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.

Early sex determination in the canine foetus by ultrasound and PCR.

Twenty bitches were seen in consultation at the Department of Reproduction at ONIRIS (College of Veterinary Medicine, Food Science and Engineering, Loire Atlantique, Nantes, France) between 25 and 50 days of gestation for early sex determination of the canine foetus using ultrasound. The genital tubercle is not visible before 26 days; between 26 and 30 days, it is visible between the pelvic limbs; between 33 and 50 days, the position of the genital tubercle enables sex determination as it migrates caudally in the female and cranially in the male. Good statistical concordance between sexing via ultrasound and sexing at birth has been established (kappa coefficient of 0.8). Macroscopic, microscopic, and histological examinations of the external genital organs were also performed on 10 foetuses at 35 days of gestation; a cartilaginous structure was visualized in the genital apparatus of the male but also in half of the females. Finally, the development of a PCR technique on the SRY gene using formaldehyde-preserved tissues has been described for the first time in this study. It served as a reference for sexing canine foetuses.

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

OBJECTIVE: To 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). STUDY DESIGN: Randomized, prospective clinical trial. ANIMALS: Healthy, sexually intact female cats (n = 60). METHODS: Cats were randomly assigned to 1 of 3 groups: Midline (n = 20), Flank (20), and Lap (20) were evaluated 1, 2, 4, 6, and 12 hours 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. RESULTS: There 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. CONCLUSIONS: Laparoscopic 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.

Early pregnancy diagnosis and monitoring in the queen using ultrasonography with a 12.5 MHz probe.

Eleven pregnancies in six queens were monitored daily from day 7 to day 28, corresponding to the end of the embryonic period, using ultrasonography with a 12.5 MHz probe. The first mating was considered as the presumed start of gestation, as has been described to be the case in 92.3% of pregnancies. The embryonic vesicles were identified on day 11, while the embryo appeared on day 15 or 16. The stage of pregnancy could be evaluated approximately by measuring the length of the embryonic vesicle or the crown-rump length of the embryo from days 11 and 17, respectively, up until the end of the embryonic phase of gestation. The visualisation of certain organs could also be used to date gestation; for example, the limbs, neural tube and stomach were visible from days 19, 20 and 26, respectively. The 12.5 MHz probe did not enable the diagnosis of gestation to be performed any earlier than with 7.5 and 10 MHz probes. However, there was a significant difference in comparison with a 5 MHz probe.

The advantages of using a combination of LDL and glutamine in comparison with TRIS egg yolk and Equex® STAMP extenders in the cryopreservation of canine semen.

Twenty semen samples taken from 5 dogs were frozen in liquid nitrogen at -196 °C in four different extenders: one control extender based on 20% egg yolk, 6% LDL alone (low density lipoproteins: the active cryoprotective principle in chicken egg yolk), 6% LDL combined with 20 mmol glutamine, and Equex® (a reference extender that we wish to compare with the LDL-glutamine combination). After thawing, spermatozoal motility was evaluated using a HAMILTON THORNE CERROS 12 image analyzer; the percentage of motile spermatozoa was 27.7% in the egg yolk extender (p<0.05), 49.9% with 6% LDL alone (p>0.05), 54.7% in the 6% LDL+20 mmol glutamine extender, and 47.9% with Equex® (p>0.05). The motility parameters (VAP, VCL, VSL and ALH) were also superior in the 6% LDL+20 mmol glutamine extender in comparison with the other extenders. Finally, the spermatozoa were generally better protected during freezing with the 6% LDL+20 mmol glutamine association than with the egg yolk, 6% LDL, or Equex extenders in terms of the flagellar plasma membrane (HOS test), DNA (Acridine orange test), and acrosome integrity (Spermac® test: no significant difference). The Equex® extender obtained the best results for the acrosome, followed by 6% LDL+20 mmol glutamine (FITC-PSA test: p<0.05 between each extender).

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.

Ovariohysterectomy in the bitch.

Ovariohysterectomy is a surgical procedure widely employed in practice by vets. It is indicated in cases of pyometra, uterine tumours, or other pathologies. This procedure should only be undertaken if the bitch is in a fit state to withstand general anaesthesia. However, the procedure is contradicated if the bitch presents a generalised condition with hypothermia, dehydration, and mydriasis. Ovariohysterectomy is generally performed via the linea alba. Per-vaginal hysterectomy can also be performed in the event of uterine prolapse, if the latter cannot be reduced or if has been traumatised to such an extent that it cannot be replaced safely. Specific and nonspecific complictions can occur as hemorrhage, adherences, urinary incontinence, return to oestrus including repeat surgery. After an ovariectomy, bitches tend to put on weight, it is therefore important to inform the owner and to reduce the daily ration by 10%.

Freezing canine sperm: comparison of semen extenders containing Equex and LDL (Low Density Lipoproteins).

Chicken egg yolk is held as an excellent cryoprotective agent for freezing canine semen. Recent advances have enabled the extraction of low density lipoproteins from egg yolk, which are responsible for the cryoprotective abilities of the latter. The objective of this article was to compare 3 semen extenders for freezing canine semen: 2 containing egg yolk (Tris egg yolk and Equex STAMP) and one containing 6% LDL. After freezing and thawing 20 ejaculates from 5 different dogs, the 6% LDL extender produced 50% mobile spermatozoa, compared with 48% with the Equex extender and 27.7% with the extender containing egg yolk alone (EY). In vitro functional tests demonstrated that the integrity of the plasma membrane (hypoosmotic test) was respected in 65-66% of spermatozoa as a function of the extender; DNA integrity was respected in more than 97% of the spermatozoa. The Equex extender provided superior acrosome integrity (FITC/PSA test): 68.4% compared with 55.1% with LDL and 53.3% with egg yolk. However, the 6% LDL extender resulted in fewer spermatozoal anomalies (Spermac test), with 54.6% normal spermatozoa compared to 53.6% for Equex and 53.3% with the egg yolk. All six of the bitches inseminated artificially via the intra-uterine route (Scandinavian technique) using semen frozen in the 6% LDL extender became pregnant. The LDL extender resulted in percentages of mobile spermatozoa and movement characteristics that were as good if not better than those obtained with the reference extenders following thawing. The 6% LDL extender appears to have the same cryoprotective qualities as the reference diluent, Equex STAMP.

Seroprevalence of Neospora caninum antibodies and its consequences for reproductive parameters in dairy cows from Dakar-Senegal, West Africa.

The aim of this study was to determine the seroprevalence of Neospora caninum antibodies and its effects on reproductive parameters in cows in intensive dairy herds in Dakar. Randomised blood samples were taken for serology from 196 cows in four herds with a history of sporadic abortion. All of the sera were assayed for antibodies against N. caninum, Candida guillermondii, Coxiella burnetii, and Chlamydophila sp. The associations between serostatus and reproductive parameters (abortion, number of inseminations to conception, and calving to conception interval) were assessed over a period of 5 years (2004-2008). The seroprevalence of N. caninum antibodies in dairy cattle was 17.9%. The local Gobra breed and crossbreeds had higher levels of N. caninum antibodies than exotic breeds (p < 0.05). For the most recent pregnancies, seropositive cows required more inseminations to establish conception than seronegative cows (p < 0.05). The results indicate that dairy cattle from Dakar are exposed to N. caninum. Neosporosis should, therefore, be systematically considered as a cause when the calving to conception interval is prolonged.

Modifications of bull spermatozoa induced by three extenders: Biociphos, low density lipoprotein and Triladyl, before, during and after freezing and thawing.

The success of artificial insemination with frozen semen implies the reduction of the deleterious effects on the cells induced by this technique. These effects can occur as early as during the first dilution in an extender, as well as at any step, during or after the freezing process. In this work, we have compared the modifications induced by Triladyl, low density lipoproteins (LDL) and Biociphos extenders, after dilution and cooling to 4 degrees C for 1, 4 and 24 h. Alterations in the cell structures were visualized by electron microscopy (EM). More than 80% of spermatozoa were injured after incubation for 4 h in Triladyl, while 3% and 47% were counted in LDL and Biociphos respectively. This latter extender was deleterious to cell membrane integrity after incubation for 4 h or longer. The ultrastructure of frozen spermatozoa was studied by EM of cryofixed-cryosubstituted samples obtained from regular 0.5 ml French straws frozen using our usual protocol. The main differences between samples concerned the size and appearance of the frozen extender veins, while very few cell defects were found to be added by the freezing process at any depth in the straws. After thawing, semen motility was twofold higher (P < 0.05) in Biociphos (64%) and LDL (61%) than in Triladyl (32%) and the cells were less altered in LDL. We concluded that the LDL extender offers a better protection for storage of frozen spermatozoa, and can probably also be used for the preservation of fresh semen for short periods.

Effects of glutamine on post-thaw motility of stallion spermatozoa: an approach of the mechanism of action at spermatozoa level.

The cryoprotective effect of l-glutamine and an approach of its mechanism of action, in preserving motility of stallion spermatozoa during the freezing-thawing process, were studied. In Experiment 1, thirty-six ejaculates were collected from six stallions (two good, two middle, and two of poor sperm freezability) and were diluted with 10 different freezing media derived from INRA 82 medium supplemented with 20 mM HEPES and 2% (v/v) centrifuged egg yolk (BM). After thawing, sperm motility was evaluated by a computer-assisted semen motility analyser. The effects of glutamine and glycerol at different concentrations on post-thaw sperm motility were studied. A possible interaction between medium and semen freezability was investigated. Only the 50 mM glutamine + 2.5% glycerol medium significantly improved sperm motility compared to classical freezing medium (2.5% glycerol). The presence of glutamine at 50 mM was not sufficient to offset the need to use glycerol in the freezing extender. The use of glutamine at a higher concentration >100 mM in the presence of 2.5% of glycerol was toxic. Reducing the glycerol proportion from 2.5% to 2 or 1.5% in the presence of glutamine at 50, 75, and 100 mM had no influence on post-thaw motility of semen of middle and good freezability. Moreover, the substitution of 2.5% glycerol by 50 mM glutamine in BM, did not significantly change the post-thaw motility of semen of good freezability. In Experiment 2, 3H-glutamine and 3H-glycerol were used to study the kinetics of penetration of glutamine and glycerol in sperm cells. The radioactivity of each radio-labelled semen pellet was measured at different times (0, 15, 30, 60, 90, 120 min), by using a Packard tri-carb 4530 apparatus. The percentages of incorporated radioactivity (%IRA) in semen pellets were calculated at different times. The %IRA of 3H-glycerol in semen pellets were significantly higher than the %IRA of 3H-glutamine. The %IRA of 3H-glycerol in semen pellets increased greatly from time 0 to 60 min, and then it is stabilized from 60 to 120 min. In contrast, the %IRA of 3H-glutamine in semen pellets increased slightly from 0 to 60 min, then it stabilized until 120 min. These experiments demonstrate that glutamine has a synergistic cryoprotective effect with glycerol on cryopreservation of stallion spermatozoa, and suggest that glutamine acts at the extra-cellular level, independently of glycerol.

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.

Investigation of Neospora sp. antibodies in aborted mares from Normandy, France.

Neospora caninum, an apicomplexan protozoan parasite, is recognized as a major cause of abortion in cattle while limited information is presently available on association between equine Neospora infections and abortions. The aim of the present study was to document prevalence of antibodies against Neospora sp. in aborted mares as a clue to the role of N. caninum in mare reproductive failure in Normandy, France. Using an agglutination test, the number of animals with elevated (>80) anti-Neospora sp. antibody titer was higher in a group of 54 aborted mares than in randomly chosen groups of 45 mares and 76 horses sampled for equine arteritis virus and Fasciola hepatica antibodies, respectively (P<0.001). N. caninum DNA was found in 3/91 fetal brains, 2/77 fetal hearts, and 1/1 placenta, and present in both brains and hearts of two fetuses. In 13 cases for which both mare serum and fetus were available, no fetal N. caninum amplification product was present while a large variation of maternal antibody titers was found. Data prompt at additional surveys of association between equine reproductive failure and Neospora sp. infection.