Djemil Bencharif

The advantages of LDL (low density lipoproteins) in the cryopreservation of canine semen.

A medium containing LDL (Low Density Lipoproteins, the cryoprotective component of chicken egg yolk) was compared with egg yolk for the preservation canine spermatozoa during the freeze-thaw process. Twenty sperm samples taken from 10 dogs were frozen in liquid nitrogen at -196 degrees C in seven different media: one control medium containing 20% egg yolk, and six test media containing 4%, 5%, 6%, 7%, 8%, and 10% LDL, respectively. Following thawing, sperm motility was assessed using a Hamilton-Thorne Sperm Analyser equipped with the CEROS 12 software. The percentage of motile spermatozoa was 55.3% in the 6% LDL medium (optimal concentration) compared with 27.7% in the egg yolk based medium (p<0.05). In comparison with the egg-yolk medium, the LDL medium also resulted in an improved preservation of spermatozoa during the freezing process (p<0.05) in terms of acrosomal integrity (FITC-PSA test), flagellar plasma membrane integrity (HOS test), and DNA integrity (Acridine Orange test). In addition, six Beagle bitches were inseminated twice, via the intra-uterine route, at an interval of 24h; 200x10(6) spermatozoa that had been previously frozen in the 6% LDL medium were used per insemination. All of the bitches became pregnant (gestation rate of 100%). In conclusion, the 6% LDL medium provides improved protection of the spermatozoa during the freeze-thaw process and a marked improvement in the motility parameters of canine spermatozoa in comparison with the control medium containing egg yolk alone. Finally, the use of LDL as a cryoprotectant for canine semen does not interfere with fertility.

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.

Effect of semen dilution to low-sperm number per dose on motility and functionality of cryopreserved bovine spermatozoa using low-density lipoproteins (LDL) extender: comparison to Triladyl® and Bioxcell®.

Artificial insemination with doses containing low-sperm numbers has been utilized to optimize the use of elite bulls. Hen egg yolk is widely used as a cryoprotective agent in semen freezing extender protecting the spermatozoa. Its action is due to the presence of low-density lipoproteins (LDL) in the hen egg yolk. The objectives of the present study were to evaluate the effects of the semen dilution to low-sperm number/dose on sperm motility and integrity of sperm plasma membrane in the cryopreservation process, using two commercial extenders (Triladyl, Bioxcell and LDL extender prepared in our laboratory, 97% purity. Fifteen ejaculates were collected from five fertile crossbred bulls (Bos taurusxBos indicus). After collection, sperm motility was examined by Computer-Assisted Semen Analysis (Hamilton Thorne), morphological sperm characteristics were evaluated by differential interference microscopy and the integrity of plasma membranes was determined using the hypo-osmotic swelling test. The semen was subsequently divided into three aliquots and diluted with the three extenders into 120 x 10(6), 60 x 10(6) and 20 x 10(6)sperm/mL, corresponding to 30 x 10(6), 15 x 10(6) and 5 x10(6) sperm/dose, respectively. This study revealed that LDL extender was more effective in preservation of motility and integrity of the plasma membrane of spermatozoa than Bioxcell and Triladyl (p<0.05), but no significant difference was observed between Triladyland Bioxcell. Therefore we can conclude that LDL extender could be used instead of Triladyl or Bioxcellat low semen concentration per dose for elite bulls, it also could be envisaged for the industry of sex-stored semen.

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

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.

Canine-chilled Sperm: Study of a Semen Extender Made with Low-density Lipoproteins from Hen Egg Yolk Supplemented with Glutamine

The objective of this study was to evaluate the effects of a combination of 6% low-density lipoproteins (LDL) and 20 mm glutamine in comparison with other extenders used for the refrigeration of canine semen: Tris egg yolk (EY) 20% and 6% LDL. The percentages of mobile spermatozoa after 4 days storage in a domestic refrigerator at +4 °C were 53.1%, 44.2% and 52.2% for the 6% LDL + 20 mm glutamine, 20% EY and 6% LDL extenders respectively for 100% of the dogs. After 7 days of storage, these percentages fell to 37.8%, 26.4% and 33.6% in the same extenders for 50% of the dogs. In vitro fertility tests were performed with all of the extenders following the mobility results. These tests were conducted on the day of sampling (D0), and 48 and 96 h after sampling. The results of the hypo-osmotic swelling test were 82.6%, 81.2% and 85.7% on D0, 75.2%, 74.1% and 78.5% on D2, and 70.8%, 71% and 76.1% on D4 for the 6% LDL + 20 mm glutamine, 20% EY and 6% LDL extenders, respectively. For the FITC/pisum sativum agglutinin (PSA) test, the results were 81.5%, 70.2% and 84.8% on D0, 78.9%, 62.3% and 84.2% on D2, and 72.7%, 59.6% and 73.7% on D4 for the 6% LDL + 20 mm glutamine, 20% EY and 6% LDL extenders, respectively. The acridine orange test was positive; in nearly 100% of cases, none of the spermatozoa had been denatured on D0, D2 and D4. The 6% LDL + 20 mm glutamine and the 6% LDL extenders are capable of preserving spermatozoa that have been stored in a domestic refrigerator at +4°C for at least 4 days. This means that the spermatozoa retain good cytoplasmic membrane integrity, had not capacitated and contained intact DNA in comparison with spermatozoa preserved in the egg yolk extender. The duration of storage is a very important consideration when faced with the problem of sending semen over ever-greater distances.

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.

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.

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%.

Organization of lipids in the artificial outer membrane of bull spermatozoa reconstructed at the air–water interface

Cryopreservation is widely used to preserve the quality of bull spermatozoa over time. Sequestration of seminal plasma proteins by low density lipoproteins and formation of a protective film around the spermatozoa membrane by low density lipoproteins were the main mechanisms proposed. However, the organization of lipids in the outer leaflet of the spermatozoa membrane has been never considered as a possible parameter. This study evaluated whether a change in the organization of the outer leaflet of the spermotozoa membrane could occur during cooling down. The organization of the main components of the spermatozoa membranes outer layer at the liquid-gas interface was analysed. Cryopreservative media (at 8° and 34°C) were used to study the miscibility of the spermatozoa membrane lipids using epifluorescence imaging and by tensiometry on Langmuir films. The results show that the four lipids: sphingomyelin, cholesterol, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PC) and plasmalogen 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphocholine (P-PC) were not fully miscible and their organization was controlled by temperature. Cholesterol and sphingomyelin form condensed domains surrounded by a mixture of PC and P-PC at 34°C while these condensed domains are surrounded by separated domains of pure PC and pure P-PC at 8°C. The organization of the outer membrane lipids, in particular the separation of PC and P-PC lipids during cooling down, must be considered to fully understand preservation of membrane integrity during cryopreservation.