Using egg yolk in a TRIS-Equex STM paste extender for freezing of dog semen is superior to egg yolk plasma, also after addition of lecithin and catalase.

We compared the results of using egg yolk plasma (EYP) instead of egg yolk (EY) in a TRIS-based Equex STM Paste freezing extender system for dog semen [25]. We also tested whether the addition of lecithin and catalase to the EYP extenders would improve results. Fractionated semen collection was done in 17 stud dogs and the sperm rich fraction diluted with different extenders in 2 steps: (I) TRIS-fructose-citric acid extender (TRIS) containing 20% egg yolk (EY) and 3% glycerol [25], (II) TRIS containing 20% egg yolk plasma (EYP) and 3% glycerol, and (III) TRIS containing 20% EYP and 0.8% lecithin (EYP-L) and 3% glycerol. After equilibration the second dilution step was done: samples with (I) were diluted with TRIS-EY with 7% glycerol and 1% Equex STM paste [25]; samples with (II) and (III) were divided in 2 aliquots each, and one part diluted with TRIS-EYP or TRIS-EYP-L, both containing 7% glycerol and 1% Equex STM paste, and the other one part with the same extenders containing additionally 300 I.U./mL catalase. After freezing and thawing, samples were analyzed by CASA and sperm chromatin structure assay (SCSA); reactive oxygen species (ROS), degree of apoptosis and zona binding ability were determined. Semen samples with TRIS-EY with a final concentration of 5% glycerol and 0.5% Equex STM paste [25] showed best post thaw progressive motility (P), most intact cells, lowest percentage of ROS, acrosome damages, dead and apoptotic cells. Curvilinear velocity (VCL), DNA fragmentation, morphological abnormalities and zona binding ability did not differ between groups. Replacement of egg yolk by EYP increased the ROS and late apoptotic cells. Addition of lecithin and catalase to EYP containing extenders decreased motility and increased complete apoptosis. We conclude that egg yolk is superior to EYP in the here investigated extenders. The TRIS-based extender [25] with EYP could not be improved by addition of lecithin and catalase; however, in-vivo fertilization capacity of the here examined extenders remains to be investigated.

Combined cryopreservation of canine ejaculates collected at a one-hour interval increases semen doses for artificial insemination without negative effects on post-thaw sperm characteristics.

A limiting factor in canine artificial insemination (AI) is the low number of insemination doses obtained per ejaculate. In this study, semen was collected from dogs (n = 28) either once and frozen directly after collection or the same dogs were submitted to a dual semen collection with a 1-hr interval and the two ejaculates were combined for cryopreservation. We hypothesized that combining two ejaculates increases semen doses per cryopreservation process without negative effects on semen characteristics. Total sperm count was lower in semen from a single semen collection in comparison with the combination of the first and second ejaculate of a dual semen collection (p < .001). The percentage of motile and membrane-intact spermatozoa determined by computer-assisted sperm analysis (CASA) in raw semen did not differ between single and combined dual ejaculates and was reduced (p < .001) by cryopreservation to the same extent in single (motility 73.7 ± 1.8%, membrane integrity 65.6 ± 2.2%) and combined dual ejaculates (motility 72.7 ± 2.3%, membrane integrity 64.6 ± 2.5%). The percentage of spermatozoa with morphological defects increased after cryopreservation (p < .001) but was similar in single and combined dual ejaculates. The CASA sperm velocity parameters decreased with cryopreservation (p < .001) but did not differ between single and combined dual ejaculates. The number of insemination doses increased from 2.7 ± 0.4 for single to 4.7 ± 0.8 for combined dual ejaculates (p < .01), based on 100 million motile spermatozoa per frozen-thawed semen dose. In conclusion, combining two ejaculates collected at short interval for one cryopreservation process increases the number of AI doses without compromising semen quality.

First detection of heat shock protein 60 and 70 in the serum of early pregnant bitches.

Heat shock proteins (HSPs) belong to a group of cellular stress proteins. Heat shock protein 10 immunoregulates and promotes growth during early gestation in humans, while HSP70 is considered to regulate autophagy and apoptosis during pregnancy and parturition. Both HSPs are detectable in the serum and placentas of early pregnant women and considered to contribute to the establishment of pregnancy. Within this pilot study we aimed (1) to assess whether HSPs 10, 60 and 70 are measurable in the serum of healthy early pregnant and non-pregnant bitches, and (2) to explore whether measurable differences between groups indicate pregnancy. Blood was collected from 31 bitches on days 7, 14 and 21 after mating. At 21 days post mating, all bitches were examined for pregnancy by ultrasonography; 23 were pregnant, and the eight non-pregnant bitches served as controls. Pregnant bitches had normal parturitions and gave birth to healthy puppies. The serum concentrations of HSPs 10, 60 and 70 were measured by electrophoresis and western blot. Serum HSP10 was not detectable. Average serum HSP70 concentration was significantly (d7, P = 0.030; d14, P = 0.023; d21, P = 0.030) lower in pregnant animals at all days investigated, while serum HSP60 was significantly lower at day 21 of gestation (P = 0.024) when compared to the controls. HSP 60 and HSP70 concentrations correlated positively (d7, r = +0.386, P = 0.021; d14, r = 0.450, P = 0.008; d21, r = +0.472, P = 0.006). We conclude that in pregnant bitches, serum concentrations of HSP60 and HSP70 are significantly decreased between days 7 and 21 of gestation, in comparison to non-pregnant bitches in early dioestrus, raising the question about intrauterine functions during the peri-implantation period.

Pre-pubertal treatment with a GnRH agonist in bitches-Effect on the uterus and hormone receptor expression.

Aim of the study was to examine the effect of deslorelin on uterine tissues of eleven pre-pubertal bitches aged 4.2 ± 0.6 m. Implants containing placebo (sodium chloride 0.9%; n = 4, G I), 4.7 mg (n = 3, GII) or 9.4 mg (n = 4, GIII) deslorelin acetate (Suprelorin® ; Virbac, France), were administered subcutaneously. Signs of oestrus, vaginal cytology, serum progesterone (P4) and estradiol-17ß (E2) concentrations were monitored until the occurrence of oestrus. Bitches were ovariohysterectomized and sections from the uterine tissue were subjected to immunohistochemistry (IHC) for detection of GnRH receptor (R), Kisspeptin (KP)10, Kisspeptin receptor (GPR54), androgen receptor (AR), oestrogen receptor (ER) α,ß, and progesterone receptor (PR). Tissue sections were scored semi-quantitatively using an immunoreactivity score (IRS) ranging from 0 to 300 (3). Since some animals were ovariohysterectomized before puberty (n = 1 from GII and n = 2 from GIII), and some in metestrus (all controls and 2 from GII and GIII each), results from these animals were separately evaluated and compared to the controls. Results: No abnormalities were seen in uterine tissues. Kisspeptin 10 expression was low in all cell types, highest IRS were seen in the vascular endothelial cells. The GPR54 was mainly detected in the luminal epithelial cells, superficial and deep uterine glands. The expression of GPR54 and ERα,ß was especially high in bitches operated prepubertally. No difference was observed between the controls and experimental bitches operated in their first metestrus. The PR and ERα,ß were exclusively expressed in superficial and deep uterine glands and luminal surface epithelial cells. The AR and GnRH-R expression was negative in all cells of all groups. We conclude that application of 4.7 or 9.4 mg deslorelin at the age of 4 months did not cause uterine disturbances. GPR54 expression might be influenced by pre-pubertal deslorelin treatment or the changings related to approaching puberty; the latter is supposed in case of ERα,ß.

Primary anestrus due to dietary hyperthyroidism in a miniature pinscher bitch.

A 2-year-old intact miniature pinscher bitch that had been on a bones and raw foods diet since birth showed no signs of estrus despite a 40-day course of cabergoline. Elevated levels of thyroxine were detected in the serum (51 nmol/L) and in the juice of the meat (183 nmol/L) fed to the dog. Change in diet and treatment with oral cabergoline resulted in signs of proestrus in 13 d, pregnancy, and normal birth of 5 puppies.

Anœstrus primaire attribuable à un hypothyroïdisme alimentaire chez une chienne Pinscher miniature. Une chienne Pinscher miniature intacte âgée de 2 ans qui avait consommé une diète composée d'os et d'aliments crus depuis la naissance ne manifestait aucun signe d'œstrus malgré une série de traitements de 40 jours à la cabergoline. Des niveaux élevés de thyroxine ont été détectés dans le sérum (51 nmol/L) et le jus de la viande (183 nmol/L) consommée par la chienne. Une modification de la diète et un traitement à la cabergoline orale ont produit des signes de pro-œstrus après 13 jours, puis la gestation et la naissance normale de 5 chiots.(Traduit par Isabelle Vallières).

Does Better Post-Thaw Motility of Dog Sperm Frozen with CLC Mean Better Zona Pellucida Binding Ability?

Even though the search for methods improving cryopreservation of canine spermatozoa led to an improvement of post-thaw quality, fertilizing results after insemination with frozen-thawed semen are still not satisfying. In this study, we focused on modification of spermatozoa membrane fluidity and investigated whether kinematic parameters as assessed by computer-assisted semen analyzer (CASA) can be improved. The primary aim of our study was to investigate whether the use of cholesterol-loaded cyclodextrins (CLC; 0.5 mg, 1 mg, 2 mg) and 2-Hydroxypropyl-ß-cyclodextrin (HBCD; 1 mg) positively influence capacitation status as examined by tyrosinphosphorylation, cholesterol efflux and zona binding assay (ZBA) of spermatozoa. The use of 0.5 mg of CLC increased the percentage of motile, progressive and rapid spermatozoa compared to the control. Addition of HBCD decreased motility and progressive motility of spermatozoa and the population with rapid movement in comparison to the control. The percentage of live spermatozoa without efflux of cholesterol compared to the control was increased when extender with 0.5 mg of CLC was used. There was no change in capacitation status. The zona binding ability of spermatozoa was significantly lower in the group with 0.5 mg of CLC than in the control. In conclusion, these results suggest that improvement of kinematic parameters does not necessarily coincide with better zona pellucida binding ability of spermatozoa.

Monitoring of canine pregnancy by considering Anti-Mullerian hormone, C-reactive protein, progesterone and complete blood count in pregnant and non-pregnant dogs.

In this study, we measured the serum concentration of anti-Mullerian hormone (AMH), C-reactive protein (CRP), progesterone (P4), and the complete blood count (CBC) in pregnant and non-pregnant bitches. The aim was to investigate the suitability of these parameters for monitoring canine pregnancy. Blood samples were collected from all bitches introduced for timed mating on the day of first mating (>5 ng/ml). The first blood sample after mating was obtained on day 12 post-copulation. The dogs whose pregnancy was confirmed on days 25 and 35, were allotted to the pregnancy positive group (G+) and those that were not pregnant were grouped as pregnancy negative (G). Ultrasonography (US) was performed on days 25, 35, 45 and 55 in pregnant (N = 13) and non-pregnant (N = 7) animals; The sonographic examinations in non-pregnant bitches were continued up to day 63, and in pregnant bitches they were also carried out one day after parturition (D+1). Blood samples were taken in parallel with these periods. Furthermore, the pregnant bitches were classified as G1A (1-2 puppies), G1B (3-4 puppies), and G1C (5-11 puppies) based on the number of puppies, and G1X (10 kg), G1Y (10-20 kg), and G1Z (>20 kg) based on their body weight. No significant difference was found between G+ and G-with regard to AMH, except on day 45, when AMH was higher in G+ (P < 0.01). On the other hand, the CRP values in the G+ exceeded those in the G-group on day 25 (38.26 vs. 15.66 mg/L, P < 0.05), on day 35 (32.54 vs. 15.97 mg/L, P < 0.05) and on day one after parturition (36.24 vs. 10.10 mg/L, P < 0.01). When puppy number was considered, it was discovered that CRP values significantly increased with puppy number on days 12 and 45 (G1A vs. G1B day 12: 4.13 vs 15.84 mg/L, P < 0.05; day 45: 12.40 vs. 25.76 mg/L, P < 0.001), and on day 35 (G1B vs. G1C: 24.18 vs. 38.87 mg/L, P < 0.01). With regards to AMH, this was only detectable on day 12 (G1A vs. G1B: 0.56 vs. 1.13 ng/mL, P < 0.05). When the body weight of the pregnant bitches was considered, bitches <10 kg had significantly higher AMH values than bitches bitch >20 kg on days 12 and 25 (day 12: 1.20 vs. 0.21 ng/mL, P < 0.01; day 25: 0.91 vs. 0.21 ng/mL, P < 0.05). This was not found in the case of CRP. The white blood cells (WBC) and the granulocytes (GRAN) were found to be higher in the G+ group (P < 0.01) on day 55, while the hematocrit (HCT) was significantly lower on day 45 (P < 0.05) and day 55 (P < 0.01). The increased GRAN was still detectable one day after parturition (P < 0.05). In conclusion, measurement of the AMH and CRP concentrations may contribute to determination of gestation stage and monitoring of the course of pregnancy; values are related to maternal body weight and number of puppies; however, AMH did not change over the course of a normal pregnancy. Sonography, the increase in CRP and complete blood count values may be beneficial for monitoring canine pregnancy. More studies are necessary to prove these findings.

Diagnostic tests in canine andrology - What do they really tell us about fertility?

Dog breeders often require breeding soundness evaluations which include andrological examinations of the genital organs, hormone measurements, and semen analyses. During the past decades, a considerable number of research results have been published, allowing diagnoses of specific andrological conditions and fertility assessment. For specific examinations, however, no standard procedures have been defined and for some parameters different reference ranges have been published. Therefore, examination results from different facilities are difficult to compare and profound conclusions regarding health and fertility of a male dog are not always possible. Conventional semen examination, however, is still useful in identifying deviations or no deviations from normality, especially if confounding factors are taken into account and if the exam is repeated in case of inconclusive findings. A standardization of examination procedures and reference ranges would help to harmonize the exchange of examination results and interpretation of the findings.

Effects of age, size and season on cryotolerance of dog semen - A retrospective analysis.

In a retrospective analysis of 508 ejaculates from 297 dogs, efficiency of semen cryopreservation and effects of age, season and dog size on characteristics of fresh and cryopreserved semen were evaluated. Volume of the sperm-rich ejaculate fraction increased to 10 years of age, decreased in 10 and 11-year old dogs (P < 0.001) but did not differ among seasons. Total sperm count was less in 10 and 11-year-old dogs (P < 0.001). The percentage of progressively motile, membrane-intact and morphologically normal spermatozoa before cryopreservation was least in 10 and 11-year-old dogs (P < 0.001). Cryopreservation resulted in less progressively motile spermatozoa (P < 0.001) with this being most pronounced in 10 and 11-year-old dogs (cryopreservation x age P = 0.004). The cryopreservation-induced decrease in morphologically normal spermatozoa (P < 0.001) was not affected by dog body weight. Number of cryopreserved AI doses differed among age groups (P < 0.001) and was less in 10 and 11-year-old dogs (median 1.5) compared with younger dogs (6-7 years, median 4.3). When ejaculates were grouped by a threshold of ≥ 35% progressively motile spermatozoa after freezing-thawing, 86.5% of all ejaculates were greater the threshold but this percentage decreased to 66% in 10 and 11-year-old dogs (P < 0.001). In conclusion, sperm cryotolerance is consistent for much of a dog´s lifespan but decreases after a certain age. Dog semen can be cryopreserved successfully throughout the year. Post-thaw semen characteristics were not different among cryopreserved ejaculates obtained from dogs differing in body weight.

Canine Spermatozoa-Predictability of Cryotolerance.

Markers of freezability allow the selection of ejaculates of good freezability. So far, most investigations were conducted in boars, bulls, rams and horses, with high economic interests triggering the efforts. The progress in dogs is comparably slow. A critical evaluation of the methods requires consideration of practicability, with most labs not even possessing a computer assisted sperm analyser (CASA); furthermore, small canine ejaculates mostly do not allow the use of large semen volumes. In dogs, modern markers of freezability no longer assess single membrane constituents or seminal plasma components but comprise tests of cell functionality and adaptability, energy metabolism, cluster analyses of kinetic and morphometric parameters, as well as DNA intactness. Identification of the most efficient combination of tests seems useful. At present, examination by CASA combined with cluster analysis of kinetic subgroups, JC-1 staining and COMET assay or staining with toluidine blue seem most appropriate; however, cell volumetry and other functional tests deserve better attention. A better understanding of spermatozoa energy metabolism might reveal new markers. This review focuses on the requirements and markers of freezability of canine semen, highlighting potential future candidates.

Prepubertal Use of Long-Term GnRH Agonists in Dogs: Current Knowledge and Recommendations.

The search for an alternative approach of estrus control (induction or suppression) in dogs is an important issue and the use of slow GnRH agonist-releasing implants has been the subject of frequent research in recent years. Studies to date demonstrate that the short- and long-term effects of deslorelin implants applicated at different time points of the prepubertal period are similar to those of adult dogs; however, there are important differences. The age of the prepubertal bitch and the dosage appear to be the main determinants of the response to deslorelin, as well as the individual metabolism of the bitch. Recent studies reported that the deslorelin-mediated long-term delay of puberty does not have negative carry-over effects on subsequent ovarian functionality, serum steroid hormone concentrations, uterine health, and fertility; however, more molecular studies are needed to determine the effects of application time of GnRH agonists on hormone concentrations and peripheral receptor expression. Furthermore, the long-term effects of delay of puberty with deslorelin on joint health, tumor development, the immune system, and social behavior deserve further investigations.

Computerized histogram analysis of the canine placenta during normal pregnancy.

In this study, the aim was to evaluate digitalized images obtained via B-mode sonography (USG) during different stages of pregnancy and to assess features of the canine placenta, typical for the different gestational periods. For this purpose, ultrasound pictures were digitalized and a computerized histogram analysis performed. Finally, 151 USG images were obtained from 37 healthy, pregnant dogs of different breeds, aged 4.4 ± 1.8 years, between days 17 and 62 of gestation, and 755 Regions of Interest (ROI) within these images were evaluated. Five ROI per picture were situated in the ventral and dorsal uterus/placenta (early stages) or exclusively the placenta (later stages). The data were grouped into GI: gestational day 17-25, GII: gestational day 26-40, GIII: gestational day 41-50 and GIV: gestational day 51-62. The mean grey value (MGV), standard deviation of grayscale value (StdDGV), minimum grey value (MinGV), maximum grey value (MaxGV), Modal/Mode grey value (ModGV), median grey value (MedGV), homogeneity (Angular Second Moment, ASM), contrast (CONT), correlation (COR), regional homogeneity (Inverse Difference Moment, IDM) and the entropy (ENT) parameters in the Region of Interest (ROI) were calculated. In the course of pregnancy, the grey value parameters, CONT and the local homogeneity (IDM) changed characteristically. The MGV increased between days 17 and 40 (p < 0.0001) and decreased towards late gestation (GIV, p < 0.0001). In parallel, the ModGV, MedGV and MaxGV values changed significantly (p < 0.0001). Similarly, CONT value significantly increased between implantation (GI) and later gestational periods, together with the increasing grayscale variation, probably indicating the massive increase in vascularisation during mid-gestation. This is supported by the fact that local homogeneity (IDM) was highest during implantation and decreased towards late pregnancy (p < 0.01). There was no difference between groups in terms of ASM, COR and ENT (p > 0.05). We conclude that some of the parameters evaluated by computational analysis increased or decreased with the progression of pregnancy, and correlated with the gestational period of bitches. Further studies will reveal whether this technique can contribute to early diagnosis of placental alterations.

Freezability of Dog Semen after Collection in Field Conditions and Cooled Transport.

Dog semen freezing is gaining popularity, but it has to be performed in equipped facilities, which can be far from the place where the stud dog lives. The aim of this study was to evaluate whether freezing dog semen after 24 or 48 h of cooled transport to an equipped laboratory was possible when semen collection was performed in the field such as in local breeding kennels. Single ejaculates from different dogs (mixed breeds and ages) were collected. In Experiment I, 10 ejaculates were conventionally frozen using the Uppsala method or frozen after 24 or 48 h of storage in a Styrofoam transport box cooled by icepacks. In Experiment II, 10 ejaculates were used to assess the influence of two extenders (Uppsala chilling extender or freezing extender 1) used for semen dilution during the 24 or 48 h storage. Motility, morphology, membrane, and acrosome integrity were analyzed as well as spermatozoa zona-binding ability. No significant differences were observed among the frozen groups, regardless of freezing time (Experiment I) or extender (Experiment II). Motility at thawing, however, decreased in absolute value at 48 h. Freezing of freshly collected semen is the gold standard, but the results obtained in this study prompt the application of freezing after cooled transport for the long-term preservation of dog semen, especially if the transport can be organized in 24 h.

Cystic endometrial hyperplasia and uterine adenocarcinoma in two miniature pigs.

Due to the increasing popularity of minipigs as pets, the frequency of presentation to the veterinarian is increasing as well. In addition to routine measures such as claw trimming, vaccination and antiparasitic treatment, diagnostics, and therapy of diseased minipigs are also common activities in the veterinary practice. In recent years, we have noticed more and more pathological changes of the female genital tract of uncastrated minipigs. There are also some case reports available in the international literature in this regard. The following case report describes the diagnostic procedures and ovariohysterectomies of 2 13-year-old female minipigs in detail. In these cases, uterine adenocarcinoma, and uterine carcinoma, respectively, combined with cystic endometrial hyperplasia (CEH) of the endometrium were diagnosed.

Expression of Anti-Müllerian Hormone and Its Type 2 Receptor in the Ovary of Pregnant and Cyclic Domestic Cats.

To evaluate the expression of AMH and its receptor AMHRII, ovaries of 33 p cats were investigated by western blot and immunohistochemistry. After ovariohysterectomy, the cats were grouped according to pregnancy stages and ovarian/placental endocrine activity: group I (n = 3, 24−29 days), II (n = 8, 32−40 days), III (n = 4, 41−46 days), IV (n = 6, 53−61 days) and according to cycle stages: V (n = 6, interestrus) and VI (n = 6, estrus). Serum progesterone- and AMH-concentration was measured. Follicle numbers did not differ between groups. The number of corpora lutea was higher in pregnant cats than in the non-pregnant cats. Serum AMH concentration was at maximum between day 30 and 50 of gestation, and was higher than in non-pregnant cats, then decreased towards term (p < 0.05). In the ovaries, AMH immunopositivity was observed in granulosa cells of secondary and antral follicles, and in interstitial cells of corpora lutea; highest percentage of immunopositive areas was detected in group III (p < 0.05). A positive correlation between the number of corpora lutea and the positive AMH signals in ovarian tissue was determined (r2 = 0.832, p < 0.05); however, only during mid-gestation (group II). Expression of AMHRII was in close co-localization with AMH and strong in the interstitial cells surrounding follicles undergoing atresia. AMHRII expression did not differ between pregnant groups but was higher compared to estrus cats (p ˂ 0.05). We conclude that AMH and AMHRII expression in the feline ovary is comparable to other species. The high serum AMH concentration and ovarian AMHRII expression between day 30 and 50 of gestation are probably related to ovarian activity and follicular atresia.

Expression of the insulin-like growth factor (IGF) gene family in feline uterus during pregnancy.

Members of the IGF gene family participate in cell differentiation and proliferation during pregnancy. We used 35 cats assigned to experimental groups (G) based on pregnancy stages: G1, pre-implantation; G2, implantation; G3, early pregnancy; G4, mid-pregnancy; G5, nonpregnant. Quantitative polymerase chain reaction (qPCR) and immunohistochemistry were used to analyze the expression of the IGF gene family. During pregnancy, expression of IGF-1 gene was significantly greater at implantation sites in the G1 and G2 groups than at placentation sites in G3 and G4 groups. IGF-2 expression was greater in the G2, G3 and G4 groups than in G1. Expression of the IGF-1R gene was significantly greater at placental sites in G3 than in G1 and G4. IGF-2R genes were expressed in all groups. Insulin-like growth factor binding proteins (IGFBPs) were expressed at intensities that depended on the stage of pregnancy; they were detected in different cell types and at different sites in the uterus. We found that members of the IGF gene family were expressed differentially in the endometrium during pregnancy. Our findings suggest that the IGF family may be a regulatory factor for pregnancy in cats.

The ABCA1 blocking agent probucol decreases capacitation in ejaculated dog spermatozoa.

BACKGROUND: The ATP binding cassette (ABC) transporters participate in the cholesterol and phospholipid transport within and through cell membranes of many cells including spermatozoa. Cholesterol efflux is important for capacitation of spermatozoa. ABCA1 expression has been assessed in canine spermatozoa previously but its role in capacitation still has to be determined. The aim of the study was to test whether inhibition of ABCA1 (1) decreases capacitation in ejaculated and epididymal canine sperm samples and (2) decreases cholesterol efflux in the same samples. Twenty-one ejaculates and sperm from 22 epididymal tails were collected from healthy dogs. Motility was measured by CASA and viability assessed after staining with SYBR-14/PI. Samples from ejaculated sperm and sperm from epididymal tails were aliquoted. One part was incubated with the ABCA1 inhibitor probucol, the other served as a negative control. In all samples, capacitation was evaluated by chlortetracyclin (CTC) assay and cholesterol was measured by cholesterol efflux assay and colorimetric enzymatic assay. RESULTS: In ejaculated sperm, blockade of ABCA1 with 100 µM of probucol/mL of sample resulted in a significantly higher percentage of uncapacitated and acrosome reacted spermatozoa (P < 0.001 and P = 0.031), capacitation was significantly decreased (35% in probucol samples vs 54.2% in controls, P < 0.001). In probucol inhibited sperm samples from epididymal tails, the percentage of capacitated spermatozoa did not differ between groups but the percentage of acrosome reacted spermatozoa increased significantly (P = 0.014). The cholesterol measurement revealed significantly lower cholesterol concentration in the probucol group when compared to the controls (P = 0.035), however only in ejaculated sperm samples. CONCLUSIONS: CTC assay and cholesterol measurement revealed significant differences between groups; we conclude that inhibition of ABCA1 significantly decreased capacitation and cholesterol efflux in ejaculated canine spermatozoa. The inhibition was not complete but ABCA1 is supposed to contribute to capacitation in canine ejaculated spermatozoa. ABCA1 is probably not important for capacitation of epididymal spermatozoa but might exert other functions during spermatozoa ripening.

Embryo-maternal communication in dogs: Immune system related factors.

In the bitch, establishment of pregnancy is believed to be mainly initiated by the free-floating embryo in the uterus that is under progesterone influence. As in other species, the active participation of the embryo is no longer questioned. Secretory products are transported to the embryo-maternal interface and contribute to extra-cellular matrix (ECM) degradation, a change in the intrauterine immune milieu towards a reduction of immune cells and a change in lymphocyte subsets, cell differentiation, angiogenesis, and the balance between proliferation and apoptosis. For cell-to-cell communication between embryo and maternal tissue, biomolecules inclusive microRNAs might be transported and exchanged via extracellular vesicles (EVs) as in other species. Maternal acceptance of the fetal allograft is vital for the establishment of pregnancy. Findings so far indicate that the embryo avoids attacks from the maternal system via passive and active mechanisms. One hypothesis is that expression or suppression of surface molecules help the canine embryo to hide from the maternal immune system on one side and to actively destroy cytotoxic immune cells on the other side; there are further clues that the canine embryo blocks activation of intrauterine leukocytes. Intracellular repair mechanisms via heat shock proteins (HSP) are candidates under investigation. The presence and function of immunomodulatory intrauterine cells like Treg cells and their interaction with the embryo have been intensely studied in other species but remains to be investigated in the canine preimplantation uterus.