Laboratory of Biotechnology and Pathology of Reproduction

Nantes, France

Laboratory of Biotechnology and Pathology of Reproduction

Nantes, France
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Bencharif D.,Laboratory of Biotechnology and Pathology of Reproduction | Amirat-Briand L.,Laboratory of Biotechnology and Pathology of Reproduction | Garand A.,Laboratory of Biotechnology and Pathology of Reproduction | Anton M.,French National Institute for Agricultural Research | And 8 more authors.
Research in Veterinary Science | Year: 2012

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). © 2011 Elsevier Ltd.


Bencharif D.,Laboratory of Biotechnology and Pathology of Reproduction | Amirat-Briand L.,Laboratory of Biotechnology and Pathology of Reproduction | Le Guillou J.,Laboratory of Biotechnology and Pathology of Reproduction | Vialtelle C.,Laboratory of Biotechnology and Pathology of Reproduction | And 5 more authors.
Revue de Medecine Veterinaire | Year: 2013

The aim of this study was to evaluate the effects of LDL-based extenders in comparison with other media used for the refrigeration of canine semen: Tris egg yolk (EY), Equex®, and INRA96®. The test extender was composed of 6% LDL. The percentage of mobile spermatozoa after 4 days storage in a domestic refrigerator at +4°C was 52.2%, 44.2%, 17.2%, and 24.5% for the 6% LDL, Tris Egg Yolk (20%), Equex®, and INRA 96® extenders respectively for 100% of the dogs. After 7 days of storage, these percentages fell to 33.6%, 26.4%, 5.5%, and 14.8 % in the same extenders for 50% of the dogs. In vitro fertility tests were performed with the two extenders that gave the best mobility results, i.e. 6% LDL and Tris egg yolk (20%). These tests were conducted on the day of sampling (D0), 48 hours, and 96 hours after sampling. The results of the HOS test were 81.2% and 85.8% for egg yolk and 6% LDL respectively at D0, 74.1% and 78.5% two days later (D2), and 71% and 76.1% at D4. The results of the FITC/PSA test were as follows: 70.2% and 84.8% on D0, 69.3% and 84.2% on D2, and 59.6% and 73.7% on D4 for the EY and 6% LDL 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 extender was capable of preserving spermatozoa that had been stored in a domestic refrigerator at +4°C for at least 4 days. This meant that the spermatozoa retained good cytoplasmic membrane integrity, had not capacitated, and contained intact DNA in comparison with the other extenders tested. The duration of storage is a very important consideration when faced with the problem of sending semen over ever-greater distances. Equex® and INRA 96® gave results that were 30% inferior; these extenders therefore seem ill-adapted for the refrigeration of canine sperm. Copyright © Revue Méd. Vét., 2013.


Bencharif D.,Laboratory of Biotechnology and Pathology of Reproduction | Amirat-Briand L.,Laboratory of Biotechnology and Pathology of Reproduction | Garand A.,Laboratory of Biotechnology and Pathology of Reproduction | Anton M.,French National Institute for Agricultural Research | And 8 more authors.
Animal Reproduction Science | Year: 2010

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. © 2010 Elsevier B.V. All rights reserved.


Belala R.,Laboratory of Biotechnology and Pathology of Reproduction | Belala R.,Blida University | Delay J.,Laboratory of Biotechnology and Pathology of Reproduction | Amirat L.,Laboratory of Biotechnology and Pathology of Reproduction | And 9 more authors.
Animal Reproduction Science | Year: 2016

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 48. h 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. © 2016 Elsevier B.V.


PubMed | Laboratory of Biotechnology and Pathology of Reproduction, IMV Technologies, French National Institute for Agricultural Research, Blida University and ONIRIS The National Veterinary
Type: | Journal: Animal reproduction science | Year: 2016

This study comprises 3 experiments exploring the possible benefits and mechanism of action of liposomes for chilling (4C) 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 (4C) of canine spermatozoa.


Gogny A.,Veterinary Hospital Center | Bruyas J.-F.,Laboratory of Biotechnology and Pathology of Reproduction | Fieni F.,Laboratory of Biotechnology and Pathology of Reproduction
Reproduction in Domestic Animals | Year: 2010

Contents: Pyometra in an inguinal hernia was diagnosed in a 10-year-old intact cross-bred bitch which had had dysorexia, depression and inguinal distension. The hernia contained caudal portions of the two uterine horns, uterine cervix and cranial part of the vagina. As the organs were enlarged and full of pus, manual attempt to push back the uterine horns and the vagina in the abdominal cavity through the inguinal canal was unsuccessful. Herniated uterine horns were ligated and cut in their median portion, so it became possible to remove the cervix and the caudal portion of the horns through the hernial orifice, and the ovaries and the cranial part of the horns through a peritoneal midline incision. This bitch was not intended for breeding purposes and, given the presence of a huge pyometra associated with an inguinal hernia, an ovario-hysterectomy was recommended. Uterine herniation should be considered as a differential diagnosis of a caudal lateral inguinal mass. When pushing the uterus back in the abdominal cavity is impossible, a surgical procedure should be performed to detect ischemia-reperfusion injury and/or a septic risk. © 2010 Blackwell Verlag GmbH.


PubMed | Laboratory of Biotechnology and Pathology of Reproduction
Type: Comparative Study | Journal: Research in veterinary science | Year: 2012

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).


PubMed | Laboratory of Biotechnology and Pathology of Reproduction
Type: Comparative Study | Journal: Animal reproduction science | Year: 2010

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.

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