Chantal Thorin

Bull semen in vitro fertility after cryopreservation using egg yolk LDL: a comparison with Optidyl®, a commercial egg yolk extender

Low-density lipoproteins (LDL) have been previously isolated and identified as the cryoprotective fraction of yolk. The effect of LDL on sperm motility after freezing-thawing has been reported, but no study has been made to assess the effect of LDL on bull semen fertility. The aim of this study was to evaluate the fertility of bull semen cryopreserved in the presence of LDL. Motility of semen cryopreserved in LDL was analyzed and compared to semen cryopreserved with Optidyl, a commercial extender containing egg yolk. To evaluate the fertilizing ability of semen, we used in vitro fertilization test, whereas acrosome and plasma membrane integrity were also evaluated. The percentage of motile spermatozoa was two fold higher after freezing in LDL than in Optidyl 54.4% versus 30.2% (P < 0.05). The cleavage rate was significantly higher after fertilization with semen frozen in LDL than with Optidyl 63.0% versus 54.8% (P < 0.05). No significant difference was observed on the blastocyst rate after in vitro culture. Integrity of the acrosome and the plasma membrane were maintained in both extenders. In conclusion, LDL preserve bull semen quality and fertilizing ability, allowing also better semen motility, after the freeze-thaw process.

In vivo fertility of bull semen following cryopreservation with an LDL (low density lipoprotein) extender: Preliminary results of artificial inseminations

A semen extender made with low density lipoproteins (LDL) has been used instead of a standard extender that is already available on the market for the cryopreservation of bovine semen. However, in order to extend its use to artificial insemination centres, in vivo fertility studies were required. Semen was taken from three bulls and frozen-thawed in two extenders: the LDL extender and a standard Tris-egg-yolk (20%) extender used by AI centres. The quality of the semen was assessed prior to artificial insemination: motility was assessed using an image analyser (Computer Assisted Semen Analysis (Hamilton Thorne)), and the integrity of the plasma membrane was assessed using the hypo-osmotic test (HOS test). For the first time, gestations were obtained following the artificial insemination of cows in the field (n=193) with semen that had been frozen-thawed in the LDL extender. No significant difference (p>0.05) was detected between the success rates of AI between the semen that had been frozen-thawed in the LDL extender (59.2%) and the control extender, Tris-20% egg yolk (65.3%). In conclusion, the in vivo fertility of semen that has been frozen-thawed in the LDL extender is maintained since gestations are obtained following AI.

Assessment of Postoperative Pain in Cats After Ovariectomy by Laparoscopy, Median Celiotomy, or Flank Laparotomy

OBJECTIVEnTo compare postoperative pain, duration of surgery, and duration of anesthesia for 3 methods of ovariectomy in cats: (1) conventional ventral median open approach (Midline), (2) right flank approach (Flank), and (3) median 2-portal laparoscopic procedure (Lap).nnnSTUDY DESIGNnRandomized, prospective clinical trial.nnnANIMALSnHealthy, sexually intact female cats (nu2009=u200960).nnnMETHODSnCats were randomly assigned to 1 of 3 groups: Midline (nu2009=u200920), Flank (20), and Lap (20) were evaluated 1, 2, 4, 6, and 12u2009hours after endotracheal extubation. Postoperative pain was scored using the 4A-vet pain scale that combines a subjective numerical pain rating and objective scoring of physiologic and behavioral variables including the response to stimulation of the surgical site. Pain scores (PS) were compared between groups.nnnRESULTSnThere was a significant difference in the PS between groups. PS for Midline and Flank were not significantly different but were both significantly higher compared with Lap. Depending on time, 5-20% of the cats had intense postoperative pain in both Midline and Flank groups. None of the Lap cats had intense postoperative pain.nnnCONCLUSIONSnLaparoscopic ovariectomy, although slower, appeared less painful compared with conventional ventral midline and flank ovariectomy. Postoperative pain did not differ significantly between midline and flank groups.

Effect of low-density lipoproteins, spermatozoa concentration and glycerol on functional and motility parameters of bull spermatozoa during storage at 4 °C

An extender has been developed with low-density lipoproteins (LDLs) that eliminates the microbial risks associated with the use of whole egg yolk. The objective of this study was to assess the effects of substituting egg yolk with LDLs for use as an extender in sperm preservation at 4 °C, as well as on spermatozoa motility, plasma membrane and acrosome integrity, at two different concentrations (80×10(6) and 240×10(6) sperm per ml) for 8 days and to evaluate glycerol toxicity in both extenders. A total of 12 ejaculates were collected from three bulls. Spermatozoa motility was examined using computer-assisted semen analysis. Plasma membrane integrity was determined using the hypo-osmotic swelling test and acrosome integrity with the fluorescein isothiocyanate-Pisum sativum agglutinin test. The semen was subsequently divided into four aliquots and diluted with Tris-egg yolk-glycerol (TEG), Tris-egg yolk without glycerol (TE), LDL with glycerol (LDL(+)) and LDL without glycerol (LDL(-)), at 80×10(6) and 240×10(6) sperm per ml. This study showed that the LDL(+) and LDL(-) extenders were more effective at preserving spermatozoa motility, plasma membrane integrity and acrosome integrity than TEG and TE (P<0.05) during 8 days of incubation. After 3 days of incubation, a toxicity of glycerol was observed in TEG, whereas no significant difference was observed between LDL(+) and LDL(-). We can therefore conclude that the LDL extender can be used to refrigerate semen at 4 °C instead of TEG and TE at 80×10(6) and 240×10(6) sperm per ml for elite bulls. This finding can be used to define a policy for the storage of high-quality bull semen.

Evaluation of the presence of equine viral herpesvirus 1 (EHV-1) and equine viral herpesvirus 4 (EHV-4) DNA in stallion semen using polymerase chain reaction (PCR)

In the horse, the risk of excretion of two major equine pathogens (equine herpesvirus types 1 (EHV-1) and 4 (EHV-4)) in semen is unknown. The objective of our study was to assess the possible risks for the horizontal transmission of equine rhinopneumonitis herpesviruses via the semen and the effect of the viruses on stallion fertility. Samples of stallion semen (n=390) were gathered from several different sources. Examination of the semen involved the detection of viral DNA using specific PCR. The mean fertility of the stallions whose sperm tested positive for viral DNA and the mean fertility of stallions whose sperm did not contain viral DNA, were compared using the Students t-test. EHV-4 viral DNA was not detected in any of the semen samples. EHV-1 DNA was identified in 51 of the 390 samples, (13%). One hundred and eighty-two samples came from 6 studs and there was significant difference (p<0.05) among the proportion of stallions whose semen tested positive for viral DNA from 0 to 55% between the studs. There was a significant difference (p<0.014) between the fertility of stallions whose semen tested positive for viral DNA and those whose semen was free from viral DNA. The stallions that excreted the EHV-1 virus in their semen appeared to be more fertile than the non-excretors, but this difference was in fact related to the breeding technique since higher proportion of excretors were found among those whose semen was used fresh rather than preserved by cooling or freezing. In conclusion, this study suggests that the EHV-1 virus may be transmitted via the semen at mating or by artificial insemination as demonstrated with other herpes viruses in other species.

In vitro bovine embryo production in a synthetic medium: Embryo development, cryosurvival, and establishment of pregnancy

The aim of this study was to develop an in vitro embryo culture medium without either fetal calf serum or BSA, using various growth factors and cytokines (GFs-CYKs; IGF-I, IGF-II, bFGF, LIF, GM-CSF, TGF-β1, and PDGF-BB), and other molecules with surfactant and embryotrophic properties, such as recombinant albumin (RA) and hyaluronan (HA). The first part of the study was dedicated to define the best combination of GFs-CYKs + RA + HA for optimal embryonic development. Next, we compared development rates and embryo quality (inner cell mass [ICM]-to-total cell number [TCN] ratio), and postthaw survival and hatching rates using this synthetic medium (T1) and a control medium: synthetic oviduct fluid + BSA + ITS (insulin, transferrin, and selenium). The blastocyst rates were significantly higher with T1 than those with the control at 7 and 8 days after fertilization. There was no significant difference in TCN or the ICM/TCN ratio between the two treatments. Survival and hatching rates 48 hours after thawing were similar for both treatments. Finally, nine embryo transfers were conducted using fresh and previously frozen Day-7 blastocysts to evaluate the in vivo viability of embryos produced in this synthetic medium; four gestations were obtained from six fresh embryos and one gestation from three frozen embryos. In conclusion, the fetal calf serum and BSA-free medium, supplemented with GFs-CYKs + RA + HA, improved embryo development and gave comparable ICM/TCN ratios and postthaw survival rates to the control with BSA. Fresh and frozen embryos produced in this medium are viable for embryo transfer. This fully synthetic method of embryo culture is a useful means of reducing the risk of disease transmission via embryo transfer.

Skeletal Muscle Regenerative Potential of Human MuStem Cells following Transplantation into Injured Mice Muscle

After intra-arterial delivery in the dystrophic dog, allogeneic muscle-derived stem cells, termed MuStem cells, contribute to long-term stabilization of the clinical status and preservation of the muscle regenerative process. However, it remains unknown whether the human counterpart could be identified, considering recent demonstrations of divergent features between species for several somatic stem cells. Here, we report that MuStem cells reside in human skeletal muscle and display a long-term ability to proliferate, allowing generation of a clinically relevant amount of cells. Cultured human MuStem (hMuStem) cells do not express hematopoietic, endothelial, or myo-endothelial cell markers and reproducibly correspond to a population of early myogenic-committed progenitors with a perivascular/mesenchymal phenotypic signature, revealing a blood vessel wall origin. Importantly, they exhibit both myogenesis inxa0vitro and skeletal muscle regeneration after intramuscular delivery into immunodeficient host mice. Together, our findings provide new insights supporting the notion that hMuStem cells could represent an interesting therapeutic candidate for dystrophic patients.

Gait characterization in golden retriever muscular dystrophy dogs using linear discriminant analysis

BackgroundAccelerometric analysis of gait abnormalities in golden retriever muscular dystrophy (GRMD) dogs is of limited sensitivity, and produces highly complex data. The use of discriminant analysis may enable simpler and more sensitive evaluation of treatment benefits in this important preclinical model.MethodsAccelerometry was performed twice monthly between the ages of 2 and 12xa0months on 8 healthy and 20 GRMD dogs. Seven accelerometric parameters were analysed using linear discriminant analysis (LDA). Manipulation of the dependent and independent variables produced three distinct models. The ability of each model to detect gait alterations and their pattern change with age was tested using a leave-one-out cross-validation approach.ResultsSelecting genotype (healthy or GRMD) as the dependent variable resulted in a model (Model 1) allowing a good discrimination between the gait phenotype of GRMD and healthy dogs. However, this model was not sufficiently representative of the disease progression. In Model 2, age in months was added as a supplementary dependent variable (GRMD_2 to GRMD_12 and Healthy_2 to Healthy_9.5), resulting in a high overall misclassification rate (83.2%). To improve accuracy, a third model (Model 3) was created in which age was also included as an explanatory variable. This resulted in an overall misclassification rate lower than 12%. Model 3 was evaluated using blinded data pertaining to 81 healthy and GRMD dogs. In all but one case, the model correctly matched gait phenotype to the actual genotype. Finally, we used Model 3 to reanalyse data from a previous study regarding the effects of immunosuppressive treatments on muscular dystrophy in GRMD dogs. Our model identified significant effect of immunosuppressive treatments on gait quality, corroborating the original findings, with the added advantages of direct statistical analysis with greater sensitivity and more comprehensible data representation.ConclusionsGait analysis using LDA allows for improved analysis of accelerometry data by applying a decision-making analysis approach to the evaluation of preclinical treatment benefits in GRMD dogs.

The benefits of liposomes for chilling canine sperm for 4 days at 4°C.

This study comprises 3 experiments exploring the possible benefits and mechanism of action of liposomes for chilling (4°C) canine sperm over a period of 4 days. In the first experiment, 20 ejaculates collected from 5 Beagle dogs were chilled in an extender containing 6% low density lipoproteins (LDL) (Control), or one of 7 extenders containing different concentrations (2, 4, 6, 8, 10, 15, 20%) of liposomes (LIPO). These ejaculates were chilled over 4 days and motility was assessed daily using a Hamilton Thorne analyzer (HTM-IVOS, 14.0). The 2% LIPO obtained the best results (p=0.038) after four days (72.55% motile spermatozoa and 31.4% progressive spermatozoa). In experiment 2, 10 ejaculates were collected from same 5 dogs and chilled in 6% LDL or 2% LIPO-based extenders. Sperm integrity characteristics were assessed prior to refrigeration and every 48h for four days (D0, D2, and D4). Acrosome integrity was assessed using the FITC-PSA test (Fluorescein IsoThiocyanate-Pisum Sativum Agglutinin), plasma membrane (PM) integrity using both the hypo-osmotic swelling test (HOSt) and SYBR14/Propidium Iodide test (SYBR14/PI), and DNA integrity using the Acridine-Orange test (AO). The 2% LIPO extender provided equivalent preservation of sperm integrity parameters to the reference extender (6% LDL). In experiment 3, a Langmuir-Blodgett trough was used to evaluate the mechanistic interactions between LDL, LIPO, prostatic fluid, and the canine spermatozoal membrane during chilling. Results indicate that LDL and LIPO interact differently with the biomimetic membrane. The most likely conclusion of these findings is that LDL and liposomes employ different protective mechanisms during the chilling (4°C) of canine spermatozoa.

Vascular Delivery of Allogeneic MuStem Cells in Dystrophic Dogs Requires Only Short-Term Immunosuppression to Avoid Host Immunity and Generate Clinical/Tissue Benefits

Growing demonstrations of regenerative potential for some stem cells led recently to promising therapeutic proposals for neuromuscular diseases. We have shown that allogeneic MuStem cell transplantation into Golden Retriever muscular dystrophy (GRMD) dogs under continuous immunosuppression (IS) leads to persistent clinical stabilization and muscle repair. However, long-term IS in medical practice is associated with adverse effects raising safety concerns. Here, we investigate whether the IS removal or its restriction to the transplantation period could be considered. Dogs aged 4–5 months old received vascular infusions of allogeneic MuStem cells without IS (GRMDMU/no-IS) or under transient IS (GRMDMU/tr-IS). At 5 months post-infusion, persisting clinical status improvement of the GRMDMU/tr-IS dogs was observed while GRMDMU/no-IS dogs exhibited no benefit. Histologically, only 9-month-old GRMDMU/tr-IS dogs showed an increased muscle regenerative activity. A mixed cell reaction with the host peripheral blood mononucleated cells (PBMCs) and corresponding donor cells revealed undetectable to weak lymphocyte proliferation in GRMDMU/tr-IS dogs compared with a significant proliferation in GRMDMU/no-IS dogs. Importantly, any dog group showed neither cellular nor humoral anti-dystrophin responses. Our results show that transient IS is necessary and sufficient to sustain allogeneic MuStem cell transplantation benefits and prevent host immunity. These findings provide useful critical insight to designing therapeutic strategies.