Final Report Summary - SHEEPREP (Advanced Studies on Improving Sheep Fertility by Using Artificial Means of Reproduction)
Advanced Studies on Improving Sheep Fertility by Using Artificial Means of Reproduction (SheepRep)
SheepRep is a Marie Curie joint exchange (FP7-IRSES) programme, which was specifically designed to provide opportunities of transfer of knowledge and skills through mobility and networking activities - i.e. primarily tranining and scientific research - to experienced and early stage researchers from the following organizations within the Academic sector: University of Agricultural Sciences and Veterinary Medicine Bucharest Romania (UASVMB), University of Medicine and Pharmacy “Carol Davila” Bucharest Romania, Universiy of Ovidius Constanta - Romania, University of Molise, Campobasso - Italy, University of Kafrelsheikh - Egypt, University of Laval, Quebec City - Canada and the Research Institute for Animal Production, Kafrelsheikh - Egypt.
The concept behind the research work performed through SheepRep was based on the following background:
• Fresh ram semen, although more fertile than stored semen, cannot be kept alive and fertile on long-term, this making it improper for transporting and using at distances away from the donor. Although the use of long-term stored semen has practical advantages within flocks, the quality and subsequent fertilizing properites of ram semen following cryopreservation are unsatisfactory.
• The factors able to enhance or deprive the adenosine triphosfate (ATP) production, especially those which govern the mitochondrial function, were shown to affect sperm motility.
• Different types of cryopreservatives and diluents used in preparing the semen before cryopreservation and after thawing were proven to affect the time that spermatozoa remain vital and motile. However, acceptable motility and viability of spermatozoa do not necessarily lead to acceptable conception rates.
• Sperm motility, viability, membrane integrity and biochemical parameters can be routinely used to assess the quality of frozen-thawed semen.
• It was reported that certain energy doses of Helium-Neon laser irradiation might enhance the quality of semen in some species. Laser irradiation seems to be acting on cell membranes, but the mechanism by which this happens is still unknown.
• There are inferences about reactive species of oxygen (ROS) and nitrogen (NO) which may induce early sperm hypercapacitation and acrosome reaction.
• The levels and transport of intracellular Ca2+ into the irradiated sperm cells in some species can influence the motility, capacitation and acrosome reaction.
• Other factors which might influence the success rate of artificial insemination can be related to on-farm effects, farmer skills, ewe condition and age, stress and livestock management.
With SheepRep, we aimed at increasing the conception rate in ewes through artificial insemination (AI) means, by using frozen-thawed semen. Our main objectives were: i) to improve the freezing technologies, ii) to evaluate the cytological and cryobiological indexes of the spermatozoa, iii) to obtain accurate biochemical and electronomicroscopical profiles of the cryopreserved cells, and iv) to test in vivo the efficiency of different preserving and AI technologies on the cryobiological parameters and reproduction indexes. Additionally, the programme encouraged international and transdisciplinary research cooperation by bringing together specialists in the field of genetics, reproduction, histology, biochemistry, biology and livestock management, from organizations established on three continents: Europe, North Africa and North America.
SheepRep started on January 1st, 2012 (launching date January 17th) and ended on 31 July 31st, 2014. The first training session was provided by the invited guest, Prof Mostafa Ibrahim from the University of Kafrelsheikh to the Romanian researchers, who was hosted by the UASVMB for a month. Professor Mostafa provided lectures on three disciplines, complimentary to the research activities that were to be carried out within the project: Genetic improvement in sheep, Principles of sheep breeding technologies and AI technologies in ewes used in Egypt.
The second training programme started on April 19th and finished on June 18th 2012, and took place at the University of Laval (Faculté des sciences de l'agriculture et de l'alimentation), under the coordination of the Canadian project responsible, Prof Janice Bailey, the consortium coordinator, Dr Laura Urdes, the Romanian study director, Prof Alin Birtoiu and the principal investigator, Prof Cristiana Diaconescu. By admirable efforts and full commitment, the Canadian coordinator managed to gather together a renowned group of experts in the field, such as herself (NB: Dr Janice Bailey), Drs Robert Sullivan, François Castonguay, Clotilde Maurice, Pierre Leclerc, Julieta Caballero, Puttaswamy Manjunath, Olivier D’Amours, François Richard, Guillermin, Janice Evans, Claude Robert, Drd Vianney Salmon, Drs François Senechal, François Potier and Carolina Cardenas, all offering the invaluable opportunity to the Romanian Marie Curie fellows to learn about the cutting edge work level that had been done in the field of assisted reproduction in human and animal medicine, as well as in related disciplines. The training programme involved five types of academic activities designed to allow for knowledge and experience sharing, as well as other assets, through: formal lectures, hands-on lab training, work package development, journal clubs and field visits. At the end of the training programme, all the participants were issued a certificate for attending the programme.
Following the two training stages, one in Romania and the other in Canada, on 1 July 2012, the research work started at the University of Ovidius, under the supervision of Prof Stela Zamfirescu, and the University of Molise, under the supervision of Prof Passarella Salvatore, as local and distance networking activities amongst Egyptian, Italian and Romanian researchers. Among the main research results achieved by the SheepRep, it is worth mentioning: i) Cysteine (5 mM) added to the diluting media for the ram semen proved to bring the best results in terms of frozen-thawed ram semen quality improvement; the motility, as well as functional and structural integrity of the plasmatic membranes, were up to 12% higher then in the controls; ii) At the research facility in Constanta, a new dilluting-preserving media, based on soybean milk with TRIS was tested with encouraging results in terms of sperm cell motility; iii) The technical procedure of irradiating frozen-thawed ram spermatic cells with Helium-Neon laser was successfully developed and encouraging results were observed in terms of improvement of the seminal qualitative parameters; iv) Following the ewes insemination with the analyzed cryopreserved samples, the fertility indexes following parturition positively correlated with the viability of the spermatic cells (by flowcitometry); v) By the study on the biochemical profile of cryopreserved ram semen, it was observed that membrane lipid peroxidation is involved in lowering sperm cell motility after freezing-thawing; vi) freezing- thawing leads to plasma membrane alterations including rupture, especially at head level, and membrane detachment or vacuolation of the head or flagellum; vii) the fertility rate was higher in normal season of reproduction when frozen semen was diluted with Tris medium associated with cysteine. Overall, the outcomes of the project emphasize the advantage of artificial insemination programmes in flocks, as they ensure genetic progress. Through artificial insemination with good quality frozen semen, any breeder can have access to and can infuse genes from the best breeders tested in their flock. Finally, by controlling breeding and delivery periods, the market demand for more meat and milk can be met year-round. Also the artificial inseminations reduce the risk of reproductive disease transmission.
The consortium members, especially the research-development partners from University of Laval, University of Kafrelsheikh and the Research Institute for Animal Production Egypt, have an excellent track record as their past endeavours led to the introduction of AI with frozed-thawed ram sperm in practice. There is a particularly strong and persistent academia-industry relationship, which is the guarantee that projects results will be transferred and implemented in sheep farms. All project partners are dedicated to the dissemination, transfer and exploitation of the project results. The articles and the workshop with practical results after IA with freezing semen of ram will be further developed throughout the –now- former project partners, even after the end of the project. There will be a constant encouragement that the practical aspects of IA to be transferred to farm breeders, as feasible and economic means of optimization of flock reproduction efficiency.
SheepRep is a Marie Curie joint exchange (FP7-IRSES) programme, which was specifically designed to provide opportunities of transfer of knowledge and skills through mobility and networking activities - i.e. primarily tranining and scientific research - to experienced and early stage researchers from the following organizations within the Academic sector: University of Agricultural Sciences and Veterinary Medicine Bucharest Romania (UASVMB), University of Medicine and Pharmacy “Carol Davila” Bucharest Romania, Universiy of Ovidius Constanta - Romania, University of Molise, Campobasso - Italy, University of Kafrelsheikh - Egypt, University of Laval, Quebec City - Canada and the Research Institute for Animal Production, Kafrelsheikh - Egypt.
The concept behind the research work performed through SheepRep was based on the following background:
• Fresh ram semen, although more fertile than stored semen, cannot be kept alive and fertile on long-term, this making it improper for transporting and using at distances away from the donor. Although the use of long-term stored semen has practical advantages within flocks, the quality and subsequent fertilizing properites of ram semen following cryopreservation are unsatisfactory.
• The factors able to enhance or deprive the adenosine triphosfate (ATP) production, especially those which govern the mitochondrial function, were shown to affect sperm motility.
• Different types of cryopreservatives and diluents used in preparing the semen before cryopreservation and after thawing were proven to affect the time that spermatozoa remain vital and motile. However, acceptable motility and viability of spermatozoa do not necessarily lead to acceptable conception rates.
• Sperm motility, viability, membrane integrity and biochemical parameters can be routinely used to assess the quality of frozen-thawed semen.
• It was reported that certain energy doses of Helium-Neon laser irradiation might enhance the quality of semen in some species. Laser irradiation seems to be acting on cell membranes, but the mechanism by which this happens is still unknown.
• There are inferences about reactive species of oxygen (ROS) and nitrogen (NO) which may induce early sperm hypercapacitation and acrosome reaction.
• The levels and transport of intracellular Ca2+ into the irradiated sperm cells in some species can influence the motility, capacitation and acrosome reaction.
• Other factors which might influence the success rate of artificial insemination can be related to on-farm effects, farmer skills, ewe condition and age, stress and livestock management.
With SheepRep, we aimed at increasing the conception rate in ewes through artificial insemination (AI) means, by using frozen-thawed semen. Our main objectives were: i) to improve the freezing technologies, ii) to evaluate the cytological and cryobiological indexes of the spermatozoa, iii) to obtain accurate biochemical and electronomicroscopical profiles of the cryopreserved cells, and iv) to test in vivo the efficiency of different preserving and AI technologies on the cryobiological parameters and reproduction indexes. Additionally, the programme encouraged international and transdisciplinary research cooperation by bringing together specialists in the field of genetics, reproduction, histology, biochemistry, biology and livestock management, from organizations established on three continents: Europe, North Africa and North America.
SheepRep started on January 1st, 2012 (launching date January 17th) and ended on 31 July 31st, 2014. The first training session was provided by the invited guest, Prof Mostafa Ibrahim from the University of Kafrelsheikh to the Romanian researchers, who was hosted by the UASVMB for a month. Professor Mostafa provided lectures on three disciplines, complimentary to the research activities that were to be carried out within the project: Genetic improvement in sheep, Principles of sheep breeding technologies and AI technologies in ewes used in Egypt.
The second training programme started on April 19th and finished on June 18th 2012, and took place at the University of Laval (Faculté des sciences de l'agriculture et de l'alimentation), under the coordination of the Canadian project responsible, Prof Janice Bailey, the consortium coordinator, Dr Laura Urdes, the Romanian study director, Prof Alin Birtoiu and the principal investigator, Prof Cristiana Diaconescu. By admirable efforts and full commitment, the Canadian coordinator managed to gather together a renowned group of experts in the field, such as herself (NB: Dr Janice Bailey), Drs Robert Sullivan, François Castonguay, Clotilde Maurice, Pierre Leclerc, Julieta Caballero, Puttaswamy Manjunath, Olivier D’Amours, François Richard, Guillermin, Janice Evans, Claude Robert, Drd Vianney Salmon, Drs François Senechal, François Potier and Carolina Cardenas, all offering the invaluable opportunity to the Romanian Marie Curie fellows to learn about the cutting edge work level that had been done in the field of assisted reproduction in human and animal medicine, as well as in related disciplines. The training programme involved five types of academic activities designed to allow for knowledge and experience sharing, as well as other assets, through: formal lectures, hands-on lab training, work package development, journal clubs and field visits. At the end of the training programme, all the participants were issued a certificate for attending the programme.
Following the two training stages, one in Romania and the other in Canada, on 1 July 2012, the research work started at the University of Ovidius, under the supervision of Prof Stela Zamfirescu, and the University of Molise, under the supervision of Prof Passarella Salvatore, as local and distance networking activities amongst Egyptian, Italian and Romanian researchers. Among the main research results achieved by the SheepRep, it is worth mentioning: i) Cysteine (5 mM) added to the diluting media for the ram semen proved to bring the best results in terms of frozen-thawed ram semen quality improvement; the motility, as well as functional and structural integrity of the plasmatic membranes, were up to 12% higher then in the controls; ii) At the research facility in Constanta, a new dilluting-preserving media, based on soybean milk with TRIS was tested with encouraging results in terms of sperm cell motility; iii) The technical procedure of irradiating frozen-thawed ram spermatic cells with Helium-Neon laser was successfully developed and encouraging results were observed in terms of improvement of the seminal qualitative parameters; iv) Following the ewes insemination with the analyzed cryopreserved samples, the fertility indexes following parturition positively correlated with the viability of the spermatic cells (by flowcitometry); v) By the study on the biochemical profile of cryopreserved ram semen, it was observed that membrane lipid peroxidation is involved in lowering sperm cell motility after freezing-thawing; vi) freezing- thawing leads to plasma membrane alterations including rupture, especially at head level, and membrane detachment or vacuolation of the head or flagellum; vii) the fertility rate was higher in normal season of reproduction when frozen semen was diluted with Tris medium associated with cysteine. Overall, the outcomes of the project emphasize the advantage of artificial insemination programmes in flocks, as they ensure genetic progress. Through artificial insemination with good quality frozen semen, any breeder can have access to and can infuse genes from the best breeders tested in their flock. Finally, by controlling breeding and delivery periods, the market demand for more meat and milk can be met year-round. Also the artificial inseminations reduce the risk of reproductive disease transmission.
The consortium members, especially the research-development partners from University of Laval, University of Kafrelsheikh and the Research Institute for Animal Production Egypt, have an excellent track record as their past endeavours led to the introduction of AI with frozed-thawed ram sperm in practice. There is a particularly strong and persistent academia-industry relationship, which is the guarantee that projects results will be transferred and implemented in sheep farms. All project partners are dedicated to the dissemination, transfer and exploitation of the project results. The articles and the workshop with practical results after IA with freezing semen of ram will be further developed throughout the –now- former project partners, even after the end of the project. There will be a constant encouragement that the practical aspects of IA to be transferred to farm breeders, as feasible and economic means of optimization of flock reproduction efficiency.