Comparative Evaluation of the In Vitro Cytotoxicity of a Series of Chitosans and Chitooligosaccharides Water-Soluble at Physiological pH.

Chitosans (CS) have been of great interest due to their properties and numerous applications. However, CS have poor solubility in neutral and basic media, which limits their use in these conditions. In contrast, chitooligosaccharides (COS) have better solubility in water and lower viscosity in aqueous solutions whilst maintaining interesting biological properties. CS and COS, unlike other sugars, are not single polymers with a defined structure but are groups of molecules with modifiable structural parameters, allowing the adaptation and optimization of their properties. The great versatility of CS and COS makes these molecules very attractive for different applications, such as cryopreservation. Here, we investigated the effect of the degree of polymerization (DP), degree of N-acetylation (DA) and concentration of a series of synthesized CS and COS, water-soluble at physiological pH, on their cytotoxicity in an L929 fibroblast cell culture. Our results demonstrated that CS and COS showed no sign of toxicity regarding cell viability at low concentrations (≤10 mg/mL), independently of their DP and DA, whereas a compromising effect on cell viability was observed at a high concentration (100 mg/mL).

Effect of DHA on the quality of In vitro produced bovine embryos.

Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid (PUFA) that improves fertility by increasing membrane fluidity. Moreover, embryos produced by donor females supplied with n-3 PUFA did not show any difference in terms of the lipid profile after 7 days of culture. The present study aimed to investigate the effects of DHA (20 and 100 µM) coupled with carnosine (5 mg/mL), an antioxidant, during oocyte maturation and embryo development on the developmental and cryosurvival rates and the number of pluripotent cells. Free fatty acid receptor-4 (FFAR4), which is able to bind DHA, was visualised by immunostaining. The addition of DHA in the in vitro development (IVD) medium decreased the percentage of pluripotent SOX2 positive cells compared with the control (8.4% vs. 10.9%) without affecting the number of cells (196.7 vs. 191.6 cells) or the developmental (20.9% vs. 23.9% blastocysts rate on D7) and cryosurvival rates (86.3% vs 86.2%). Such a decrease in pluripotent cells, relevant to the differentiation of the first lineage within the inner cell mass, represents an improvement in the embryo quality. On the contrary, embryos without any pluripotent SOX2-positive cells would not be able to achieve gestation. Future studies should follow up these results by carrying out embryo transfers to assess the beneficial effects of DHA supplementation.

Effects of the donor factors and freezing protocols on the bovine embryonic lipid profile†.

Embryo lipid profile is affected by in vitro culture conditions that lead to an increase in lipids. Efforts have been made to optimize embryo lipid composition as it is associated with their quality. The objective of this study was to evaluate whether the diet supplementation of donor cows (n-3 or n-6 polyunsaturated fatty acids), or the slow freezing protocols (ethylene glycol sucrose vs. glycerol-trehalose), or the physiological stage of the donor (nulliparous heifers vs. primiparous lactating cows) may impact the bovine embryo lipid profile. Lipid extracts of 97 embryos were individually analyzed by liquid chromatography-high resolution mass spectrometry, highlighting 246 lipids, including 85% being overabundant in cow embryos compared to heifer embryos. Among 105 differential lipids, 72 were overabundant after ethylene glycol sucrose protocol, including a single glycerophosphate PA(32:1) representing 27.3% of the significantly modulated lipids, suggesting that it is degraded when glycerol-trehalose protocol is used. No lipids were different according to the n-3 or n-6 supplementation of the donor cows. In conclusion, the embryonic lipid profile was mainly affected by the physiological stage of the donors and the slow freezing protocols. The overabundance of lipids in lactating cow embryos and the resulting lower quality of these embryos are consistent with the lower pregnancy rate observed in cows compared to heifers. Unlike glycerol-trehalose protocol, ethylene glycol sucrose freezing allowed to preserve glycerophospholipids, potentially improving the slow freezing of in vitro-produced embryos. Further studies are required to modulate embryo quality and freezability by modulating the lipidome and by integrating all stages of embryonic production.

Lipid profile of bovine grade-1 blastocysts produced either in vivo or in vitro before and after slow freezing process.

Currently, in vitro embryo production (IVP) is successfully commercially applied in cattle. However, the high sensitivity of embryos to cryopreservation in comparison to in vivo (IVD) embryos slows the dissemination of this biotechnology. Reduced cryotolerance is frequently associated with lipid accumulation in the cytoplasm mainly due to in vitro culture conditions. The objective of this study was to evaluate the lipid composition of biopsied and sexed embryos, produced either in vivo or in vitro from the same Holstein heifers before and after a slow freezing protocol. Lipid extracts were analysed by liquid chromatography-high resolution mass spectrometry, which enabled the detection of 496 features. Our results highlighted a lipid enrichment of IVP embryos in triglycerides and oxidised glycerophospholipids and a reduced abundance in glycerophospholipids. The slow freezing process affected the lipid profiles of IVP and IVD embryos similarly. Lysophosphatidylcholine content was reduced when embryos were frozen/thawed. In conclusion, the embryonic lipid profile is impacted by IVP and slow freezing protocols but not by sex. Lysophosphatidylcholine seemed highly sensitive to cryopreservation and might contribute to explain the lower quality of frozen embryos. Further studies are required to improve embryo freezability by modulating the lipidome.

Insights into Species Preservation: Cryobanking of Rabbit Somatic and Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) are obtained by genetically reprogramming adult somatic cells via the overexpression of specific pluripotent genes. The resulting cells possess the same differentiation properties as blastocyst-stage embryonic stem cells (ESCs) and can be used to produce new individuals by embryonic complementation, nuclear transfer cloning, or in vitro fertilization after differentiation into male or female gametes. Therefore, iPSCs are highly valuable for preserving biodiversity and, together with somatic cells, can enlarge the pool of reproductive samples for cryobanking. In this study, we subjected rabbit iPSCs (rbiPSCs) and rabbit ear tissues to several cryopreservation conditions with the aim of defining safe and non-toxic slow-freezing protocols. We compared a commercial synthetic medium (STEM ALPHA.CRYO3) with a biological medium based on fetal bovine serum (FBS) together with low (0-5%) and high (10%) concentrations of dimethyl sulfoxide (DMSO). Our data demonstrated the efficacy of a CRYO3-based medium containing 4% DMSO for the cryopreservation of skin tissues and rbiPSCs. Specifically, this medium provided similar or even better biological results than the commonly used freezing medium composed of FBS and 10% DMSO. The results of this study therefore represent an encouraging first step towards the use of iPSCs for species preservation.

French veterinarians' opinions and practices regarding early neutering of cats: a convenience sampling survey interpreted in an international context.

BACKGROUND: Although data are still needed, early neutering of cats appears to be as safe as neutering at the traditional age of six months or older and beneficial to the individual and the population. The aim of this observational study was to obtain an overview of veterinarians' opinions and practices about feline early neutering (ie, until the age of four months). METHODS: In this retrospective work, a web survey was distributed to French practitioners. RESULTS: A total of 609 veterinarians returned the survey. Majority of the veterinarians (56 per cent) reported never performing early neutering, 42 per cent reported performing it inconsistently, and 2 per cent reported consistently performing it. When carried out, it was mainly on kittens from commercial breeding, following breeders' request, and performed at the age of three months. An overwhelming majority (93 per cent) of veterinarians performing early neutering reported no incidents. Veterinarians who did not practise early neutering neutered mostly (81 per cent) kittens from four to six months of age. CONCLUSION: In conclusion, early neutering is still not a common practice among French veterinarians. Opinions differed as to its advantages and disadvantages, although reported incidents were scarce. Veterinarians who do not perform early neutering reported a lack of interest in this practice rather than reluctance.

Assessment of Optimized Frozen/Thawed Semen Samples in Canines with the New A-Kinase Anchor Protein 4 Precursor Biomarker.

Optimizing thawing conditions are required to maximize recovery of semen capacities after cryopreservation. This study aimed to assess the effect of thawing procedures on motility recovery and maintenance of semen capacities over time. A fractional factorial design approach was used to reduce the number of repetitions and simultaneously analyze the different interactions. Thirty canine frozen semen samples were thawed under different thawing conditions. Motility and velocity parameters were recorded using a computer-assisted semen analyzer up to 6 hours after thawing. Ten quadratic models were found to be significant, with the most significant effects observed on the velocity parameters. Second, this study allowed us to evaluate the effect of the proAKAP4 marker on frozen/thawed semen, with regard to motility recovery. ProAKAP4 is the precursor of A-kinase anchor protein 4. It has been studied in several species as a marker to assess sperm quality. In our study, the expression of proAKAP4 was determined using flow cytometry. No correlation was found between post-thaw motility parameters and proAKAP4 levels. However, the effects of thawing temperature, incubation time, and straw size were significant and similar to those observed for velocity parameters.

Prenatal Diagnosis of Megacystitis and Hydronephrosis in an Equine Fetus: How Transabdominal Ultrasonography Can Help to Diagnose Fetal Abnormalities.

Premature udder development constitutes an alarm signal in pregnant mares. The objective of this clinical case report is to highlight the importance of transabdominal ultrasound examination of the fetus in these cases and to report a unique case of prenatal diagnosis of obstructive uropathy based on the observation of severe fetal hydronephrosis and megacystitis in utero. A 4-year-old French chaser primiparous mare was referred for evaluation of premature udder development during the ninth month of pregnancy. The mare had clinical signs within normal limits, a developed and sensitive udder with secretions, and no vulvar discharge. Transrectal examination revealed the presence of an immobile fetus. Combined uteroplacental thickness was within normal limits. Transabdominal ultrasound revealed a single live fetus in posterior presentation with several abdominal abnormalities. Unilateral hydronephrosis and megacystitis lead to a hypothetical diagnosis of fetal multiple urinary tract malformation with outflow obstruction. Treatment was discontinued and the mare was monitored. Abortion occurred spontaneously a week later. Postmortem examination revealed a ruptured bladder of abnormally large dimensions and a severely distended left kidney without parenchyma (filled with free urine) and lack of permeability in the left ureter and urethra. Postmortem diagnosis was consistent with our prenatal ultrasonographic diagnosis. Even though described during human pregnancy with various etiologies and severity, prenatal diagnosis of fetal hydronephrosis and megacystitis has not been reported in equine veterinary medicine before. These malformations need to be characterized more precisely in the future. This case highlights the importance of transabdominal ultrasonography to detect equine fetal abnormalities.

A retrospective internet-based survey of French cat breeders about early-age neutering.

OBJECTIVES: The aim of this study was to analyse cat breeders' application of and opinions concerning early-age (ie, <16 weeks old) neutering in cats in France. METHODS: This study analysed a retrospective cohort. A web survey was designed for French cat breeders and was published in June 2017 on the Facebook page of Livre Officiel des Origines Félines, the official feline studbook for purebred cats born in France. The early-age cat neutering habits, opinions and expectations of breeders were collected. RESULTS: A total of 999 breeders returned the questionnaire. Almost half of the breeders (49%) reported consistently requesting neutering of kittens before selling them, 32% claimed that they never requested it and the remaining 19% reported that they inconsistently requested it. When performed, 83% of breeders had kittens neutered at 12 weeks of age; for a large majority of these breeders (94%), the neutering was undertaken on their own initiative. Various reasons for neutering were provided, including the prevention of uncontrolled breeding, short- and long-term welfare benefits for neutered cats, and practical and economic reasons. Reported incidents related to early-age neutering were scarce. Nearly half of breeders who did not apply early-age neutering to their kittens cited a veterinarian's unwillingness to perform the surgery as a cause. CONCLUSIONS AND RELEVANCE: A large majority of surveyed French cat breeders supported early-age neutering that would occur before kittens were sold, most of the time at the age of 3 months. These breeders reported high satisfaction with early-age neutered cats, with a low number of incidents and accidents reported.

Comparison Between an Animal-Derived Product Medium and a Chemically Defined Medium for Ram Sperm Cryopreservation.

Animal-derived products are widely used in sperm cryopreservation for their cryoprotective properties. These components, however, tend to be replaced because of sanitary risks. STEMALPHA.CRYO3 (Ref. 5617; Stem Alpha, Saint-Genis-l'Argentière, France), called "CRYO3," is a chemically defined preservation medium currently used for freezing human tissue and adult stem cells. The aim of this study was to evaluate the effect of a CRYO3-based medium on ram sperm freezing regarding in vitro parameters and in vivo fertility. Semen from nine Charolais rams was collected using an artificial vagina, then split and frozen using two media: a CRYO3-based medium or a control medium containing egg yolk (10%) and milk (45%). Sperm membrane integrity (propidium iodide [PI]/SYBR-14 and calcein AM/ethidium homodimer-1), acrosome integrity (FITC-PNA/PI), and mitochondrial membrane potential (JC-1) were assessed using flow cytometry, while functional membrane integrity was assessed using a hypo-osmotic swelling test and motility parameters, evaluated by computer-assisted sperm analysis. Pregnancy rates, prolificacy, and the average daily weight gain (DWG) of lambs were evaluated after performing 195 laparoscopic inseminations. The control medium showed significantly higher results than CRYO-based medium for all in vitro parameters, except for linearity and straightness (motions parameters). Conversely, field trials showed no significant difference between the control medium and the CRYO3-based medium for pregnancy rates (72.2% and 67.9%, respectively), prolificacy (1.8 and 1.6, respectively), and the DWG (0.34 and 0.35 kg/d, respectively). This preliminary study showed that CRYO3 cannot replace egg yolk and milk in freezing extenders for commercial purposes. However, as laparoscopic inseminations allowed a 67% pregnancy rate, CRYO3-based medium remains an option for international transport or long-term storage of genetic diversity.

Rapid cooling of rabbit embryos in a synthetic medium.

Embryo cryopreservation media usually contain animal-derived products, such as bovine serum albumin (BSA). These products present two major disadvantages: an undefined variable composition and a risk of pathogen transmission. We aimed to evaluate the effect of replacing BSA in rabbit embryo rapid cooling "freezing" and warming media with a chemically defined medium with no animal-derived products: STEM ALPHA. Cryo3 ("Cryo3"). A total of 1540 rabbit morulae were divided into three cryopreservation groups (group 1: BSA, group 2: 20% Cryo3 and group 3: 100% Cryo3) and a fresh controls group. After rapid cooling, embryos were cultured (in vitro approach), or transferred into synchronized does (in vivo approach). In the in vitro approach, post-warm survival rates obtained with 100% Cryo3 (94.9%) were superior to BSA (90.8%) and 20% Cryo3 (85.6%). The blastocyst formation rate was similar between BSA, 20% Cryo3 and 100% Cryo3 groups (85.1, 77.9 and 83.3%, respectively), as was the expansion/hatching rate (63.1, 63.4 and 58.0%, respectively) and embryo mitochondrial activity. In the in vivo approach, pregnancy (80.0, 68.0 and 95.2%, respectively), implantation (40.5, 45.9 and 44.8%, respectively), and live-foetus rates (35.6, 35.5 and 38.1%, respectively) were similar between the three groups. To conclude, Cryo3 can replace BSA in rabbit embryo rapid cooling "freezing" and warming media.

Milk Fat Globules Hamper Adhesion of Enterohemorrhagic Escherichia coli to Enterocytes: In Vitro and in Vivo Evidence.

Enterohemorrhagic Escherichia coli (EHEC; E. coli) are food-borne agents associated with gastroenteritis, enterocolitis, bloody diarrhea and the hemolytic-uremic syndrome (HUS). Bovine milk glycans have been shown to contain oligosaccharides which are similar to host epithelial cell receptors and can therefore prevent bacterial adhesion. This study aimed to describe interactions between EHEC O157:H7 EDL933 and O26:H11 21765 and milk fat globules (MFGs) in raw milk and raw milk cheese, and the impact of MFGs on EHEC strains adhesion to the intestinal tract in vitro and in vivo. Both EHEC serotypes clearly associated with native bovine MFGs and significantly limited their adhesion to a co-culture of intestinal cells. The presence of MFGs in raw milk cheese had two effects on the adhesion of both EHEC serotypes to the intestinal tracts of streptomycin-treated mice. First, it delayed and reduced EHEC excretion in mouse feces for both strains. Second, the prime implantation site for both EHEC strains was 6 cm more proximal in the intestinal tracts of mice fed with contaminated cheese containing less than 5% of fat than in those fed with contaminated cheese containing 40% of fat. Feeding mice with 40% fat cheese reduced the intestinal surface contaminated with EHEC and may therefore decrease severity of illness.

Ice nucleating agents allow embryo freezing without manual seeding.

Embryo slow freezing protocols include a nucleation induction step called manual seeding. This step is time consuming, manipulator dependent and hard to standardize. It requires access to samples, which is not always possible within the configuration of systems, such as differential scanning calorimeters or cryomicroscopes. Ice nucleation can be induced by other methods, e.g., by the use of ice nucleating agents. Snomax is a commercial preparation of inactivated proteins extracted from Pseudomonas syringae. The aim of our study was to investigate if Snomax can be an alternative to manual seeding in the slow freezing of mouse embryos. The influence of Snomax on the pH and osmolality of the freezing medium was evaluated. In vitro development (blastocyst formation and hatching rates) of fresh embryos exposed to Snomax and embryo cryopreserved with and without Snomax was assessed. The mitochondrial activity of frozen-thawed blastocysts was assessed by JC-1 fluorescent staining. Snomax didn't alter the physicochemical properties of the freezing medium, and did not affect embryo development of fresh embryos. After cryopreservation, the substitution of manual seeding by the ice nucleating agent (INA) Snomax did not affect embryo development or embryo mitochondrial activity. In conclusion, Snomax seems to be an effective ice nucleating agent for the slow freezing of mouse embryos. Snomax can also be a valuable alternative to manual seeding in research protocols in which manual seeding cannot be performed (i.e., differential scanning calorimetry and cryomicroscopy).

Lymphodepletive effects of rabbit anti-pig thymocyte globulin in neonatal swines.

Lymphodepletive agents play important role in different clinical applications or experimental transplant studies. In order to facilitate preclinical pediatric transplant studies, we have developed the rabbit anti-pig thymocyte globulin (pATG) and studied its effects in neonatal swines. In vitro assays showed that pATG can bind to lymphocytes and neutrophils in a dose-dependent manner and lyse peripheral blood mononuclear cells by apoptosis and complement-dependent cytotoxicity. In vivo, pATG as a monotherapy was administered at different doses (2.5, 5, 20, 40 and 80mg/kg) in newborn pigs. Results showed that pATG induced a dose-dependent but transient T-cell depletion in peripheral blood. Lymphodepletion was also observed in lymph nodes, spleen and thymus. Pharmacokinetic studies showed dose-related cell-bound pATG on lymphocytes, as well as the presence of free pATG in the serum. Both cell-bound and free pATG levels decreased gradually after administration. Interestingly, adjuvant mycophenolate mofetil (MMF) given at 1g/m2/day for 1week successfully maintained pATG-induced T-cell depletion. In conclusion, pATG administration can cause transient T-cell depletion in neonatal pigs and this effect can be maintained by MMF. Therefore, we have developed an original immunosuppressive regimen that can be used for transplantation studies in swine model.

Genomic Rearrangements and Functional Diversification of lecA and lecB Lectin-Coding Regions Impacting the Efficacy of Glycomimetics Directed against Pseudomonas aeruginosa.

LecA and LecB tetrameric lectins take part in oligosaccharide-mediated adhesion-processes of Pseudomonas aeruginosa. Glycomimetics have been designed to block these interactions. The great versatility of P. aeruginosa suggests that the range of application of these glycomimetics could be restricted to genotypes with particular lectin types. The likelihood of having genomic and genetic changes impacting LecA and LecB interactions with glycomimetics such as galactosylated and fucosylated calix[4]arene was investigated over a collection of strains from the main clades of P. aeruginosa. Lectin types were defined, and their ligand specificities were inferred. These analyses showed a loss of lecA among the PA7 clade. Genomic changes impacting lec loci were thus assessed using strains of this clade, and by making comparisons with the PAO1 genome. The lecA regions were found challenged by phage attacks and PAGI-2 (genomic island) integrations. A prophage was linked to the loss of lecA. The lecB regions were found less impacted by such rearrangements but greater lecB than lecA genetic divergences were recorded. Sixteen combinations of LecA and LecB types were observed. Amino acid variations were mapped on PAO1 crystal structures. Most significant changes were observed on LecBPA7, and found close to the fucose binding site. Glycan array analyses were performed with purified LecBPA7. LecBPA7 was found less specific for fucosylated oligosaccharides than LecBPAO1, with a preference for H type 2 rather than type 1, and Lewis(a) rather than Lewis(x). Comparison of the crystal structures of LecBPA7 and LecBPAO1 in complex with Lewis(a) showed these changes in specificity to have resulted from a modification of the water network between the lectin, galactose and GlcNAc residues. Incidence of these modifications on the interactions with calix[4]arene glycomimetics at the cell level was investigated. An aggregation test was used to establish the efficacy of these ligands. Great variations in the responses were observed. Glycomimetics directed against LecB yielded the highest numbers of aggregates for strains from all clades. The use of a PAO1ΔlecB strain confirmed a role of LecB in this aggregation phenotype. Fucosylated calix[4]arene showed the greatest potential for a use in the prevention of P. aeruginosa infections.

Canine placenta: A promising potential source of highly proliferative and immunomodulatory mesenchymal stromal cells?

In veterinary medicine, therapeutic mesenchymal stromal cells (MSC) have been traditionally isolated from adult bone marrow or adipose tissue. Neonatal tissues, normally discarded at birth from all species have become an alternative source of cells for regenerative medicine in the human clinic. These cells have been described as being more primitive, proliferative and immunosuppressive than their adult counterparts. Our objective was to examine if this phenomena holds true in dogs. Little information exists regarding canine neonatal MSC characterisation. In this study, we were able to both isolate, phenotype and assess the differentiation and immunomodulatory properties of MSC from canine foetal adnexa allowing us to compare their characteristics to their more well-known bone marrow (BM) cousins. Neonatal tissues, including amnion (AM), placenta (PL), and umbilical cord matrix (UCM) were collected from 6 canine caesarean sections. Primary cells were expanded in vitro for 5 consecutive passages and their proliferation measured. BM-MSC were isolated from 5 control dogs euthanised from other studies and grown in vitro using an identical protocol. All MSC lines were systematically evaluated for their ability to differentiate into 3 mesodermal lineages (adipocyte, osteocyte and chondrocyte) and phenotyped by cytometry and qPCR. In addition, the enzymatic activity of the key immunomodulatory marker indoleamine 2,3-dioxygenase (IDO) was evaluated for each MSC line. MSC displaying a fibroblastic appearance were successfully grown from all neonatal tissues. PL-MSC exhibited significantly higher proliferation rates than AM- and UCM-MSC (p=0.05). Cytometric analysis showed that all MSC express CD90, CD29, and CD44, while no expression of CD45, CD34 and MHC2 was detected. Molecular profiling showed expression of CD105 and CD73 in all MSC. Low levels of SOX2 mRNA was observed in all MSC, while neither NANOG, nor OCT4 were detected. All MSC differentiate into 3 mesodermal lineages. Following inflammatory stimulation, the activity of the immunomodulatory enzyme IDO was significantly higher in neonatal MSC compared to BM-MSC (p=0.009). Our results show that canine foetal adnexa cells share very similar properties to their adult equivalents but upon stimulation show significantly higher IDO immunomodulatory activity. Further studies will be needed to confirm the potential therapeutic benefits of these cells.

Alfaxalone for total intravenous anaesthesia in bitches undergoing elective caesarean section and its effects on puppies: a randomized clinical trial.

OBJECTIVE: To evaluate the effects and reliability of alfaxalone constant rate infusion (CRI) in comparison to isoflurane to maintain anaesthesia in bitches undergoing elective caesarean section. STUDY DESIGN: Prospective, randomized, 'blinded' clinical trial. ANIMALS: Twenty-two client-owned bitches and 94 puppies. METHODS: Bitches were randomly assigned to receive an alfaxalone CRI [0.2 mg kg(-1)  minute(-1) intravenously (IV), and once the last puppy was delivered, the dose was halved; n = 11] or 2% (vaporizer dial setting) isoflurane (n = 11) for maintenance of anaesthesia. All dogs were induced with alfaxalone (3 mg kg(-1) ) IV. Additional alfaxalone (0.3 mg kg(-1) IV) was administered if the depth of anaesthesia was inadequate and the total dose was calculated. Bitches were mechanically ventilated. Analgesia was administered after the delivery of puppies. Physiological variables were recorded every 5 minutes. The bitches' recovery times were also recorded. Quality of induction and recovery were evaluated. Puppies' vigour was evaluated with a modified Apgar score at 5 and 60 minutes after birth. Puppies' survival rates at 24 and 48 hours and at 15 days were recorded. Data were analysed using an anova, Student's t-test or Wilcoxon rank-sum test. RESULTS: The rescue dose of alfaxalone was higher (p = 0.01); bitches' recoveries were longer (p < 0.001) and puppies' Apgar scores were significantly lower at 5 and 60 minutes (p < 0.001 and p = 0.003, respectively) with alfaxalone than with isoflurane. However, no significant differences were found for puppies' survival between groups. CONCLUSIONS AND CLINICAL RELEVANCE: Alfaxalone CRI seems to be a possible protocol for puppies and bitches undergoing elective caesarean sections. However, bitches recovered more slowly and puppy Apgar scores were lower in comparison to isoflurane.

Oral cyclosporine A in neonatal swines for transplantation studies.

The purpose of this study is to define the optimal dose of oral cyclosporine A (CsA) microemulsion in newborn swine for transplantation studies and to describe its pharmacokinetics and acute renal effects in short-term administration. Thirteen neonatal pigs were randomized into four groups: one control and three groups with CsA administration at 4, 8 and 12 mg/kg/d for 15 days (D). Blood samples were collected on D 0, 2, 4, 9 and 14 to determine the changes of the CsA trough concentrations, the creatinine (Cr) and blood urea nitrogen (BUN) serum concentrations. On D 14, blood samples were collected every hour from 1 h to 10 h after CsA administration to determine the area under the curve (AUC). On D 15, kidneys were removed for histological analysis. We observed a stabilization of CsA trough concentrations from D 4 to D 14. On D 14, in the three treated groups, CsA trough concentrations were 687 ± 7, 1200 ± 77 and 2211 ± 1030 ng/ml, respectively; AUC (0-10 h) were 6721 ± 51 ng·h/ml in group 4 mg/kg/d, 13431 ± 988 ng·h/ml in group 8 mg/kg/d and 28264 ± 9430 ng·h/ml in group 12 mg/kg/d. Cr concentrations were not significantly different among the four groups; but compared to control group, BUN concentrations of the three treated groups increased significantly. CsA was well tolerated; neither acute, severe adverse event nor renal histological abnormality was observed. In conclusion, a 15-d course of oral CsA treatment ranged from 4 to 12 mg/kg/d is safe for newborn pigs, which need much lower CsA dose than adult pigs to reach comparable trough level and AUC. As immunosuppressive therapy in newborn pigs, we recommend a CsA dose of 4 mg/kg/d to achieve a trough blood concentration between 400 and 800 ng/ml.

Electroejaculation and semen buffer evaluation in the microbat Carollia perspicillata.

Scientific interests and conservation needs currently stress the necessity to better understanding bat reproductive biology. In this study, we present the first, safe, inexpensive, and reliable method to obtain sperm from a microbat species (Carollia perspicillata) by electroejaculation. This method revealed to be highly efficient (100% success rate). We obtained ejaculates composed of two characteristically different fractions. We compared three buffers and recommend using an Earle's balanced salt solution as a semen extender. Earle's balanced salt solution provided significant repeatable measure of swimming ability (intraclass correlation coefficient: 0.74, P < 0.01) and proportion of motile sperms (intraclass correlation coefficient: 0.08, P = 0.01) and allowed sperm to maintain optimal swimming capacity over time. None of the buffers could dissolve all the coagulated sperm. Although the trypsin buffer freed a larger fraction of spermatozoa in the ejaculate, it impaired swimming ability without improving motility, viability, and stamina. We thus argue that the sperm population analyzed with Earle's balanced salt solution is a representative of the ejaculate. Finally, we found that the mean sperm velocity of C perspicillata (78.8 µm/s) is lower than that predicted by regressing sperm velocity on relative testes mass, a proxy of sperm competition. The question as to whether C perspicillata is an outsider for sperm velocity, or whether bats evolved yet another unique mechanism to cope with sperm competition deserves more investigations.

A chemically defined medium for rabbit embryo cryopreservation.

This study evaluates a new synthetic substitute (CRYO3, Ref. 5617, Stem Alpha, France) for animal-based products in rabbit embryo cryopreservation solutions. This evaluation was performed using two approaches: a thermodynamic approach using differential scanning calorimetry and a biological approach using rabbit embryo slow-freezing. During the experiment, foetal calf serum (FCS) was used as a reference. Because FCS varies widely by supplier, three different FCS were selected for the thermodynamic approach. The rabbit embryo slow-freezing solutions were made from Dulbecco's phosphate buffer saline containing 1.5 M Dimethyl Sulfoxide and 18% (v.v(-1)) of CRYO3 or 18% (v.v(-1)) of FCS. These solutions were evaluated using four characteristics: the end of melting temperature, the enthalpy of crystallisation (thermodynamic approach) and the embryo survival rates after culture and embryo transfer (biological approach). In the thermodynamic approach, the solutions containing one of the three different FCS had similar mean thermodynamic characteristics but had different variabilities in the overall data with aberrant values. The solution containing CRYO3 had similar thermodynamic properties when compared to those containing FCS. Moreover, no aberrant value was measured in the solution containing CRYO3. This solution appears to be more stable than the solutions containing a FCS. In the biological approach, the in vitro embryo survival rates obtained with the solution containing CRYO3 (73.7% and 81.3%) and with the solution containing a FCS (77.6% and 71.9%) were similar (p = 0.7). Nevertheless, during the in vivo evaluation, the implantation rate (21.8%) and the live-foetuses rate (18.8%) of the CRYO3 group were significantly higher than the implantation rate (7.1%, p = 0.0002) and the live-foetuses rate (5.3%, p = 0.0002) of the FCS group. The pregnancy rate was also higher in the CRYO3 group compared to the FCS group (81.3% and 43.8%, respectively, p = 0.066). We conclude that CRYO3 can be used as a chemically defined substitute for animal-based products in rabbit embryo cryopreservation solutions.