Short-term clinical and hormonal effects of a deslorelin implant on late-prepubertal bitches - Based on flare-up signs.

Gonadotropin-releasing hormone (GnRH) agonists offer an alternative to surgical sterilization in prepubertal dogs, preserving ovarian and uterine functions. However, the clinical and hormonal effects of GnRH agonist application during the late-prepubertal stage remain insufficiently understood. This study aimed to investigate the clinical effect (flare-up) and hormonal changes, specifically serum progesterone (P4) and estradiol (E2) levels, in bitches treated with 4.7 mg deslorelin acetate (DA) implants (Suprelorin®, Virbac, F) during the late prepubertal period. Sixteen clinically healthy kangal cross-breed bitches, aged 7-8 months, with a mean body weight of 20.5 ± 0.8 kg, were implanted with DA. Estrus signs were monitored daily, and blood and vaginal cytological samples were collected every other day for four weeks. Cytological changes were analyzed for overall and superficial cell index. Six out of sixteen DA-treated bitches (EST group; n = 6) exhibited clinical proestrus 8.6 ± 0.6 days after implant insertion. The mean serum concentrations of P4 and E2 at the onset of estrus were 1.38 ± 0.32 ng/ml and 37.38 ± 10.07 pg/ml, respectively. Notably, all non-estrus (N-EST group; n = 10) bitches demonstrated an increase in superficial cell index, in addition to expected cytological changes observed in the EST group. On the 18th day post-implantation, the EST group exhibited a significantly higher number of superficial cells compared to the N-EST group (p < 0.001). DA implantation resulted in cytological profile alterations accompanied by a slight increase in estrogen concentrations in all dogs. However, the flare-up response exhibited significant variability, differing from that observed in adult dogs. This study highlights the importance of meticulous timing and breed-specific considerations when utilizing DA for puberty manipulation in late-prepubertal bitches. The observed cytological and hormonal changes in response to DA implants provide valuable insights, but the variability in flare-up responses warrants further investigation.

Inhibition of the cholesterol transporter ABCA1 by probucol decreases capacitation and tyrosine phosphorylation of dog spermatozoa, and is dose dependent.

The ATP binding cassette (ABC) transporter molecule ABCA1 participates in the cholesterol transport within and through cell membranes. We recently demonstrated that in dog spermatozoa, capacitation could be decreased with probucol (PRO), an ABCA1 specific antagonist. In this study, a dose-effect relationship of PRO on dog sperm capacitation, tyrosine phosphorylation and cholesterol efflux from the sperm plasma membrane was investigated. A total of 16 ejaculates from dogs of different breeds, aged 2-4 years were used. Sperm motility and membrane integrity in the main fraction was determined by CASA. Samples were stained with a boron dipyrromethene difluoride (BODIPY) fluorophore (P9672, Sigma- Aldrich, A) diluted in DMSO at a final concentration of 0.4 µM. All samples were divided into 5 aliquots, with 0, 100, 250, 500 and 1000 µM of PRO. After incubation at 37 °C for 2 h, PI was added and flow cytometry performed. All aliquots were examined for capacitation and acrosome reaction by using the CTC assay and tyrosine phosphorylation (TP). Membrane integrity was measured in all aliquots to investigate the effect of PRO on cell membranes. Membrane integrity did not differ between controls (0 µM), and 100, 250 and 500 µM PRO, but decreased with 1000 µM PRO (p < 0.05). Increasing PRO concentration decreased the percentage alive cells with cholesterol efflux per PRO group (0 µM: 77.8 ± 10.6%, 100 µM: 63.7 ± 11.7%, 250 µM: 52.1 ± 12.9%, 500 µM: 37.7 ± 11.6%, 1000 µM: 33.1 ± 14.4%; p < 0.05), decreased head and entire tail phosphorylated cells (0 µM: 34.6%, 1000 µM: 5.1% p < 0.05); and decreased the percentage capacitated cells (maximum with PRO 500 µM: capacitated vs. control: 54.2 ± 17% vs 25 ± 7.7%, p < 0.05). Conclusion: PRO decreased the cholesterol efflux, and decreased tyrosine phosphorylation and capacitation in a dose-dependent manner. This suggests a strong involvement of the ABCA1 transporter in different functional aspects of sperm capacitation in dogs.

Canine post-thaw sperm quality can be predicted by using CASA, and classification and regression tree (CART)-analysis.

In individual dogs, despite good quality of raw sperm, some parameters are significantly changed after thawing, which cannot be predicted. We therefore investigated whether motility parameters objectively obtained by CASA, membrane integrity (MI), cell morphology or a combination are suitable to improve the prediction of bad post-thaw quality. For this purpose 250 sperm analysis protocols from 141 healthy stud dogs, all patients introduced for sperm cryopreservation, were evaluated and a Classification and Regression Tree (CART) -analysis performed. The sperm was routinely collected, analysed, and frozen by using a modified Uppsala system. After thawing, data were routinely examined by using CASA, fluorescent microscopy for membrane integrity (MI) and Hancock's fixation for evaluation of cell morphology. Samples were sorted by post-thaw progressive motility (P) in good (P > / = 50%, n=135) and bad freezers (P⟨50%, n=115). Among bad freezers, 73.9% showed in addition post-thaw total morphological abberations of >40% and/or MI ⟨50%. Bad freezers were significantly older than good freezers (p⟨0.05). Progressive motility (P), velocity curvilinear (VCL), mean coefficient (STR), and linear coefficient (LIN) were potential predictors for post-thaw sperm quality since specifity was best (85.8%) and sensitivity (75.4 %) and accuracy (80.4 %) good. For these objectively measured raw sperm parameters, cut-off values were calculated allowing prediction of bad post-thaw results with high accuracy: P = 83.1 % VCL = 161.3 µm/sec, STR = 0.83 %, and LIN = 0.48 %. Raw sperm samples with values below these cut off values will have below average post-thaw quality with a probability of 85.8%. We conclude that VCL, P, STR and LIN are potential predictors of the outcome of sperm cryopreservation, when combined.

A guide to the clinical management of attrition.

Attrition is an enigmatic condition often found in older individuals and often as a result of bruxism which can take place as a result of either day bruxism, night bruxism or both. Various studies and systemic reviews clearly shown that tooth wear is an age-related phenomena and the last Adult Dental Health Survey showed that 15% of participants showed moderate wear and 3% severe wear with 80% of patients over 50 years of age showing signs of wear. This review examines current theories around the aetiological factors contributing to attrition together with the clinical management of attrition focusing on minimal intervention where possible.

Expression of GnRH receptor in the canine corpus luteum, and luteal function following deslorelin acetate-induced puberty delay.

The goals of this study were as follows: (Experiment 1) to examine the basic capability of canine corpora lutea (CL) to respond to GnRH by assessing expression of gonadotropin-releasing hormone receptor (GnRH-R) in luteal samples collected throughout the luteal lifespan from non-pregnant dogs, and (Experiment 2) to investigate the effects of pre-pubertal application of the GnRH agonist deslorelin acetate on luteal function following the first oestrus. Mature CL were collected during the mid-luteal phase (days 30-45) from treated and control bitches. Transcript levels of several factors were determined: estrogen receptors (ESR1/ERα, ESR2/ERß), progesterone (P4)-receptor (PGR), prolactin receptor (PRLR), PGE2-synthase (PTGES) and PGE2 receptors (PTGER2/EP2, PTGER4/EP4), vascular endothelial growth factor (VEGFA) and VEGF receptors (VEGFR1 and VEGFR2), cyclooxygenase 2 (COX2/PTGS2), steroidogenic acute regulatory protein (STAR) and 3ß-hydroxysteroid dehydrogenase (3ßHSD). Additionally, levels of Kisspeptin 1 (Kiss1) and its receptor (KISS1-R) were evaluated. Although generally low, GnRH-R expression was time dependent and was elevated during early dioestrus, with a significant decrease towards luteal regression. In deslorelin-treated and control dogs, its expression was either low or frequently below the detection limit. EP2 and VEGFR1 were higher in the treated group, which could be caused by a feedback mechanism after long-term suppression of reproductive activity. Despite large individual variations, 3ßHSD was higher in the deslorelin-treated group. This, along with unchanged STAR expression, was apparently not mirrored in increased luteal functionality, because similar P4 levels were detected in both groups. Finally, the deslorelin-mediated long-term delay of puberty does not have negative carry-over effects on subsequent ovarian functionality in bitches.

Morphological, histological and molecular investigations on canine uterine tissue after ovariectomy.

In the present study, we investigated the course of atrophy in canine uterine tissue and the expression of estrogen receptors (ER) and progesterone receptors (PR) within 6 months after ovariectomy (OE). In nine primipar bitches of different breeds, bilateral OE and removal of one horn was performed. Six months after surgery, the remaining uterine tissue was removed. The tissue was examined for signs of inflammation and proliferation, and for expression of ER, PR and Ki67 by means of immunohistochemistry (IHC); furthermore transcription of vascular endothelial growth factor (VEGF), transforming growth factor-ß (TGF-ß), epithelial growth factor (EGF), platelet activating factor (PAF), tumor necrosis factor α (TNFα), and their specific receptors was determined by means of RT-qPCR. Serum concentrations of estrogen and progesterone were measured immediately before the first and second operation. Six month after OE, no inflammation was seen in any uterine tissue, the thickness of the stump was decreased in most bitches. Protein expression of Ki67 revealed high individual differences after the second operation. Concentration of both hormones was not significantly changed, the estrogen concentration always revealed high individual differences. The expression of ER was significantly decreased in stromal and smooth muscle cells of the uterine tissue (p < 0.01), and the expression of PR in stromal cells only (p < 0.05). The gene expression of growth factors did not change significantly between first and second operation. We conclude that complete atrophy did not occur within 6 months after OE, instead, a high percentage of uterine cells still expressed ER and PR, rendering the stump susceptible to hormone treatments.

Kisspeptin-10 and the G protein-coupled receptor 54 are differentially expressed in the canine pregnant uterus and trophoblast cells.

Uterine tissue was collected from bitches after ovariohysterectomy at different times after ovulation. Samples were assigned to four groups: metestrous non-pregnant, day 10-12, n = 4; pre-implantation, day 10-12, n = 9; post-implantation, day 18-25, n = 13; mid-gestation, day 30-40, n = 7. RT-qPCR detection was performed for kiss1 and the G protein-coupled receptor 54 (GPR54, specific receptor for kisspeptin). In addition, immunohistochemistry was performed for detection of kisspeptin-10 (KP-10), GPR54, as well as pan-cytokeratin and vimentin. The latter two were included to differentiate the different placental cell types. The percentage of positive stained cells was evaluated, and an immunoreactivity score (IRS) was obtained by multiplying the labelling intensity score (0-3) with the percentage of immunolabelled cells (range: 0-300). In non-pregnant and pre-implantation tissues, gene expression was highly variable for kiss1 and GPR54. Expression of GPR54 was higher before embryo adhesion than during post-implantation and mid-gestation (p < .05), whereas there was no difference found between groups for kiss1. Except during the pre-implantation period, KP-10 expression was higher in the non-pregnant uterus compared to all gestational periods investigated, indicating a pregnancy-related downregulation. In the pre-implantation period, KP-10 was present in larger vessels only, whereas the presence of GPR54 in vessels was found in all samples, with most labelling in the post-implantation period. KP-10 was present in superficial uterine glands, GPR54 in superficial and deep uterine glands of the post-implantation uterus. In myocytes, the highest staining for KP-10 was seen in the non-pregnant uterus, whereas the highest staining for GPR54 was seen in post-implantation and mid-gestation. Syncytiotrophoblast cells stained for both KP-10 and GPR54 in post-implantation and mid-gestation, with maximum intensity for GPR54 in the latter. We conclude that KP-10 and GPR54 are expressed in the canine uterus and trophoblast cells. However, during pregnancy, expression of both proteins seems to be differentially regulated.

Seminal Plasma Characteristics and Expression of ATP-binding Cassette Transporter A1 (ABCA1) in Canine Spermatozoa from Ejaculates with Good and Bad Freezability.

The composition of seminal plasma and the localization of the ATP-binding cassette transporter A1 (ABCA1) in spermatozoa from good and bad freezers were compared to frozen-thawed spermatozoa from the same dog. Ejaculates were obtained from 31 stud dogs, and the sperm-rich fraction (SRF) was kept for analysis. One aliquot was used for the analysis of concentration, progressive motility (P; CASA), viability (V; CASA) and leucocyte count, and the analysis was performed by flow cytometry (FITC-PNA/PI), SCSA and HOST. In seminal plasma, concentration of albumin, cholesterol, calcium, inorganic phosphate, sodium, potassium, zinc and copper was measured. Semen smears were prepared and evaluated for the expression of ABCA1. The remainder of each ejaculate was frozen. After thawing, the quality assessment was repeated and further smears were prepared. According to post-thaw semen quality, dogs were assigned to good freezers (n = 20) or bad freezers (n = 11), the latter were defined as < 50% progressive motility and/or > 40% morphologically abnormal sperm and/or < 50% viability. Bad freezers were older than good freezers (5.3 vs 3.4 years, p < 0.05). In bad freezers, the percentage of sperm with ABCA1 signal in the acrosome was lower (26.3% vs 35.7%, p < 0.01) and the percentage of sperm with complete loss of ABCA1 signal higher (46.7% vs 30%, p < 0.01); the percentage of dead spermatozoa was higher (36.1% vs 25.5%, p < 0.05), and the concentration of cholesterol and sodium in seminal plasma was lower than in good freezers (p < 0.05). We conclude that in thawed bad freezer sperm, an increase in acrosome damages coincided with an increased loss of cholesterol transporters and cell death, and a lower cholesterol concentration in seminal plasma. Follow-up studies revealed whether a relation exists between these findings.

The cholesterol transporter ABCA1 is expressed in stallion spermatozoa and reproductive tract tissues.

In the present study, we assessed the presence of the ATP-binding-cassette (ABC) transporter molecules ABCA1 in spermatozoa of adult stallions and in testicular and epididymal tissue of prepubertal and adult stallions. For this purpose, semen samples from six fertile Shetland pony stallions aged 4 to 19 years were collected. Semen was collected from each stallion on three consecutive days. Ejaculates were analyzed immediately after collection, and only ejaculates meeting minimal requirements for fertile stallions were further evaluated. ABCA1 immunosignal was localized after staining of semen smears with different antibodies and counterstaining with Fluorescein isothiocyanate (FITC)-peanut agglutinin (PNA) and 4',6-Diamidin-2-phenylindol (DAPI). In a total of three samples, capacitation and acrosome reaction were induced by means of capacitation medium and progesterone substitution, respectively. Testicular and epididymal tissues were obtained from five prepubertal stallions aged 8 to 12 months and five adult stallions aged 4 to 9 years. For quantitative RT-PCR (qPCR), testicular and epididymal tissue of another seven adult (aged 1.5-14.5 years) and five prepupertal stallions (6-8 months) was used. For immunohistochemistry, sections from the caput, corpus, and cauda of the testes and epididymes were stained with the same specific antibodies as for immunocytochemistry. In stallion spermatozoa, strong immunosignal for ABCA1 was detected in the acrosomal area, the equatorial zone, and the principle piece of the flagellum but not in the caudal part of the head and the midpiece. In damaged or acrosome-reacted spermatozoa the FITC-PNA signal vanished together with the ABCA1 signal in most spermatozoa. In testicular tissue, strong immunostaining for ABCA1 was mainly visible in the heads and flagella of round spermatids and weaker signals in late spermatids and released spermatozoa. No staining was assessed in the Sertoli cells and spermatogonia of adult stallions, whereas strong signals in Leydig cells were present in prepubertal stallions. In prepubertal stallions, the ABCA1 messenger RNA level in testicular tissue was significantly higher than in adult stallions. We conclude that the ABCA1 transport molecule is present in adult and prepubertal stallion spermatozoa as well as testicular and epididymal tissue. ABCA1 is supposed to contribute to cholesterol transport and to support capacitation; however, this remains to be proven by functional studies. Species-specific differences concerning the localization inside the spermatozoa membrane are alike.

GnRH and its receptor (GnRH-R) are expressed in the canine placenta and uterus.

In reproductive tissues, GnRH participates in the regulation of cell growth and proliferation by direct binding to the GnRH-R, which is essential for embryo implantation. However, there is no study on the expression and cellular localization of GnRH and GnRH-R in the canine uterus and placenta. Therefore, bitches were ovariohysterectomized 10 to 12 days after mating (vaginal cytology and progesterone measurement), the uteri were flushed, and if embryos were detectable, bitches were allocated to the embryo positive group (E-pos.; preimplantation, n = 5). Other bitches were operated at later stages and, dependent on the gestational age, either allotted to the post-implantation group (Day 18-25 after mating, n = 9), or the mid-gestation group (Day 30-40 after mating, n = 3). Dogs negative in embryo flushing served as controls (E-neg.; controls, n = 5). Samples of the entire uterine wall were taken from the middle of the horn in E-neg. and E-pos. groups, and from placental and interplacental uterine sites in post-implantation and mid-gestation groups. GnRH-R expression was localized at the mRNA and protein levels by immunohistochemistry and in situ hybridization. The expression of GnRH and GnRH-R mRNA was assessed by semiquantitative polymerase chain reaction. Additionally, both GnRH and GnRH-R mRNA were expressed in all tissues examined until mid-gestation. Relative expression of GnRH was higher than that of GnRH-R (P < 0.05). During the post-implantation stage, GnRH-R expression was significantly higher in uteroplacental than in interplacental tissues. In the uterus, GnRH-R stained strongly in the surface and glandular epithelial cells, and seemed to be weaker in myometrium and stroma. Placental signals were predominantly localized in fetal trophoblast cells and to a lesser extent in maternal decidual cells. These findings suggest a local regulatory function of GnRH during early canine pregnancy.

Clinical use of deslorelin implants for the long-term contraception in prepubertal bitches: effects on epiphyseal closure, body development, and time to puberty.

Long-acting GnRH agonists have been used both for canine estrus induction and prevention. The objective of the study was to investigate the use of a deslorelin implant as a long-term and reversible contraceptive in prepubertal bitches with special regard to the time of epiphyseal closure. Thirteen healthy, crossbreed, medium-sized prepubertal female dogs were used in this study. An implant containing 9.4 mg (G1, n = 5) and 4.7 mg (G2, n = 4) deslorelin acetate (Suprelorin) or a placebo (sodium chloride 0.9%; G3, n = 4) was inserted subcutaneously in the interscapular region. Estrus was monitored once daily by physical and sexual behavioral changes. Body development, vaginal cytology, and serum progesterone and estradiol 17ß concentration were monitored weekly for the first 5 weeks, and then every 3 weeks throughout the treatment period. Radiographic examinations were performed monthly to determine the epiphyseal closure. Half of the deslorelin-treated bitches (G1: n = 2 and G2: n = 2) came into estrus during the 83-week observation period. All animals in the control group showed estrus between the 39th and 64th weeks of observation. Time to puberty averaged 82.7 ± 8.9 and 61.9 ± 9.7 weeks in the deslorelin-treated (G1 and G2) and the control bitches, respectively (P < 0.02). Both deslorelin implants (9.4 and 4.7 mg) can be used efficiently for the long-term prevention of estrus in prepubertal bitches; however, epiphyseal closure is clearly delayed which was without any clinical effect in the present study.

Expression of steroid hormone receptors in the genital structures of a true hermaphrodite pug dog.

Hermaphroditism is a rare and a not well-understood disordered sexual development (DSD) in dogs. The objective of the study was to analyse the sex steroid hormone receptor (STHR) expression patterns in the internal genital structures, because the responsiveness of the different tissue types to the steroid hormones may have a key role in pathological alterations based on DSDs. Furthermore, the adhesion molecule ß-catenin was investigated by means of immunohistochemistry because of its important role in development, tissue integrity and disease. Molecular sexing was performed via PCR targeting DBX/DBY genes to identify the pug dog as a true XX hermaphrodite. The portions of uterine tissue revealed comparable expression patterns for STHRs as investigated in normal female reproductive tissue. In the male parts, ß-catenin showed strong expression in the Sertoli cells of the seminiferous tubules; this was in contrast to normal testicular tissue. Likewise, the layers of smooth muscle actin-positive cells surrounding the seminiferous tubules were reduced in the hermaphrodite. The results of this study deepen the knowledge of tissue characteristics in a hermaphrodite dog and highlight the importance of early diagnosis because the STH responsiveness in maldeveloped reproductive tissue might lead to serious problems for the dog.

Suppression of fertility in pre-pubertal dogs and cats.

Pre-pubertal gonadectomy in dogs and cats is still controversially discussed because some consequences cause health problems. Nevertheless, postponement of puberty, that is, prevention of an increase in sexual hormones and thereby prevention of their manifold effects, is of major importance, not only in controlling overpopulation but also to preserve the genetic base for future breeding stock and pets. Therefore, alternatives for surgical suppression of fertility in pre-pubertal animals were critically reviewed. As a promising alternative, the slow-release GnRH agonist deslorelin and other GnRH analogues have been investigated. In female dogs and cats, puberty could be significantly postponed without initial flare-up effect and without disturbance of body development. First trials to delay puberty in female and male cats by application of a 4.7-mg deslorelin implant 24 h after birth so far are promising. In female dogs, a previous investigation showed that when the implant was inserted at the age of 4 months, the initial flare-up effect was prevented. Body development was normal in the studies reviewed here, and with the 9.4-mg implant, puberty was significantly delayed until the age of 21 months or older. In one study, bitches either received a 4.7- or a 9.4-mg implant at the age of 4 months and the epiphyses were mostly closed before the time of first oestrus. Using a 4.7-mg deslorelin implant in pre-pubertal male dogs significantly postponed puberty, and age at puberty was >2 years when a 9.4-mg implant was used. However, further investigations are required, especially concerning the effect of different GnRH agonist dosages and resorption rates on the duration of postponement of puberty as well as long-term effects in both dogs and cats.

The effects of aglepristone alone and in combination with cloprostenol on hormonal values during termination of mid-term pregnancy in bitches.

This study was designed to assess endocrine changes associated with termination of mid-term pregnancy after use of two different protocols. For this purpose we compared the effects of aglepristone (AGL) alone and in combination with cloprostenol (CLO) on serum concentrations of progesterone (P4), estradiol (E2) and relaxin (RLN) measured at short-term intervals during the abortion period in bitches. Fourteen pregnant bitches between day 25 and 32 of gestation were used in the study. In the AGL group (n=7), aglepristone was administered solely (10mg/kg body weight (BW), subcutaneously, once daily on two consecutive days) whereas in the AGL-CLO group (n=7), aglepristone (dosage as in AGL group) and cloprostenol (1µg/kg BW, subcutaneously, same with aglepristone) were combined. All pregnancies were successfully terminated 5.2±1.6 days after initiation of treatments, which was significant in both groups (P>0.05). At the time of the start of abortion (SA) and the end of abortion (EA), the mean P4 concentrations were 26.6±7.3 and 12.0±6.4ng/ml in AGL group, and 2.7±0.7 and 0.9±0.1ng/ml, in AGL-CLO group, respectively (P<0.01). Serum E2 concentrations were significantly higher (P<0.05) in AGL group at 42, 48, 54h and SA after initiation of treatment. In the AGL-CLO group, serum RLN concentrations did not significantly change from the initiation of treatment to EA (P>0.05). However, markedly higher RLN concentrations (P<0.05) were observed in the AGL group at 48h (1.5±0.7ng/ml) and at SA (1.6±0.5ng/ml). The results of the present study indicate that changes in the hormonal concentrations affect the mechanism of abortion in different ways. Further in depth studies investigating changes in the expression of hormone receptors inside the ovary, endometrium and placenta might be helpful to our understanding of the endocrinological differences observed in this study.

Immunolocalization of the cholesterol transporters ABCA1 and ABCG1 in canine reproductive tract tissues and spermatozoa.

The mammalian sperm membrane undergoes cholesterol efflux during maturation and fertilization. Although ATP-binding cassette (ABC) transporters are known to transport cholesterol through cell membranes in other organs, their presence in canine testis, epididymis and sperm has not been proven to date. Hence, the aim of the present study was to localize the ABC transporters ABCA1 and ABCG1 in canine testicular and epididymidal tissue as well as in spermatozoa membranes. To this end, semen samples from 12 dogs as well as testicles and epididymides of four young and healthy dogs were prepared for immunohistochemistry, respectively. Capacitation and acrosome reaction (AR) were induced in aliquots of the semen samples before immunostaining to assess changes in the expression of ABCA1 and ABCG1. Evaluation by confocal microscopy revealed the presence of both ABCA1 and ABCG1 in canine testicles and of ABCA1 in the epididymides. In spermatozoa, only ABCA1 immunoreactivity was detected, mainly in the region of the acrosome and midpiece. After induction of capacitation, ABCA1 signal persisted in the acrosome but disappeared after AR, indicating a loss of ABCA1 with the loss of the acrosome. We conclude that ABCA1 and ABCG1 are expressed in canine testis, whereas only ABCA1 is expressed in epididymis and spermatozoa membrane, both transporters probably contributing to the regulation of membrane cholesterol content.

Clinical evaluation of different applications of misoprostol and aglepristone for induction of abortion in bitches.

The aim of the present study was to compare the clinical and endocrinological effects of different applications of misoprostol (MIS) and aglepristone (AGL) for the induction of abortion in bitches. For this purpose, 28 healthy pregnant bitches from different breeds, ages, body weights (Body weigt, BWs, 10-40 kg), and between Days 25 to 35 of gestation were used. Bitches were randomly assigned to four groups. In group 1 (GI, n = 7), AGL (10 mg/kg BW, s.c. on 2 consecutive days); in group 2 (GII, n = 7), AGL (as in GI), intravaginal MIS (IVag, 200 µg for bitches with ≤20 kg BW, 400 µg for bitches with >20 kg BW, daily intravaginally until completion of abortion); in group 3 (GIII, n = 7), AGL (as in GI), ICVag (as in GII), per os MIS (400 µg for bitches with ≤20 kg BW, 800 µg for bitches with >20 kg BW, daily orally, until completion of abortion); in group 4 (GIV, n = 7), AGL (as in GI), per os MIS (as GIII) were used. Clinical, vaginal, and ultrasonographic examinations were performed daily until abortion was completed. For measurement of serum progesterone, blood samples were collected in all groups immediately after the first AGL administration and every other day until completion of abortion. No statistical differences were found between groups concerning the duration until completion of abortion after treatment (nonsignificant); however, in GII, one bitch completed abortion 2 days after the start of treatment.

Cooled storage of canine semen: in vitro effects of different concentrations of an antibiotic combination on growth of mollicutes.

The aim of this study was to examine effects of an antibiotic combination at different concentrations on growth of mycoplasma and ureaplasma during cooled storage of canine semen (n = 20). Semen aliquots were diluted with Tris-citric acid-fructose-egg yolk extender containing either 1.0 g/l streptomycin and 0.6 g/l benzylpenicillin (control) or a combination of gentamycin, tylosin, lincomycin and spectinomycin (GTLS-1: 0.25, 0.05, 0.15 and 0.3; GTLS-2: 0.5, 0.1, 0.3 and 0.6; GTLS-3: 1.0, 0.2, 0.6 and 1.2 g/l). Samples were assessed for motility and membrane integrity by computer-assisted sperm analysis immediately after dilution and at 24, 48 and 72 h of cooled storage. Morphologically, normal spermatozoa were determined, and bacterial culture was performed at 24 and 72 h. Mycoplasma spp. were detected in 14 of 20 ejaculates (70%) with severe growth in 12 samples. A reduction but not total elimination of mycoplasma growth occurred in all GTLS extenders with the most pronounced reduction in group GTLS-3 (control vs GTLS-1 and GTLS-2 p < 0.05, control vs GTLS-3 p < 0.001). Ureaplasmas were detected in four ejaculates, and growth was reduced to the same extent in GTLS and control extender. Progressive motility in all groups, total motility in groups GTLS 1-3 and percentage of membrane-intact spermatozoa in groups GTLS 2 and 3 decreased slightly (p < 0.05) over time. In conclusion, dilution of canine semen with GTLS extender has no major detrimental effects on spermatozoa during cooled storage. It reduced the growth but did not totally eliminate mycoplasmas and ureaplasmas from cooled-stored dog semen.

Measurement of alkaline phosphatase in canine seminal plasma--an update.

In dogs, diagnosis of incomplete ejaculation and azoospermia can be made by measuring the activity of the enzyme alkaline phosphatase (AP) in seminal plasma. However, even though upper cut-off value of 5000 IU/l is given in the literature, results by different assays may vary considerably. Furthermore, no data exist concerning the stability of the enzyme during storage of frozen seminal plasma, and no recommendations for pre-analytic dilutions can be found. During the present study, we compared results from a conventional large scale wet chemistry analyzer to a widely used dry chemistry point of care system (POC) and established a best practice for pre-analytical dilutions. Furthermore, stability of enzyme activities in seminal plasma during storage at -18 °C for 24 h was evaluated. The average activity of AP in the 2nd fraction of normal ejaculates measured by Reflotron® was 107,328 IU/l. After 24 h of frozen storage, activities did not differ significantly (96,844 IU / l, p > 0.05). Fresh and frozen samples were analysed in parallel by the POC and conventional chemistry analyser, and the results compared that did not reveal a significant difference (p > 0.05). A dilution of seminal plasma with physiologic saline 1:100 prior to analysis was sufficient for the qualitative information whether AP activity is below or above 5000 IU/l. Present data show that AP measurement by a POC dry chemistry system is sufficiently accurate in diluted seminal plasma for the diagnosis of azoospermia and that seminal plasma can be stored frozen for 24h before analysis.