Supplementation with MitoTEMPO before cryopreservation improves sperm quality and fertility potential of Piedmontese beef bull semen.

The purpose of this study was to improve the quality of frozen-thawed Piedmontese bull semen by incorporating MitoTEMPO (MT) in extended semen before cryopreservation. Semen was collected from 4 fertile bulls, using an artificial vagina, once weekly for 6 consecutive weeks. Semen samples were pooled, diluted with Bullxcell® extender, and supplemented with different concentrations of MT (0 as control, 5, 10, 20, 40, and 80 µM) before cooling, equilibration, and freezing procedures. The frozen-thawed semen was assessed for motility, vitality, acrosome intactness, plasma membrane integrity, DNA integrity, apoptosis, mitochondrial membrane potential, intracellular ROS level and in vitro fertilizing capability. The results showed that MT at concentrations of 10, 20, and 40 µM improved the total, progressive, and rapid motility directly after thawing while, at the highest tested concentration (80 µM), it decreased the progressive and rapid motility after 1, 2, and 3 h of incubation. The sperm kinetics including STR and LIN were noticeably increased at concentrations of 10, 20, and 40 µM directly after thawing (0 h), whereas the MT effect was variable on the other sperm kinetics during the different incubation periods. MitoTEMPO improved the sperm vitality at all tested concentrations, while the acrosomal and DNA integrity were improved at 20 µM and the mitochondrial membrane potentials was increased at 80 µM. The cleavage and blastocyst formation rates were significantly increased by using semen treated with 20 µM MT compared with controls. These findings suggest a potential use of MT mainly at a concentration of 20 µM as an additive in the cryopreservation media of bull semen to improve sperm quality.

The vaginal microbiota of healthy female cats.

The vaginal microbiota of the queen (i.e., female cat) has never been described using culture independent methods. The objectives of the present research were to describe the vaginal microbiota of healthy domestic shorthair queens using both 16S rRNA sequencing and culture, and to assess the effects of age, living environment, and reproductive season on its composition. Thirty queens undergoing elective ovariectomy were included in the study. The vaginal samples were collected just before surgery, from animals under general anaesthesia. Two consecutive mini-swabs were introduced in the queens' vaginal tract. A preliminary study with 10 healthy queens aimed to negate sampling order's effect. Two consecutive samples for sequencing (5 queens, 10 swabs) and culture (5 queens, 10 swabs) were collected, confirming a match (100 % in culture, Bray-Curtis P = 0.96 in sequencing). The experiment included 20 queens that were prospectively grouped based on age (prepubertal N = 10, adult N = 10), living environment (indoor N = 10, outdoor N = 10), and time of the year, whether during the reproductive season (N = 10) or during seasonal anoestrous (N = 10). Bacteria were identified through metataxonomic analysis, amplifying the V1-V2 regions of 16S rRNA gene, and through standard culture followed by MALDI-TOF MS. The feline vaginal microbiota is dominated by Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteria. Escherichia-Shigella, Streptococcus, and Pasteurella were the most abundant genera. Although culture underestimated bacterial richness and diversity compared to sequencing, Escherichia and Streptococcus were the most isolated bacteria. No bacterial growth was observed in 15 % of samples (N = 3/20), whereas growth of one or two bacterial species was observed in 64.7 % (N = 11/17) and 35.3 % (N = 6/17) of cases, respectively. No differences in terms of alpha (Kruskal-Wallis rank sum test P = 0.65) and beta diversity (Bray-Curtis, Unweighted and Weighted UniFrac analyses P > 0.5) were observed. Although a difference in alpha diversity based on phylogenetic tree (P = 0.02) was detected between indoor and outdoor queens. In conclusion, mixed and monoculture of Escherichia coli, Streptococcus canis, Staphylococcus felis, and Enterococcus spp. are normal findings within the cat vagina. Age and reproductive season do not influence the feline vaginal microbiota, whereas further research is needed to elucidate the role of the living environment.

In vitro effects of different ozone preparations on microorganisms responsible for endometritis in the mare.

Infectious endometritis is considered one of the major causes of infertility and it can affect up to 60% of barren mares. It is characterized by the presence of one or more microorganisms in the reproductive tract and it is treated with the administration of antibiotics, ecbolic agents and uterine lavages. Ozone, thanks to its antimicrobial properties that are based on its high oxidative potential, could represent an effective alternative treatment for endometritis. The aim of this study was to test in vitro the bactericidal and fungicidal properties of different ozone formulations, either as gas (experiment 1) or dissolved in two liquid matrices (experiment 2), specifically distilled water or oil (Neozone 4000, Cosmoproject, Parma, Italy), onto 6 different species of microorganisms isolated from mares with clinical endometritis, namely Escherichia coli, Staphylococcus aureus, Streptococcus equi subsp. Zooepidemicus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans. In the first experiment, 3 clinical antibiotic-resistant strains per each species were exposed to different conditions: to O2O3 gas mixtures (15 and 40 µg/ml for 1, 3 and 5 min), to 100 % O2 or left untreated. The results showed a reduction of the microbial count of over 99,9% for every pathogen, time and concentration of O2O3 gas mixtures tested. Furthermore, gaseous ozone showed both a time-dependant effect (5 vs 3 vs 1 min of exposure) and a concentration-dependant effect (40 vs 15 µg/ml) at 1 and 3 min, while after 5 min no differences were observed. In the second experiment, minimum inhibitory concentration (MIC), and minimum bactericidal/fungicidal concentration (MBC, MFC) of ozonated distilled water and ozonated oil were evaluated. Ozonated oil showed a bactericidal/fungicidal activity against all the strains tested (MIC range 12.5-25 % v/v, MBC/MFC range 12.5-50 % v/v) while ozonated distilled water didn't show an observable antimicrobial effect, discouraging its use as an antimicrobial agent for the treatment of endometritis. The results of this in vitro study indicate that both gaseous ozone and ozonated oil exerted remarkable antimicrobial activities and are promising alternative treatments for infectious endometritis, even when caused by antibiotic-resistant bacteria, and encourage further experiments in an effort to scale down or even prevent the use of antibiotics in equine reproduction.

Influence of Intrauterine Fluid Detection, Number of Transfers and Age of the Recipient on Pregnancy Rate and Early Embryonic Loss in a Commercial Embryo Transfer Program.

The selection of the recipient mare is one of the most important factors involved in the success of equine embryo transfer. The aim of this study was to determine whether the age of the recipient, the number of transfers and the detection of intrauterine fluid during the follicular phase or after ovulation can affect pregnancy rate at 14 and 45 days (PR 14 and PR 45) or early embryonic loss (EEL). A total of 1222 ETs were included in the study. Mares receiving the first embryo of the year had a higher PR 14 and 45 days compared to mares at the third transfer (78.8% and 70.1% vs. 65.6% and 54.1%, respectively). The detection of intrauterine fluid post ovulation negatively affected PR 14 (60.5% vs. 77.6%) and should therefore be considered an abnormal finding, probably being a sign of uterine inflammation or delayed uterine clearance. On the contrary, the age of the recipient mare and detection of fluid during follicular phase did not affect PR 14. Only the age of the recipient mare influenced the EEL, since mares aged 10-13 years had a higher EEL compared to mares aged 3-5 years (15.6% vs. 6.4%). Embryo size and grade affected PR 14 and 45.

Birth weight, puppies' growth and health with limited-ingredient novel protein diet vs standard diet in late pregnancy.

Great attention has been given in the last years to the protein source of dog food, and commercial limited-ingredient diets with a single protein are available also for late pregnancy. This work compared the effect of a fish-based limited ingredient diet (LID), and of a standard mixed-protein diet (Mixed), fed to the bitches from the last three weeks of pregnancy and to the puppies at weaning, on birth weight, growth and health of the puppies. From a breeder's records, the weight of 22 Lagotto Romagnolo (LR) and 10 Appenzeller Cattle Dog (ACD) bitches on the day of mating, and of their 199 puppies, were extracted. The effect of diet on puppies' weight on day 0, 6, 30 and 60 was analyzed, considering litter size and sex. The analyses were repeated on puppies' weights normalized on the relative dam's non-pregnant bodyweight. Birth weight was available for 146 puppies, 82 LR and 64 ACD. Median birth weight of LR puppies was 287.5 g (170-400 g); sex ratio was 1.11 (males/females, N = 80). Median birth weight of ACD puppies was 390 g (240-525 g); sex ratio 1.15 (males/females, N = 58). Diet did not significantly affect birth weight in both breeds; however, it showed a significant effect on normalized birth weights (LR, P = 0.016; ACD, P = 0.034), with higher values for LID. At day 30, ACD puppies showed significantly higher weights with the Mixed diet (P = 0.002), and, at day 60, diet significantly affected the normalized weight in both breeds (LR, P = 0.019; ACD, P = 0.001), with higher values for the Mixed type. LID may help the dam to invest the energy in the growth of her litter, however, the same diet resulted in lower puppies' weights around weaning, compared to the Mixed diet. Although our results should be confirmed with larger numbers of animals and more breeds, they set some points worth to be further investigated. The choice of a limited-ingredient single-protein diet can affect litter weight and weight at weaning. Whether, administered to dams and puppies, it can prevent later pathologies, like chronic gastrointestinal diseases or food allergies, is a field of research deserving full attention.

Challenging the Hypothesis of in Utero Microbiota Acquisition in Healthy Canine and Feline Pregnancies at Term: Preliminary Data.

At present, there are no data on the presence of bacteria in healthy canine and feline pregnancies at term. Here, we investigated the uterine microbiome in bitches (n = 5) and queens (n = 3) undergoing elective cesarean section in two facilities. Samples included swabs from the endometrium, amniotic fluid, and meconium, and environmental swabs of the surgical tray as controls. Culture and 16S rRNA gene sequencing were used to investigate the presence of bacteria. Culture was positive for 34.3% of samples (uterus n = 3, amniotic fluid n = 2, meconium n = 4, controls n = 0), mostly with low growth of common contaminant bacteria. With sequencing techniques, the bacterial abundance was significantly lower than in environmental controls (p < 0.05). Sequencing results showed a species-specific pattern, and significant differences between canine and feline bacterial populations were found at order, family, and genus level. No differences were found in alpha and beta diversities between feto-maternal tissues and controls (p > 0.05). Dominant phyla were Bacteroidetes, Firmicutes, and Proteobacteria in different proportions based on tissue and species. Culture and sequencing results suggest that the bacterial biomass is very low in healthy canine and feline pregnancies at term, that bacteria likely originate from contamination from the dam's skin, and that the presence of viable bacteria could not be confirmed most of the time.

Safety and Effects of a Commercial Ozone Foam Preparation on Endometrial Environment and Fertility of Mares.

Mares' subfertility represents a complex diagnostic and therapeutic challenge and both clinical and subclinical endometritis are considered major causes of impaired fertility. Thanks to its properties, ozone has a big potential as a treatment for equine endometritis. Therefore, the aim of this study is to describe the safety and the effects on endometrium and reproductive parameters of mares of a commercial ozone foam preparation (Riger Spray®). Twenty-four mares were treated during estrus: ozone group with an intrauterine instillation of ozone foam preparation (OG, n=16) and control group with 20 ml of lactated Ringer's solution (CG, n=8). Samples for endometrial cytology were collected before the ozone treatment (T0), after 24 h (T1), after one week (T2), two weeks (T3), and when the subsequent estrous phase was detected (T4). Furthermore, samples for histological examination and uterine swab for bacteriological examination were collected at T0 and T4. At T1, a statistically significant increase of endometrial inflammation in the OG mares compared to T0 (P<.05) and to CG at same time point (P<.05) was observed, but it was already resolved at T2. No differences in endometrial inflammation in CG, biopsy grade before and after the treatment in the two groups, number of mares pregnant at the end of the season and number of mares pregnant at the first cycle were observed. However, the number of inseminations required for pregnancy tended to be lower (P=.0711) in the OG (1.69±0.06) than in CG mares (2.60±0.89).

The evolution of dam-litter microbial flora from birth to 60 days of age.

BACKGROUND: Early bacterial colonization in puppies is still a poorly understood phenomenon. Although the topic is of considerable interest, a big gap in knowledge still exists on the understanding of timing and features of neonatal gut colonization. Thence, the purpose of this study was to evaluate the relationship between dam and litter microbial flora, in vaginally delivered puppies, from birth to two months of age. Bacteria were identified using MALDI-TOF, an accurate and sensitive method, and cluster analysis of data provided a new insight on the investigated topic. METHODS: Six dam-litter units of two medium size breeds were enrolled in the study. Vaginal and colostrum/milk samples were collected from dams after delivery and 48h post-partum, while rectal samples were taken from dams and puppies after delivery and at day 2, 30 and 60 (T2, T30 and T60, respectively) post-partum. Bacterial isolation and identification were performed following standard techniques, then the data were analyzed using a new approach based on bacterial genus population composition obtained using a wide MALDI-TOF screening and cluster analysis. RESULTS: Forty-eight bacteriological samples were collected from the dams and 145 from their 42 puppies. Colostrum/milk samples (n = 12) showed a bacterial growth mainly limited to few colonies. Staphylococci, Enterococci, E. coli, Proteus spp. were most frequently isolated. All vaginal swabs (n = 12) resulted in bacteria isolation (medium to high growth). Streptococci, Enterococci, E. coli were the most frequently detected. E. coli, Proteus mirabilis, Enterococcus spp., Streptococcus spp. were often obtained from dams' and puppies' rectal swabs. Clostridia, not isolated in any other sampling site, were rarely found (n = 3) in meconium while they were more frequently isolated at later times (T2: n = 30; T30: n = 17; T60: n = 27). Analysis of the bacterial genus pattern over time showed a statistically significant reduction (P < 0.01) in the heterogeneity of microbial composition in all time points if compared to birth for each dam-litter unit. These results were confirmed with cluster analysis and two-dimensional scaling. CONCLUSION: This novel data analysis suggests a fundamental role of the individual dam in seeding and shaping the microbiome of the litter. Thus, modulating the dam's microbiota may positively impact the puppy microbiota and benefit their health.

Trends in Small Animal Reproduction: A Bibliometric Analysis of the Literature.

Small animal reproduction (SAR) is a main research field in veterinary medicine and bibliometric analyses are useful to investigate trends in specific research areas. The objective of the present study was to conduct a bibliometric analysis of the literature of the last decade on SAR. A search equation was created, and documents were retrieved from the Web of Science database. Documents were manually revised, categorized and R software version 4.1.2 with Bibliometrix R package version 3.1 and MS Excel were used to perform the analyses. The included documents (n = 1470) were mainly research articles (78%). The top countries for the number of documents and citations were Brazil, United States, Italy, Poland, and Korea. These also account for the most prolific authors and institutions. Analyses by author keywords, categories, and recent reviews of the literature suggest that research on the canine species is more abundant than research on the feline one and that reproductive biotechnologies are a main research focus. Some clinical topics are still considered niche or neglected themes (e.g., semen collection in tomcats, neonatology). However, heterogeneity and ambiguity in keywords and categories are undeniable. This study offers interesting insights, providing definitions for main keywords in the field of SAR.

A retrospective study and survival analysis on bitches with mammary tumours spayed at the same time of mastectomy.

The aim of the present study was to retrospectively assess whether spaying at the same time of mastectomy increased disease-free survival (DFS) in bitches with mammary tumours and to investigate the utility of clinical data when designing a surgical plan that includes gonadectomy. Characteristics of 225 bitches carrying 489 tumours were retrieved. Of the116 bitches that underwent surgery, 52 bitches underwent mastectomy and ovariectomy, 46 bitches underwent mastectomy alone, whereas 18 bitches were already spayed. Analysis by Kaplan-Meier and in-between groups comparisons using Student's T, Chi-square, and one-way ANOVA tests were performed. DFS was longer for bitches that underwent ovariectomy and mastectomy compared to those that were left intact (p = .00064) or were already spayed (p = .0098). Spaying status affected tumour size (spayed: 2.75 cm ± 2.72; intact: 1.76 cm ± 2.04; p = .039), but not malignancy (p > .05). Differences in age were detected between animals with benign and malignant tumours (years: 9.1 ± 2.8 and 10 ± 2.3; p = .004), with multiple and single tumours (years: 10.18 ± 2.6 and 9.3 ± 2.8; p = .007), and between purebred and mixed breed bitches (years: 10.46 ±1.78 and 9.27 ±2.68; p = .005). Malignant tumours were larger than benign ones (2.17 cm±2.31 and 1.34 cm ±1.82; p = .005) and size increased according to the degree of malignancy. DFS was shorter for animals presenting tumours >2 cm in size (p < .006) and with tumours in the first pair of thoracic mammary glands (p = .00009). Gonadectomy should be suggested to owners of intact bitches carrying mammary tumours and age, size of the tumour, and location should be carefully considered when performing surgery.

Does Bacteria Colonization of Canine Newborns Start in the Uterus?

The assumption that requires the uterus to be a sterile environment to sustain a successful pregnancy has been recently challenged in humans, and is still under debate. The aim of this study was to assess whether bacteria can be isolated from the pregnant uterus and from amniotic fluid and meconium of healthy canine fetuses at term, delivered through cesarean section. Fifteen dams of different breed, age and parity, undergoing either elective (n = 10) or emergency (n = 5) cesarean section after a healthy pregnancy, were included in the study. Swabs for bacterial culture were collected from the uterus, and from amniotic fluid and meconium. Bacteria were isolated from all the sampled sites and materials, irrespective of cesarean type. In most cases, different bacteria were isolated from the different sites. Acinetobacter spp., coagulase-negative Staphylococci and Bacillus spp. were frequently found while Pseudomonas aeruginosa, Micrococcus spp., Moraxella spp., Macrococcus spp., Glutamicibacter spp., Stenotrophomonas spp. and Psychrobacter spp. were only occasionally identified. Our data show that uterus and fetuses may not be sterile in healthy term canine pregnancies.

Plants and zootoxins: Toxico-epidemiological investigation in domestic animals.

An epidemiological study on animal poisoning due to plants and zootoxins has been carried out by the Poison Control Centre of Milan (CAV) in collaboration with the University of Milan (Italy). During the period January 2015-March 2019, the CAV received 932 calls on animal poisonings, 12.66% (n = 118) of which were related to plants and zootoxins. Among these, 95 enquiries (80.51%) concerned exposures to plants and 23 (19.49%) to zootoxins. The dog was the species most frequently involved (67.80% of the calls, n = 80), followed by the cat (26.27%, n = 31). As for the plants, several poisoning episodes were related to glycoside-, alkaloid-, oxalate- and diterpenoid-containing species. Cycas revoluta, Euphorbia pulcherrima and Hydrangea macrophylla were the most often reported plants. The outcome has been reported for half of the episodes (51.58%, n = 49) and it was fatal for 3 animals (6.12%). Regarding the zootoxins, the majority of the enquiries were related to asp viper (Vipera aspis), but exposures to pine processionary moth (Thaumetopoea pityocampa), common toad (Bufo), fire salamander (Salamandra), and jellyfish (phylum Cnidaria) were also reported. The outcome was known in 65.22% of the cases with just one fatal episode. This epidemiological investigation depicts an interesting overview on the issue of plant and zootoxin exposures in domestic animals, highlighting the relevance of these agents as causes of animal poisoning and providing useful information for prevention and diagnosis.

In vitro copper oxide nanoparticle toxicity on intestinal barrier.

The use of CuO nanoparticles (NPs) has increased greatly and their potential effects on human health need to be investigated. Differentiated Caco-2 cells were treated from the apical (Ap) and the basolateral (Bl) compartment with different concentrations (0, 10, 50 and 100 µg/mL) of commercial or sonochemically synthesized (sono) CuO NPs. Sono NPs were prepared in ethanol (CuOe) or in water (CuOw), obtaining CuO NPs differing in size and shape. The effects on the Caco-2 cell barrier were assessed via transepithelial electrical resistance (TEER) evaluation just before and after 1, 2 and 24 hours of exposure and through the analysis of cytokine release and biomarkers of oxidative damage to proteins after 24 hours. Sono CuOe and CuOw NPs induced a TEER decrease with a dose-dependent pattern after Bl exposure. Conversely, TEER values were not affected by the Ap exposure to commercial CuO NPs and, concerning the Bl exposure, only the lowest concentration tested (10 µg/mL) caused a TEER decrease after 24 hours of exposure. An increased release of interleukin-8 was induced by sono CuO NPs after the Ap exposure to 100 µg/mL and by sono and commercial CuO after the Bl exposure to all the concentrations. No effects of commercial and sono CuO NPs on interleukin-6 (with the only exception of 100 µg/mL Bl commercial CuO) and tumor necrosis factor-α release were observed. Ap treatment with commercial and CuOw NPs was able to induce significant alterations on specific biomarkers of protein oxidative damage (protein sulfhydryl group oxidation and protein carbonylation).

Plants and mushrooms associated with animal poisoning incidents in South Africa.

BACKGROUND: There is extensive literature on animal poisoning from plants and mushrooms worldwide; however, there is limited account of poisoning from South Africa. METHODS: This study sought to describe and provide an overview of animal poison exposures in South Africa. Poisoning episodes reported to the Poisons Information Helpline of the Western Cape, jointly run by the Poisons Information Centres at the Red Cross War Memorial Children's Hospital and Tygerberg Hospital over a period of approximately 2.5 years, from June 2015 to November 2017, were analysed to identify exposure patterns, severity and clinical outcomes. RESULTS: Alien plant species accounted for most cases (n=10) of reported poison exposures. Among the 26 recorded animal poisoning episodes, the dog was the most commonly implicated species (n=24), whereas just two enquiries were related to other animals (one rabbit and one cow). There were 20 plant cases and 6 mushroom cases (all dogs). There was only one fatal case involving cycad in a dog. CONCLUSION: Features of animal poisoning in South Africa were similar to those in other countries. The reported cases of animals exposed to poisonous plants and mushrooms could represent only a fraction of the actual exposures. Since most reported cases involved taxa that could not be identified to species level, more attention should be paid in case reporting and in animal poisoning prevention, engaging the public to enable people to recognise potentially hazardous plants and reduce the risk of poisoning in animals.

Beauvericin and Enniatins: In Vitro Intestinal Effects.

Food and feed contamination by emerging mycotoxins beauvericin and enniatins is a worldwide health problem and a matter of great concern nowadays, and data on their toxicological behavior are still scarce. As ingestion is the major route of exposure to mycotoxins in food and feed, the gastrointestinal tract represents the first barrier encountered by these natural contaminants and the first structure that could be affected by their potential detrimental effects. In order to perform a complete and reliable toxicological evaluation, this fundamental site cannot be disregarded. Several in vitro intestinal models able to recreate the different traits of the intestinal environment have been applied to investigate the various aspects related to the intestinal toxicity of emerging mycotoxins. This review aims to depict an overall and comprehensive representation of the in vitro intestinal effects of beauvericin and enniatins in humans from a species-specific perspective. Moreover, information on the occurrence in food and feed and notions on the regulatory aspects will be provided.

Suspected environmental poisoning by drugs, household products and pesticides in domestic animals.

Animal poisoning by chemicals (pesticides and household products) and drugs is a frequent occurrence and special attention should be paid to this phenomenon to improve prevention and treatment strategies and because of the fundamental role that animals may play as bioindicators. From January 2017 to March 2019 the Poison Control Centre of Milan (CAV) in collaboration with the University of Milan, collected and analyzed epidemiological data on animal poisoning. During this period, the CAV received a total of 442 enquiries on domestic animal poisoning episodes and, among these, 80.3 % were related to chemicals and drugs. Pesticides and drugs were the two major causes of poisoning (34.1 % and 33.5 %, respectively), followed by household products (29.3 %) and other causative agents (3.1 %, n = 11). In conclusion, these findings can provide useful information for the identification and monitoring of known and emerging toxicants, with positive repercussions on human, animal and environmental health.

Indoor Companion Animal Poisoning by Plants in Europe.

Indoor plant poisoning poses serious threats to companion animals. One of the major reasons of this kind of hazard can be identified in the increased amount of time that the pets spend indoor, sharing the domestic environment with their owners. In this review, the toxic houseplants most commonly associated with companion animal poisoning in Europe and well-documented in the literature are emphasized. An analysis of the major and emerging plant species accountable for companion animal poisoning is proposed, in order to provide a framework of the factors influencing these incidents. Indeed, knowing the way substances may induce toxic effects in companion animals can be useful in allowing easier diagnosis and treatment processes. In conclusion, the Authors argue that a better characterization of the phenomenon, as well as of its extent, would be allowed by the availability of a centralized system for the data collection. Furthermore, better information and awareness on the issue may help developing a focused corrective approach to prevent indoor pet poisoning in Europe.

Effect of relaxin on semen quality variables of cryopreserved stallion semen.

The aim of the study was to ascertain effects of different concentrations of relaxin added to extender medium during the pre-freezing incubation periods on quality variables of stallion frozen-thawed spermatozoa. Semen samples collected from three stallions were filtered, diluted with skim milk, and centrifuged at 600g for 10 min. Sperm pellets were suspended in BotuCrio freezing medium to a final concentration of 50 × 106 sperm/mL. The diluted semen was divided into five experimental groups supplemented with 0 (control), 12.5, 25, 50, or 100 ng/mL of relaxin. The semen samples were transferred into 0.5 mL straws, equilibrated at 5 °C for 30 min, and placed in liquid nitrogen (LN2) vapour for 15 min before being plunged into LN2. After thawing, sperm samples were evaluated for motility and velocity variables, mitochondrial membrane potential, apoptosis, and plasma membrane and DNA integrities. For sperm motility variables, there were dose- and time-dependent effects, with the largest values recorded when 12.5 and 25 ng/mL relaxin were used for 0-120 min of incubation. Furthermore, at all of the concentrations at which there were evaluations, relaxin additions to semen diluent led to a marked improvement in sperm mitochondrial membrane potential and a lesser percentage of apoptotic cells compared to the control group. Plasma membranes and DNA integrities were not affected by relaxin supplementations to the diluent. In conclusion, supplementation of relaxin in extender before semen cryopreservation, especially at 12.5 and 25 ng/mL, had a positive effect on the sperm quality variables.

Indoor poisoning of companion animals by chemicals.

Episodes of indoor exposure to toxic compounds in companion animals are frequent. Toxico-epidemiological data on this matter are fundamental not only to take proper corrective actions but especially because domestic animals represent crucial sentinels for environmental toxicants. In this review we will present an overview on indoor poisoning of companion animals by chemicals in Europe, providing information on trends and emphasizing emerging indoor contaminants. In general, pesticides are the most frequent class of toxicants involved in indoor animal poisoning episodes in Europe. Among them, insecticides (in particular anticholinesterase compounds) and anticoagulant rodenticides are frequently implicated, but molluscicides (i.e. metaldehyde) together with household products have also shown a growing or at least stable trend in the poisoning episodes. Considering the emerging molecules, neonicotinoids (imidacloprid and acetamiprid) and glyphosate formulations have been pointed out, whereas with regard to households, besides the well-known ethylene glycol, attention should be paid to emerging products such as sodium polyacrylate polymeric hydrogels, xylitol, glowsticks and liquid laundry detergent capsules. The environmental contamination by chemical households is a global challenge and bioindicators like pets may be valuable tools to perform a comprehensive monitoring and risk assessment.