In mammalian species there are significant physiological responses of the female reproductive tract to the deposition of sperm. These are particularly notable in species where sperm are deposited directly into the uterus, and function both to facilitate sperm transport to the sperm reservoir, and to eliminate introduced contaminants. In the bitch, sperm are deposited into the vagina and are rapidly transported through the open cervix. Sperm are then distributed around the uterus by uterine contractions such that transportation to the tip of the uterine horns occurs within 1 min of the start of mating. The main sperm reservoir appears to be the distal part of the utero-tubal junction which forms a pre uterine tube reservoir. Sperm remain attached here by their heads to uterine epithelium and remain viable. In non-capacitating conditions sperm slowly detach from this site and this seems important to replenish the uterine tube reservoir, where sperm may re-attach to the epithelium. Post-ovulatory signals trigger capacitation changes and subsequent hyperactivated motility that is associated with detachment of sperm from both reservoirs; thus facilitating fertilization. After mating, a physiological post-mating uterine inflammatory response occurs, evidenced by an influx of polymorphonuclear neutrophils, increased uterine contractions, an increased uterine artery blood flow and a decrease of the resistance index indicating a short-duration vasodilation. Disturbance of this tightly regulated system has the potential to impact fertility by a failure of elimination of the introduced contaminants (such that a clinically-significant post-breeding endometritis ensues) but also by impairing sperm transport. (C) 2020 The Authors. Published by Elsevier Inc.

The main objective of this study was to assess the semen quality of Piétrain boars originating from Belgian AI centers and to correlate these results with their meat quality traits. Freshly diluted semen doses from 140 boars originating from 10 artificial insemination (AI) centers were used and stored for five days at 17 °C. Motility was assessed daily using a computer assisted semen analyzer (Hamilton-Thorne), while morphology and concentration were assessed on the day of semen collection (Day 0) by eosin-nigrosin staining and the Bürker counting chamber, respectively. These data were correlated with the lean meat percentage, loin eye depth and backfat thickness using linear mixed models taking into account the clustering of boars within each AI center and the daily measurements for each semen dose. The mean values (± SD) on Day 0 were: motility 79.7 ± 8.2%, live sperm 91.5 ± 4.3%, live normal sperm 83.6 ± 7.4%, and concentration 29.0 ± 10.6 (×106 sperm/mL). The average five-day motility across all AI centers was 77.7 ± 8.9%. None of the assessed semen quality traits were associated with lean meat percentage. Motility and progressive motility on Day 0 were positively associated with backfat thickness (P < 0.05), while no overall negative associations were elucidated between the latter semen quality traits and loin eye depth. The percentages of live and normal live sperm were not correlated with backfat thickness nor loin eye depth. To conclude, selection of terminal Belgian Piétrain boars for reduced backfat thickness might negatively influence semen motility, whereas selection for increased lean meat percentage and loin eye depth would not necessarily compromise semen quality traits.

The maintaining of the epididymis at lower temperatures during storage and transport improves sperm quality. Our study aimed to test whether epididymis storage temperature (post-mortem) and sperm cryopreservation affect sperm kinetics, membrane integrity, mitochondrial potential and fertility capacity. Thirty-six epididymides were collected from 18 bulls after slaughter and divided into two groups: at 4 or 34°C for 2-3 hr. The sperm was collected from the epididymis cauda. The evaluation consisted of computer-assisted sperm analysis (CASA), SYBR14/PI/JC1 to evaluate membrane integrity, mitochondrial membrane potential (MMP) and measurement of lipid peroxidation (TBARS). The sperm was then frozen using an automatic device. After thawing, sperm samples were evaluated by the same variables and further in vitro fertilization rates. Cryopreservation negatively affected sperm motility in samples stored at 4 and 34°C. Nevertheless, the 4°C samples yielded higher rates of blastocyst formation. Pre-freeze sperm motility, progressive motility and velocity were higher in sperm from epididymis stored at 4°C while post-thaw sperm motility, progressive motility and velocity remained the same among samples from epididymis stored at 4 or 34°C. However, with regard to the kinetic patterns, samples collected from epididymis stored at 34°C had lower values when compared to those stored at 4°C prior the cryopreservation process. Our results indicate that epididymis handling conditions after cryopreservation may affect sperm quality after thawing, especially due to compromised MMP in sperm collected from epididymis stored at higher temperatures.

Ejaculates collected from breeding bulls during the summer season under tropical or subtropical conditions are often reported to have a lower fertilization potential. We recently reported that sperm cells at post-meiotic stages of development were more susceptible to heat stress (scrotal insulation technique). In post-meiotic stages of sperm cell development, extensive incorporation of histone variants and hyperacetylation confirms unstable chromatin. The unstable forms of chromatin are more vulnerable to heat stress and affect sperm DNA-protamine condensation. By using a special stain and nuclear morphometric assessment, Chromomycin A3 and Fourier harmonic analysis, respectively, we observed protamine-deficient spermatozoa with altered chromatin condensation in the heat-stressed ejaculates. In a recent study, we investigated the patterns of global DNA methylation in pronuclear development and fertilization potential of such altered chromatin condensed spermatozoa. To this end, 1239 in vitro-matured oocytes were fertilized with spermatozoa of 3 groups (i.e. standard IVF control, non-heat-stressed control, and heat-stressed spermatozoa). Data were analysed by means of ANOVA. The results showed that heat-stressed spermatozoa with altered chromatin condensation perturb the dynamics of DNA methylation reprogramming in the paternal pronucleus (i.e. disordered the active demethylation followed by a de novo methylation pattern during one cell stage). In addition, there was a tendency for a decrease in the size of both paternal and maternal pronuclei developed after fertilization with heat-stressed spermatozoa in comparison with standard IVF control spermatozoa, leading to lower fertilization rates (54 v. 82%). Future studies should focus on the mechanisms of perturbed DNA demethylation in the paternal genome after in vitro fertilization of oocytes with spermatozoa having altered chromatin condensation.

Heat stress has long been recognised as a cause of subfertility in farm animals. The objectives of the present study were to elucidate the effect of heat stress on sperm function and involvement of the mitogen-activated protein kinase (MAPK) 14 signalling pathway. Spermatozoa incubated for 4 h at a physiological temperature (38.5°C) exhibited significantly (P<0.05) reduced motility, plasma membrane integrity and mitochondrial potential compared with non-incubated spermatozoa; the reductions in these parameters were more severe following incubation at a hyperthermic (41°C) temperature (P<0.01). Percentages of fertilisation and embryo development were highly affected in spermatozoa incubated at 41°C compared with non-incubated spermatozoa (P<0.01). Similarly, embryo quality was adversely affected by sperm incubation at 41°C, as indicated by a higher apoptotic cell ratio in Day 7 blastocysts compared with that in the non-incubated control group (14.6% vs 6.7%, respectively; P<0.01). Using SB203580 (10 µgmL(-1)), a specific inhibitor of the p38 MAPK pathway, during sperm hyperthermia reduced MAPK14 activation (24.9% vs 35.6%), increased sperm motility (45.8% vs 26.5%) and reduced DNA fragmentation (16.9% vs 23.4%) compared with the untreated control group, but did not improve subsequent fertilisation and embryo development. In conclusion, heat stress significantly affects the potential of spermatozoa to penetrate oocytes, as well as subsequent embryo development and quality. Notably, the data show that the MAPK14 signalling pathway is largely involved in heat-induced sperm damage. However, further research is needed to elucidate other signalling pathways possibly involved in heat-induced sperm damage.

Shortly after penetration of the oocyte, sperm DNA is actively demethylated, which is required for totipotent zygotic development. Aberrant DNA methylation is thought to be associated with altered chromatin condensation of spermatozoa. The objectives of this study were to investigate the dynamics of DNA methylation reprogramming in the paternal pronucleus and subsequent fertilisation potential of heat-stressed bull spermatozoa having altered chromatin condensation. Hence, bovine zygotes (n = 1239) were collected at three different time points (12, 18 and 24 h post insemination, hpi), and stained with an antibody against 5-methylcytosine. Fluorescence intensities of paternal and maternal pronuclei were measured by ImageJ. DNA methylation patterns in paternal pronuclei derived from heat-stressed spermatozoa did not differ between time points (P > 0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24 hpi, respectively. Moreover, heat-stressed spermatozoa showed a highly reduced (P < 0.01) fertilisation rate compared with non-heat-stressed or normal control spermatozoa (53.7% vs 70.2% or 81.5%, respectively). Our data show that the normal pattern of active DNA demethylation followed by de novo methylation in the paternal pronucleus is perturbed when oocytes are fertilised with heat-stressed spermatozoa, which may be responsible for decreased fertilisation potential.

Gonadectomy is the most commonly used method for permanent contra- ception in small animals. The irreversibility of the method is however a main drawback for its use in valuable breeding animals. Moreover, several negative side effects can be observed after surgical castration. Therefore several non-surgical methods were developed. This paper describes the current non-surgical methods of contraception used in female dogs and cats. They include hormonal procedures, such as application of progestins, androgens and GnRH analogues in order to prevent the ovarian cycle. Another method is the use of 4-vinylcyclohexene diepoxide, an industrial chemical destroying primordial and primary ovarian follicles. Further prospective possibilities consist in immunocontraception and in the elaboration of a safe and effective vaccine with reversible effect. Finally the use of several abortive drugs, such as aglepristone, PGF2 and dopamine agonists are presented.

During the last decades, in vitro fertilization (IVF) has become a routine technique in most domestic animals. However, in the dog the technique has lagged behind, with to date not a single pup born after IVF. In cats, healthy kittens have been born, but in fewer numbers than in cattle and horses. In pet animals, research in reproduction has mainly been focused on contraception, although recently, the introduction of new drugs especially marketed for cats and dogs will probably expand fertility research in carnivores towards the previously neglected area of assisted reproduction. In particular, the dog remains a real challenge for the reproductive biologist, due to the low meiotic capacity of canine follicular oocytes. In cats, oocyte maturation is less of a problem and embryo production rates comparable to those of cattle can be achieved. The domestic cat is a valuable model for endangered felids and it can even be used as a recipient for wild felid embryos. In this short review, we list some of the problems associated with the implementation of IVF in dogs and cats in relation to their reproductive characteristics, and we discuss the state-of-the-art of IVF in several other domestic species such as cattle, horses and pigs.

Canine sperm transport, distribution, storage and detachment is a complex, dynamic and highly regulated process. Transport of sperm within the bitch's reproductive tract is rapid and is influenced by the method of semen deposition (natural mating or artificial insemination) and by the timing of breeding in relation to the day of ovulation. The fertile lifespan of spermatozoa in the reproductive tract of the bitch is considerably longer than in most other domestic species, and the main sperm reservoirs appear to be the uterine crypts and the distal part of the uterotubal junction, where spermatozoa attach by their heads to uterine epithelium. While several in vitro studies demonstrated prolonged motility and viability of canine spermatozoa after coincubation with uterine tube explants, spermatozoal storage has not been documented in the canine uterine tube isthmus or ampulla in vivo. Several factors, including exposure to progesterone, solubilized zona pellucida proteins and post-ovulation uterine tube fluid, appear to trigger membrane events resulting in capacitation-like changes with subsequent motility pattern changes (transitional and hyperactivated) that are associated with sperm detachment. After mating or insemination, a normal low-magnitude post-mating uterine inflammatory response occurs, evidenced by an influx of polymorphonuclear neutrophils (PMNs), increased uterine contractions and an increased uterine artery blood flow. Recently, it was also shown that normal dogs with cystic endometrial hyperplasia develop a more significant endometritis, show fewer mating-induced uterine contractions, a decreased ability of spermatozoa to bind to uterine explants in vitro and a slower uterine clearance after mating.

Soon after fertilization, mammalian zygotes need proper DNA methylation reprogramming, at which time the epigenetic marks that the oocyte and sperm have acquired during gametogenesis are erased to allow totipotent zygotic development. Aberrant epigenetic marks in the paternal genome are thought to be associated with altered chromatin condensation in spermatozoa of suboptimal quality. We have recently reported that heat stress on bulls during germ cell development, especially at the spermiogenesis stage, altered sperm chromatin condensation. The objective of this study was to investigate dynamic DNA methylation reprogramming in the male pronucleus after fertilization of oocytes with sperm known to have altered chromatin conformation. To evaluate dynamic DNA methylation reprogramming, zygotes collected at 3 different time points [i.e. 12, 18, and 24 h post-insemination (hpi)] were immunocytochemically investigated using an antibody against 5-methylcytosine (5mC). The total fluorescence intensity of the male pronuclei (n = 89, ≥25 in each group) was measured by ImageJ and data were analyzed by ANOVA. The DNA methylation pattern in male pronuclei when oocytes were fertilized with heat-stressed sperm did not change between time points (P > 0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24 hpi, respectively. The results of this study indicated that dynamic DNA methylation reprogramming patterns such as DNA demethylation followed by de novo methylation in the male pronucleus soon after fertilization were altered when oocytes were fertilized with heat-stressed sperm. In conclusion, altered sperm chromatin conformation due to heat stress perturbs dynamic DNA methylation reprogramming in the male pronucleus, which may hamper nuclear totipotency and embryo survival.

Dystocia may occur in all dog breeds, but the English bulldog is predisposed because of its conformation and specifi c problems such as anasarca pups. In this study, the frequency of abnormal pups and breeding problems in English bulldogs was investigated by a questionnaire containing information on 39 bitches in total. In 74.4% of these bitches, artifi cial insemination was performed. At the end of gestation, 25.8% of the bitches suffered from respiratory problems and partial anorexia. The average duration of gestation was 58.7 days counted from the fi rst day of mating. Caesarean section was performed in 94.8% of the cases, with natural delivery in only 5.2% of the bitches. In this study, the average litter size was six pups. Thirteen percent of the pups were stillborn, 8.2% of the pups alive were deformed, with palatoschisis (38.8%) and anasarca (27.7%) being the most commonly observed abnormalities. Ten percent of the pups died before the age of weaning.

Vaginale cytologie bij de teef is gemakkelijk uit te voeren in de praktijk, de staalname is vrij eenvoudig en het is een relatief goedkope techniek met vele toepassingsmogelijkheden. Doordat de resultaten bovendien snel beschikbaar zijn, kan het praktijkdierenartsen helpen bij het beoordelen en het opvolgen van het cyclusstadium van een teef. Vaginale cytologie kan ook voor andere doeleinden gebruikt worden, zoals het voorspellen van het exacte partustijdstip, het bevestigen van een (ongewenste) dekking en het vaststellen van vaginitis, verlengde loopsheid, ovarieel restsyndroom, problemen post partum of vaginale neoplasie. Het optimale moment van dekking of inseminatie kan niet met vaginale cytologie bepaald worden en dient te worden uitgevoerd door middel van vaginoscopie en/of progesteronbepaling.

Dystokie is een probleem bij alle hondenrassen, maar bij de Engelse buldog is het risico hoger omwille van zijn bijzondere conformatie en specifieke problemen zoals waterpups. In deze studie werd de frequentie van abnormale pups en voortplantingsproblemen onderzocht bij de Engelse buldog met behulp van een enquête bij 39 teven. Bij 74,4% van deze teven werd kunstmatige inseminatie uitgevoerd. Bij 25,8% van de teven werden ademhalingsproblemen en anorexia waargenomen aan het eind van de dracht. De dracht duurde gemiddeld slechts 58,7 dagen vanaf de eerste dekking. In 94,8% van de bevallingen werd een keizersnede uitgevoerd, spontane geboorte kwam slechts bij 5,2% voor. De gemiddelde nestgrootte in deze studie was zes pups. Dertien percent van de pups werd dood geboren, 8,2% van de levende pups had een afwijking waarbij palatoschisis (38,8%) en waterpups (27,7%) het meeste voorkwamen. Tien percent van de pups stierf vóór de speenleeftijd.

Dystocia may occur in all dog breeds, but the English bulldog is predisposed because of its conformation and specific problems such as anasarca pups. In this study, the frequency of abnormal pups and breeding problems in English bulldogs was investigated by a questionnaire containing information on 39 bitches in total. In 74.4% of these bitches, artificial insemination was performed. At the end of gestation, 25.8% of the bitches suffered from respiratory problems and partial anorexia. The average duration of gestation was 58.7 days counted from the first day of mating. Caesarean section was performed in 94.8% of the cases, with natural delivery in only 5.2% of the bitches. In this study, the average litter size was six pups. Thirteen percent of the pups were stillborn, 8.2% of the pups alive were deformed, with palatoschisis (38.8%) and anasarca (27.7%) being the most commonly observed abnormalities. Ten percent of the pups died before the age of weaning.

Neonatal isoerythrolysis in kittens can only occur when the queen has blood type B and the tomcat blood type A or AB. During the first 24 hours after birth, kittens ingest colostral antibodies, which reach the systemic circulation through the bowel. Kittens with blood type A absorb antibodies, which destroy their erythrocytes. Kittens that develop symptoms, such as anemia, hemoglobininuria or icterus, should be given a blood transfusion. However, the survival rate is very low, and prevention is of utmost importance. This can be done by determining the blood type of queens and tomcats intended for breeding with breeds at risk. Ideally, a queen with blood type B and a tomcat with blood type A or AB should not be mated. However, if this combination occurs, the kittens should be removed from the mother during the first 24 hours after birth. Subsequently, they have to be fed with artificial milk and be given oral or subcutaneous plasma from a well-immunized cat with blood type A. Neonatale iso-erytrolyse bij de kat