Canine semen cryoconservation: Emerging data over the last 20 years.

Canine semen cryoconservation was used since 1969, and this process is still nowadays in progress. This review aims to have an overview of two factors leading to a successful freezing-thawing semen. The success and efficiency of freezing process can be measured by the post-thawing sperm mobility. The first factor is the best extender used as a cryoprotectant to have a similar osmolarity and pH compared to the seminal plasma to enable sperm survival. Historically, chicken egg yolk was used since 1940, but due to microbial risks and to the presence of granules (which interfere with counting dead spermatozoa and inhibits a spermatozoal respiration), despite these disadvantages, egg yolk is considered an excellent cryoprotectant for sperm of different animal species. The low-density lipoproteins (LDL), contained in EY, when used at a concentration of 6% in a freezing medium associated with 20 mM of glutamine, show a mobility up to 54.5%, which is the best combination found. However, the sperm protection mechanism by LDL during freezing-thawing process only begins to be decrypted. But extraction protocols of LDL are not efficient for an industrial use. Therefore, egg yolk plasma is used within liquid or lyophilized state, and offering similar efficiency as the 6% LDL middle. The equilibration step, in which the diluted sperm is placed for a variable period of time at a temperature of +4°C, before freezing it. The studies show that 6 hr is the optimal duration for the canine sperm equilibration. The future of canine sperm cryopreservation is expected in liposome use and synthetic substances, which mimics LDL role.

A comparison of liquid and lyophilized egg yolk plasma to low density lipoproteins for freezing of canine spermatozoa.

The current study aimed to explore the potential usefulness of liquid or lyophilized egg yolk plasma (EYP) as a substitute for low-density lipoproteins (LDL) for cryopreservation of canine spermatozoa. In the first experiment, a total of 20 ejaculates harvested from six Beagles were frozen in extenders containing 6% LDL (control) or liquid or lyophilized EYP at one of three concentrations (20%, 40% or 60%). Motility parameters were assessed 10 min after thawing using computer-assisted sperm analysis. For both liquid and lyophilized EYP, the 40% concentration yielded motility similar (p > 0.05) to that observed with the control extender. In the second experiment, 12 ejaculates collected from the same six dogs were frozen in 6% LDL (Control), 40% liquid EYP or 40% lyophilized EYP extenders. Spermatozoal membrane integrity (hypo-osmotic swelling test [HOSt] and SYBR14/propidium iodide [PI] staining), acrosome integrity (FITC-Pisum sativum agglutinin staining) and DNA integrity (acridine orange staining) characteristics were evaluated 10 min after thawing. Both liquid and lyophilized 40% EYP-based extenders successfully preserved all assessed integrity parameters as efficiently as the control. Results of this study suggest that lyophilized EYP is a viable alternative to LDL in freezing extenders for dog semen.

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.