealing increased levels of, for example, PGR and PRLR gene expression, thereby implying its involvement in the progesterone-dependent decidualization in the canine uterus. In summary, our in vitro model with immortalized DUS cell line could serve as an ideal and unique model to study the underlying molecular and endocrine mechanisms of canine decidualization.

Immunolocalization of E-cadherin and ß-catenin in the cyclic and early pregnant canine endometrium.

Putative changes in E-cadherin and ß-catenin during implantation in dogs are of interest to study, as they are relevant proteins for epithelial integrity. E-cadherin and ß-catenin were immunolocalized in the canine endometrium during the estrous cycle and early pregnancy, using monoclonal antibodies. Both proteins were detected in all types of endometrial epithelia (surface epithelium [SE], superficial glandular, and deep glandular epithelia) at all stages of the estrous cycle and in early placental structures. E-cadherin depicted a gradient of intensity apparently being lowest in the SE to progressively increase toward the deepness of the endometrial glands, regardless of the stage of estrous cycle. The overall immunostaining was, however, weaker at diestrus. In pregnant samples, the trophoblast was conspicuously immunolabeled compared with the endometrial surface lining epithelium. In the latter, the cytoplasmic pattern predominated over the membrane-bound, as was also seen in the decidual cells of the placental labyrinth. In the early placenta, only trophoblast cells and lacunae retained membrane signals. ß-Catenin membrane labeling appeared relatively constant throughout the cycle, although a tendency toward a decrease in intensity was detected at the secretory stages. In addition, a dislocation of the immunoreaction from membrane to the cytoplasm was observed in both the SE and the glandular epithelia at particular stages of the cycle. In early pregnancy, a loss of the membranous pattern was observed in the SE and labyrinth, but neither on trophoblast nor in lacunae. The results show the existence of a softening of the adherens junctional complex in progestagen-dominated stages favoring embryo-maternal interactions and endometrial invasion during canine implantation.

Changes in c-erbB-2 Immunoexpression in Feline Endometrial Adenocarcinomas.

Human epidermal growth factor receptor type 2 (c-erbB-2), an oncoprotein with potential prognostic marker and therapeutic use, is overexpressed in several human and animal tumours. But information regarding this molecule in feline tumours is scarce. This study aimed to assess the changes in the immunohistochemical expression of c-erbB-2 in feline endometrial adenocarcinomas (FEA) compared to normal endometrium. An immunohistochemistry assay using a specific antibody against c-erbB-2 was performed in FEA samples (n = 34) and in normal endometrium in the follicular (FS; n = 12) and luteal (LS; n = 11) stages. In FEA, the c-erbB-2 immunoexpression was assessed in neoplastic epithelial cells whilst in normal endometria it was individually evaluated in the surface and the superficial and deep glandular epithelia (SE, SGE and DGE, respectively). In FS and in LS, all the epithelia were positive for c-erbB-2; positivity was higher in the SE and the SGE than in DGE. Twenty of the 34 FEA samples (58.8%) were positive for c-erbB-2 immunolabelling. Nevertheless, its expression was higher in all the epithelia in the FS compared to FEA (p ≤ 0.0001) or the LS (p = 0.016). The results presented herein suggest that c-erbB-2 molecule is differently expressed in the feline endometrium through the oestrous cycle and though it may also be involved in feline endometrial carcinogenesis, a question remains unanswered on the importance of additional pathways of epithelial proliferation in the neoplastic changes in feline endometrium.

Fertility in Adult Bitches Previously Treated with a 4.7 mg Subcutaneous Deslorelin Implant.

The absence of fertility problems in male dogs after a single treatment with deslorelin acetate (Suprelorin(®)) is well acknowledged. However, reports on the application of deslorelin in the bitch and information concerning fertility after implant treatment are still limited. In this retrospective study, data concerning induced and spontaneous oestruses of 39 bitches from 17 breeds, treated with deslorelin acetate implants (4.7 mg Suprelorin(®), Virbac, France), were retrieved to assess post-treatment fertility (ovulation rate, pregnancy rate and litter size). Animals were grouped according to treatment characteristics: group 1 (Gr1) - females submitted to oestrus induction, showing natural oestruses afterwards (n = 19); group 2 (Gr2) - females re-implanted with 4.7 mg deslorelin acetate to re-induce oestrus, showing subsequent spontaneous post-implant oestruses (n = 7); and group 3 (Gr3) - females submitted to a 4.7 mg deslorelin acetate implant for oestrus suppression, evaluated at subsequent spontaneous post-implant oestruses (n = 13). Comparison of fertility traits between induced and post-treatment spontaneous oestruses in Gr1 and Gr2 (short treatments), or between spontaneous oestruses after long-treatment schedules (Gr 3) revealed a slightly better performance in spontaneous cycles compared with induced cycles: ovulation rate post-treatment was 97.1%, 94.1% and 94.4% and the pregnancy rate post-treatment was 91.2%, 88.9% and 84.6% for groups 1, 2 and 3, respectively. Nevertheless, fertility in induced and post-treatment oestruses was considered normal. Moreover, the individual litter size did not differ within groups between induced and spontaneous cycles. From these findings, we concluded that treatment with 4.7 mg deslorelin implants did not compromise the bitches' fertility in subsequent oestruses.

Reproductive Patterns in the Non-Breeding Season in Asinina de Miranda Jennies.

This study aims to characterize the reproductive patterns in Asinina de Miranda jennies during the non-breeding season. Reproductive activity was surveyed in 12 females, aged between 3 and 18 years old, using ultrasound and teasing with a jack. The animals were monitored from September to April, six in each consecutive year. Of these 12 females, nine showed disruption to the normal pattern of ovarian activity during the non-breeding season. Loss of normal cyclicity included anoestrus (41.7%), silent ovulatory oestrus (25%), and persistence of corpus luteum (8.3%). Only three females maintained a regular cyclic pattern with oestrous behaviour during the non-breeding season. Anoestrus began in early November and lasted for an average of 147 ± 28 days (113-191 days), ending near to the spring equinox. Onset of silent oestrous cycles began more erratically, between October and February. In both groups the first behavioural ovulation of the year occurred around the time of the spring equinox. Disrupted reproductive activity was preceded by a shorter oestrous cycle only in females entering anoestrus. The mean follicle size in the first ovulation of the year was larger than in the reproductive season (44.7 ± 2.45 mm vs 39.2 ± 3.60 mm) in anoestrous jennies with protracted oestrus. Though age and body condition score (BCS) were associated, changes in BCS below a threshold of four points (for anoestrus) and five points (for silent oestrus) contributed greatly to disruption of reproductive cycles. BCS in females with regular oestrous cycles during the winter season remained unchanged or exceeded five points prior to the winter solstice.

Resident Macrophages and Lymphocytes in the Canine Endometrium.

Resident immune cells play a major role in endometrial immunity and in tissue homoeostasis. This study aimed to analyse the distribution of macrophages, B and T lymphocytes (respectively, Mø, B-Lym and T-Lym) in the canine endometrium throughout the oestrous cycle and in late involution (at the proestrus stage post-parturition). An immunohistochemistry technique was used on samples from 50 post-pubertal healthy female dogs, of which five in late post-partum. The distribution of resident immune cells was analysed in three endometrial layers (superficial, intermediate and basal areas). Mø, B-Lym and T-Lym were demonstrated to reside in the endometrium in all the stages of the canine cycle; their numbers being considerably higher during late involution. T-Lym were scattered in the stroma or amidst the glandular epithelium, constituting the predominant immune cell population in anestrus and proestrus, but decreased in number at all other stages. Endometrial B-Lym remained fairly constant during the canine cycle, although its numbers were higher in late involution. Mø counts were higher during anestrus compared to the other stages, the cells being displaced into the superficial endometrial layer. Mø demonstrated the highest level in late involution samples, forming small aggregates below the surface epithelium. The number of immune cells was not normally distributed, suggesting the influence of individual factors, such as age or parity, not explored herein due to limited sample availability. Still, this study provides important information for the interpretation of endometrial biopsies in dogs and for the understanding of the increased susceptibility to uterine infection during dioestrus found in the bitch.

Immunohistochemical expression of cyclooxygenase-2 (COX-2) in feline endometrial adenocarcinoma and in normal and hyperplastic endometria.

Cyclooxygenase-2 (COX-2) is overexpressed in several human and animal neoplasms, including the human endometrial carcinoma. It has been suggested as a prognostic marker and a potential therapeutic target. This study aimed to (i) clarify histological aspects of feline endometrial adenocarcinomas (FEA) of the papillary serous type and (ii) characterize COX-2 immunohistochemical expression in normal, hyperplastic and neoplastic endometrium in this species. Archived paraffin-embedded tissue samples of 33 FEA, eight cystic endometrial hyperplasias (CEHs) and 21 samples of normal, healthy endometrium in the follicular (FS; n = 10) and luteal (LS; n = 11) stages were evaluated. Histological evaluation of haematoxylin and eosin-stained sections of the FEA revealed a papillary proliferation of neoplastic cells of serous type, accompanied by clear and multinucleated cells. Other architectural arrangements mainly included solid and tubular growth. Randomly distributed areas of necrosis within the tumours were commonly observed. Invasion of the myometrium, of the serosa and of the vascular and/or lymphatic vessels was not constant features. The mean number of mitoses was higher in FEA compared to non-neoplastic endometrium. COX-2 scores were lower in FEA (p = 0.003) and CEH (p = 0.05) when compared to normal epithelium (NE). The loss of the membrane apical reinforcement in epithelial cells was observed in FEA samples, which was accompanied by the dislocation of COX-2 labelling into the cytoplasm and the perinuclear area; in contrast, in epithelial cells in the healthy and hyperplastic endometria, the immunoreaction showed the characteristic pattern of apical membrane reinforcement, suggestive of the membrane polarization. COX-2 epithelial scores were higher in the FS than in the LS. No differences were found in stromal COX-2 expression between normal, CEH and FEA groups, but it was higher in the LS than in the FS. In summary, loss of COX-2 compartmentalization in neoplastic epithelial cells might be one of the molecular events underlying endometrial carcinogenesis.

Characterization of the estrous cycle of Asinina de Miranda jennies (Equus asinus).

This study aims to characterize the estrous cycle of Asinina de Miranda jennies in the breeding season, on the basis of data collected from serial ultrasonographic examination and serum progesterone determinations in 14 females during a total of 33 cycles. The length of the interovulatory interval was 23.8 ± 0.55 days, the diestrus and estrus lasting 17.9 ± 0.46 days and 6.65 ± 0.30 days, respectively. Age and body condition score (BCS) affected the length of the interovulatory intervals; BCS also influenced the diestrus length and the time in heat after ovulation (P > 0.05). The incidence of single, double, and triple ovulations was 57.58%, 36.36%, and 6.06%, respectively. Multiple ovulations affected neither the length of the interovulatory interval nor the individual cycle stages (P > 0.05) but lengthened the interval from the beginning of estrus to the last ovulation (P = 0.01). When combined with age, higher BCS affected the ovulation rate (P = 0.001). Deviation of the dominant follicle occurred around Day 8.7 (Day 0 = ovulation) when both single and multiple ovulations were considered. The dominant follicle was larger at divergence in single ovulators (19.18 ± 0.97 mm) compared with that in multiple ovulators (18.05 ± 1.16 mm). The overall maximum follicular diameter before ovulation was smaller in multiple ovulatory cycles than that in single ovulatory cycles (37.2 ± 0.83 mm vs. 40.2 ± 1.41 mm, respectively; P = 0.03). The daily growth rate of dominant follicles was independent of the ovulation rate (P > 0.05) for the intervals before and after the estrus onset. The dominant follicle size and the follicle growth rates were independent of BCS (P > 0.05). Data collected in this study revealed resemblances between Mirandese and other Iberian and Brazilian breeds with regard to estrous cycle characteristics.

Temporal changes in neutral endopeptidase/CD10 immunoexpression in the cyclic and early pregnant canine endometrium.

CD10 is a multifunctional transmembrane neutral endopeptidase (NEP) that is considered to be a reliable marker of ectopic human endometrial stroma. Available information on NEP/CD10 protein expression in animal endometria is scarce. This study focused on the immunolocalization of NEP/CD10 in the canine uterus and on its temporal changes during the estrous cycle and early pregnancy (Days 11 to 23 post-LH surge) in healthy females. NEP/CD10 expression was found in the canine endometrial stroma in all stages of the estrous cycle, showing cyclic differences both in intensity and in distribution pattern. A small population of negative stromal cells in subsurface position was also observed. This population shared some morphological characteristics with the human predecidual cells, which became positive in progesterone-associated stages of the cycle. In addition, positive immunolabeling was also observed in canine myometrial stroma. In early pregnancy, the basal glandular epithelia and the syncytium cords remained negative to this marker contrasting with the trophoblast and the lacunar epithelium. A weak to moderate intensity of immunolabeling was observed in the decidual cells, whereas stromal immunolabeling was more intense at the delimitation of the syncytium cords. In conclusion, CD10 is consistently expressed in the canine endometrial stroma and myometrium but not in the endometrial epithelia. The characteristic pattern seen in early pregnancy also suggests a role for this molecule in the process of embryo invasion at implantation.

Pedigree and herd characterization of a donkey breed vulnerable to extinction.

Most donkey and local horse breeds are vulnerable to extinction as mechanization of agriculture progress throughout the world. The present study analyzed the pedigree and herd records of the donkey Asinina de Miranda breed (RAM), identifying genealogical and human factors that may affect the breed genetic diversity in the future and suggesting suitable strategies to breed preservation, early on the conservation program. The breeding rate was very low, with a ratio of foaling/live animals of 0.23 (178/760). The estimated number of founders and ancestors contributing to the reference population was 128 and 121. The number of founder herds in the reference population was 64, with an effective number of founder herds for the reference population of 7.6. The mean age of herd owners was 65.50 ± 0.884 years, with a negative association among the herd size and owner's age (P<0.001). In contrast, the size of the herd and the ownership of a male were both positively associated (P<0.001) with the herd number of in-born foals. Both the owners' age and the herd location (RAM home region v. dispersal region) were negatively associated with the foaling number (P<0.001). The main identified risk factors were: low breeding rates; low number of males and their unequal contribution to the genetic pool; unequal contribution of the herds to genetic pool; and advanced age of herd owners.

Collection of field reproductive data from carcasses of the female Eurasian lynx (Lynx lynx).

Information about reproductive physiology in the Eurasian lynx (Lynx lynx) would generate knowledge that could be useful in the management of the Swedish lynx population based on the knowledge about their reproductive potential and population development. Age-related differences in ovulation and implantation rates would affect the reproductive output and the development of the population. The aims of this study were to evaluate a protocol for collection of reproductive data from carcasses by comparisons with published field data and to generate data about reproduction in the Swedish lynx. Reproductive organs from 120 females that were harvested between March 1 and April 9 from 2009 to 2011 were collected and evaluated macroscopically for placental scars. Females had their first estrus as yearlings but did not have their first litter until the next season. Pregnancy rates were lower in 2-year-old females than in females aged 3 to 7 years but did not differ significantly from females aged 8 to 13 years (54.5%, 95.6%, and 75.0%, respectively). CL from the present season were morphologically distinctly different from luteal bodies from previous cycles (LBPC). All females ≥3 years had macroscopically visible LBPC, whereas only 67% of 22 to 23 months old females had one to three LBPC and no females <1 year of age had LBPC. Females aged 34 to 35 months had up to eight LPBC, whereas the highest number of LBPC counted in females ≥3 years of age was 11. These data would be in agreement with only one estrus per season and LBPC from at least three previous reproductive seasons in older females. The number of LBPC was significantly correlated with the weight of the ovaries rs = 0.648, P < 0.001) and the age of the animals (rs = 0.572, P < 0.001). Uterine weight differed significantly with the stage of the reproductive cycle and was highest for mature females in the luteal phase of the cycle. The estrous period, defined as occurrence of ovarian follicles lasted from March 5 to April 1 in this material. In conclusion, this study confirms that useful information about lynx reproduction can be collected from reproductive organs retrieved after the death of the animals. Continuous monitoring of lynx reproductive organs would therefore make a valuable contribution to collection of field data, gathering information that can be useful for the management of lynx populations and potentially for the lynx as an indicator of environmental disturbances.

Feline endometrial adenocarcinoma in females <1 year old: a description of four cases.

Uterine neoplasms of epithelial origin are rare in cats and most often are described in older females. Yet, in less than 2 years, four ovariohysterectomy specimens were submitted from different practices to the Laboratory of Histology and Anatomical Pathology, at UTAD (Vila Real, Portugal), that emitted a diagnosis of feline endometrial adenocarcinoma. Untypically, all the females were aged <1 year old at the surgery. Access to the clinical files was requested to document the clinical features of the four cases, including any complementary data available, to construct the present case reports. The clinical situation developed with discrete signs, but vulvar discharge was present in three cases, ranging from bloody to brownish or colourless, and from purulent to mucous. The females were in dioestrus, although the oestrus remained unperceived in most cases. In this study, the four clinical situations are described and discussed on the basis of available literature, highlighting the aspects that may impair an early diagnosis and that may favour the progression of the disease and also that age should not be an excluding criteria when analysing the differential diagnosis list.

Priapism associated with lumbar stenosis in a dog.

Priapism, a persistent long-lasting involuntary erection of the penis, is uncommon in dogs. In this report, the case of a 13-year-old male Pointer, referred to our services due to persistent exposition of the penis, is described. This condition was consecutive to an intermittent priapism situation lasting for several days, which has been initially attributed to the inflammation and haematoma associated with a perianal bite. The owners became unable to retract the penis into the prepuce. At presentation, the dog was anorectic for 48 h, intolerant to manipulation, and showed poor body condition and unsteady locomotion. During physical evaluation, a marked engorgement of the local vessels in the prepuce and penis was found. An abdominal X-ray was asked under the suspicion of a neurogenic origin for the clinical situation, which showed evidences of spondylosis. After discussion of the clinical condition, the owners asked for euthanasia. The necropsy confirmed the engorgement of the regional vessels deriving from the pudendal arteries and blood accumulation within all the cavernous spaces, accompanied by congestion and thrombosis within the erectile structures of the penis. No significant changes were observed in the pelvic organs that could be at the origin of priapism. The lumbar-sacral spinal regions were carefully inspected and evidenced signs of L7-S1 stenosis due to spondylosis. The case presented herein is a rare situation of priapism of neurogenic origin in a dog. Necropsy findings suggest that it was consecutive to cauda equina compression due to lumbar spinal stenosis.

Relationship between ultrasound measurements of body fat reserves and body condition score in female donkeys.

Several methods have been developed to monitor body fat reserves of farm animals and body condition scoring (BCS) is generally assumed to be the most practical. Objective methods, such as real time ultrasonography (RTU), are accepted methods for measuring fat reserves in several farm species but there is no published information about the use of RTU to monitor body fat reserves in donkeys. The aim of the present study was to determine the relationship between RTU measurements and BCS in female donkeys (jennies) (n=16) with a BCS of 3-7 on a 9 point scale. Ultrasound images were captured using an Aloka 500-V scanner equipped with a 7.5 MHz probe and subcutaneous fat (SF, range: 1.0-14.0mm) and thoracic wall tissue (TD, range: 5.6-21.4mm) depths measurements were determined. A significant correlation was found between BCS and all RTU measurements (0.65

Localization of tumor necrosis factor in the canine testis, epididymis and spermatozoa.

Tumor necrosis factor (TNF), formerly known as Tumor necrosis factor alpha is now regarded as a natural component of the mammalian seminal plasma (SP). Although not completely clarified, its functions in the SP have been associated with paradoxal roles, such as sperm survival in the female genital tract, while at high levels negatively affect sperm survival and fertility potential. Recently, it has been discovered that canine inseminated spermatozoa display a strong immunoreaction for TNF when lining the female endometrium. As a continuation of this finding, the present work aimed at documenting TNF localization in the canine testes and epididymis and in freshly ejaculated spermatozoa (SPZ) through immunohisto- or cytochemistry. Immunoreaction for TNF was found in all samples used. In the dog testis, TNF immunoexpression was limited to the seminiferous tubules, where late round spermatids (SPD) showed weak intensity of immunostaining, while elongating and elongated SPD evidenced moderate and the residual bodies a strong intensity. In the epididymis, a gradual progressive increase of TNF immunolabelling was found throughout the epididymal regions, ranging from a weak intensity at the caput epididymis to a moderate intensity at the cauda. TNF immunolabelling was found in mature SPZ during the epididymal transit and also in freshly ejaculated SPZ, which showed a strong midpiece immunolabelling. Data presented here provide important information on expression of TNF in spermatozoa, which is acquired by the SPZ during their formation at the testis. It further provides the basis for subsequent studies on the physiological importance of cytokines in sperm function.

Uterine prolapse with associated rupture in a Podengo bitch.

A case of uterine prolapse coexisting with uterine horn rupture in a 3-year-old Portuguese Podengo bitch, which is an uncommon occurrence, is described. The female was presented with a history of recent parturition, with delivery of four healthy puppies that were normally tended and nursed. The situation developed after an uneventfully pregnancy, and no direct causative factor was identified. The duration of the prolapse was unknown, but considered to be recent because of the swollen reddish appearance of the tubular everted mass. No foetus was found in the uterus or the abdominal cavity. The female was presented in good physical condition, without signs of shock or haemorrhage. During surgical treatment, the uterus was replaced to its normal position followed by ovary-hysterectomy at 12 h from admittance.

Hypospadias in a male (78,XY; SRY-positive) dog and sex reversal female (78,XX; SRY-negative) dogs: clinical, histological and genetic studies.

Hypospadias is rarely reported in dogs. In this study we pre-sent 2 novel cases of this disorder of sexual development and, in addition, a case of hereditary sex reversal in a female with an enlarged clitoris. The first case was a male Moscow watchdog with a normal karyotype (78,XY) and the presence of the SRY gene. In this dog, perineal hypospadias, bilateral inguinal cryptorchidism and testes were observed. The second case, representing the Cocker spaniel breed, had a small penis with a hypospadic orifice of the urethra, bilateral cryptorchidism, testis and a rudimentary gonad inside an ovarian bursa, a normal female karyotype (78,XX) and a lack of the SRY gene. This animal was classified as a compound sex reversal (78,XX, SRY-negative) with the hypospadias syndrome. The third case was a Cocker spaniel female with an enlarged clitoris and internally located ovotestes. Cytogenetic and molecular analyses revealed a normal female karyotype (78,XX) and a lack of the SRY gene, while histology of the gonads showed an ovotesticular structure. This case was classified as a typical hereditary sex reversal syndrome (78,XX, SRY-negative). Molecular studies were focused on coding sequences of the SRY gene (case 1) and 2 candidates for monogenic hypospadias, namely MAMLD1 (mastermind-like domain containing 1) and SRD5A2 (steroid-5-alpha-reductase, alpha polypeptide 2). Sequencing of the entire SRY gene, including 5'- and 3'-flanking regions, did not reveal any mutation. The entire coding sequence of MAMLD1 and SRD5A2 was analyzed in all the intersexes, as well as in 4 phenotypically normal control dogs (3 females and 1 male). In MAMLD1 2 SNPs, including 1 missense substitution in exon 1 (c.128A>G, Asp43Ser), were identified, whereas in SRD5A2 7 polymorphisms, including 1 missense SNP (c.358G>A, Ala120Thr), were found. None of the identified polymorphisms cosegregated with the intersexual phenotype, thus, we cannot confirm that hypospadias may be associated with polymorphism in the coding sequence of the studied genes.

Bilateral ovulation fossa inclusion cysts in Miranda jennets.

Multiple cysts ranging from 2-111 mm were noted bilaterally in the ovulation fossa of 11 mature Miranda jennets. These ovulation fossa inclusion cysts (OFICs) were lined by a simple low to columnar epithelium that included many ciliated cells. Although most cases were incidental findings, two of the jennets were presented with reduced fertility. Extensive cyst formation could have been responsible for the reproductive problems because they replaced most of the ovarian parenchyma. Due to their close proximity to the ovulation fossa, the OFICs may have mechanically interfered with passage of eggs into the oviduct. OFICs are histologically common in equids, but are reported uncommonly as gross lesions in either mares or jennets. Ovarian inclusion cysts are associated with neoplasia in women; however, these OFICs showed no evidence of epithelial hyperplasia or cellular atypia and no evidence of independent growth, therefore they were considered to be non-neoplastic. The bilateral occurrence and high incidence of OFICs in Miranda jennets, a breed with limited genetic variability, suggests that the lesion has a genetic causation.

Tumour necrosis factor in the canine endometrium: an immunohistochemical study.

Tumour necrosis factor (TNF), a pleiotropic cytokine that regulates cell growth and differentiation as well as the synthesis of other cytokines, has been identified in the uterus of several species describing a cyclic pattern, eventually under ovarian steroid regulation. Information is yet limited on the presence of TNF protein in the canine endometrium during the oestrous cycle and early pregnancy. This study depicts the temporal immunolocalization of TNF in the bitch endometrium along the oestrous cycle and changes associated with the early steps of embryo invasion. TNF immunolabelling was found in both the stromal fibroblasts and epithelial components of the canine endometrium in all stages studied. Stromal immunostaining was more intense than that of the epithelia, in all the stages of the oestrous cycle. In addition, a tendency for a decrease in the surface epithelium intensity score was found in early dioestrus. A positive glandular content was only observed in anoestrus and proestrus stages. In early pregnancy (days 13-16), TNF immunolabelling was detected at the embryo-maternal surface, in the syncytium cords and the trophoblast, as well in the endometrial stroma and the basal endometrial glands, but not in the lacunar epithelium. The overall TNF immunoreactivity was higher in early pregnancy samples in comparison with those of the early dioestrus and dioestrus stages, suggesting it plays a role during implantation.