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Intra-uterine injection of equine umbilical cord mesenchymal stem cells in the mare
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Abstracts / Journal of Equine Veterinary Science 43 (2016) S56eS82
insemination technique itself does not have an influence on PBIE; hysteroscopic insemination causes an increase in the inflammatory response after 8h and delayed uterine clearance. Key Words: insemination, hysteroscopy, androcoll
47 Intratesticular application of allogenic mesenchymal stem cells in stallions P. Mello-Papa, P.N. Guasti, C.P. Freitas-Dell’Aqua, L.R.P. Andrade, L.F.M. Chaves Silva, E.A.B. Araujo, S.N. Oliveira, L. Maia, N.G. Nakazato, B. De Vita, F.O. Papa, F.C. Landim-Alvarenga, M.A. Alvarenga ~o Paulo State University, UNESP, Botucatu, SP, Brazil Sa In horses, bone marrow and adipose tissue are the main sources of mesenchymal stem cells (MSCs) for therapeutic use. Despite the high potential to restore damaged tissues, its use in reproductive disorders and testicular degeneration in stallions has not been studied. The aim of the present study was to evaluate the scrotal surface temperature (SST), testicular volume (TV) and sperm motility after intratesticular application of allogenic bone marrow derived MSC from a single donor stallion. Obtained cells were processed, cultured and frozen until 15 days before the intratesticular application. After thawing, the MSCs were plated and expanded to confluence to provide 20 106 cells for each animal. For intratesticular application, 18 adult stallions (3-4 yr old) were divided into two groups: 5mL sterile phosphate saline buffer (Control; n¼9) and 5mL of the cell suspension (MSC; n¼9; 10 106 cells/testicle). The injection procedure was performed in the medial curvature of each testicle.The TV was calculated as length X width X height X 0.53 cm3, the SST was obtained using an infrared thermographer (Flir Systems; oC) and the sperm motility was assessed by computer-assisted sperm analyzer (CASA, HTM IVOS). The parameters were monitored at the moment of application (M0) and one day after application (M1)for TV, SST and sperm motility. After 48h of application (M2), only the SST was measured. Parametric data were examined by ANOVA followed by Tukey’s test and non-parametric data by Kruskal-Wallis followed by Dunn test with a significance level if p< 0.05.No difference was observed in the Control group for SST in M0 (29.9±1.2), M1 (29.4 ± 1.0) and M3 (29.6 ± 1.0); for TV in M0 and M1 (136.1 ± 22.2 vs 150.9 ± 73.1), respectively. However, on MSC, the M1 (29.5±0.78) showed a significant increase of SST when compared to M0 (28.4 ± 0.97) (p< 0.05). The SST showed similar results at M0 and M2 (29.3 ± 0.56). No difference was observed in MSC group, for TV in M0 and M1 (162.9 ± 50.3 vs 140.1 ± 36.5), and for sperm motility at M0 and M1 (78.2 ± 11.8 vs 75.0 ± 17.3). In conclusion, the injection of MSCs into testicles did not affect the TV and sperm motility. The intratesticular application of MSCs increased the SST at 24h, however, after 48h returned to physiological, suggesting that this procedure can be safely used for scientific and/or therapeutic purposes. Studies are in progress to determine the effect of MSCs injection on spermatogenesis.
Key Words: stem cells, inflammatory reaction, testicle
48 Intra-uterine injection of equine umbilical cord mesenchymal stem cells in the mare D. Casarini 1, A. Josson 1, N. Saulnier 2 1 VetAgro Sup, 69280 Marcy L’Etoile, France; 2 Vetbiobank, 69280 Marcy L’Etoile, France
Numerous reviews describe the relationship between fertility and specific pathological changes in the mare’s endometrium, but more studies are needed to show if endometrial pathology can be treated topically. Therefore, we designed a preliminary study whereby 2 reproductive healthy Standardbred mares of 8 and 10 yrs old were used to investigate if an increasing dose of equine umbilical cord mesenchymal stem (eUCMS) cells may have an effect on the mare’s endometrium. The eUCMS cells were donated to us by Vetbiobank (France) after being fully characterised following ISCT (International Society for Cellular Transplantation) recommendations. Reported immunophenotype is CD90+/29+/ 44+/105+/MHC2-/CD45- This type of cells are known to differentiate into mesodermal lineage upon induction. As soon as each mare passed transition, she was examined 2 to 3 times weekly by transrectal ultrasound from April to October. Prior to the study, an endometrial culture, uterine cytology and endometrial biopsy were performed during diestrus followed by 1 ml of PGF2 alpha IM. Both mares received between 5 and 30 million eUCMS cells in 20 ml PBS intra-uterine (IU) during 3 estrous cycles each. All IU infusions of eUCMS cells were done when the mare showed estrus with a minimum of a 38 mm follicle and a grade 1 endometrial edema (grade 1 for minimal and grade 4 for maximal) Twenty four hours after eUCMS cells infusion, a uterine swab for culture and a uterine lavage for cytology were performed using between 0.5 and 1 L Ringers Lactate to search for bacteria and neutrophils. In two of the six cultures one contaminant (Granulicatella adiacens) and one weak growth of Streptococcus equi subspecies zooepidemicus were isolated; all other four cultures remained negative. All six endometrial cytologies remained negative through the entire study. At the end of the study an endometrial biopsy was performed on each mare. The results of these biopsies, a grade 1, remained unchanged. Both mares were subsequently inseminated with 1 billion motile sperm with >90% total motility and 14 days later were diagnosed pregnant. We concluded that IU infusions of eUCMS cells, even at 30 million, did not cause any inflammation or irritation of the endometrium which would interfere with fertility. Further research is needed to investigate if the IU use of eUCMS cells would be of benefit in post breeding endometritis, metritis, pyometra or endometriosis. Key Words: mare, stem cells, uterus
49 Plasma membrane lipid profile from resistant and sensitive spermatozoa of Mangalarga Marchador stallions after cooling at 5 C C. Ramires Neto 1, K.R.A. Belaz 2, Y. F. R. Sancler Silva 1, M.J. Sudano 3, D. Zampieri 2, A. Tata 2, M.N. Eberlin 2, C.P. FreitasDell'aqua 1, E.M.S. Santana 1, F.O. Papa 1, M.A. Alvarenga 1 1 ~o Animal e Radiologia Veterina ria da Departamento de Reproduça ria e Zootecnia, UNESP, Botucatu, Faculdade de Medicina Veterina ~o Paulo, Brazil; 2 Instituto de Química, Universidade Estadual de Sa ~o Paulo, Brazil; 3 Universidade Federal do Campinas, Campinas, Sa Pampa, Bage, Rio Grande do Sul, Brazil Equine cooled semen is routinely used for artificial insemination. However, a large proportion of stallions present sperm cells with poor tolerance to the cooling process. The lipid composition of the sperm plasma membrane has been suggested as a factor affecting sperm to tolerate low temperatures. Therefore, the aim of this study was to evaluate and compare membrane phospholipid profiles from stallions presenting sperm resistant or less tolerant to cooled-storage at 5 C. Two ejaculates from each of 20 Mangalarga Marchador stallions were used. All stallions had high fertility (more than 60% with fresh semen) when using fresh
Abstracts / Journal of Equine Veterinary Science 43 (2016) S56eS82
insemination technique itself does not have an influence on PBIE; hysteroscopic insemination causes an increase in the inflammatory response after 8h and delayed uterine clearance. Key Words: insemination, hysteroscopy, androcoll
47 Intratesticular application of allogenic mesenchymal stem cells in stallions P. Mello-Papa, P.N. Guasti, C.P. Freitas-Dell’Aqua, L.R.P. Andrade, L.F.M. Chaves Silva, E.A.B. Araujo, S.N. Oliveira, L. Maia, N.G. Nakazato, B. De Vita, F.O. Papa, F.C. Landim-Alvarenga, M.A. Alvarenga ~o Paulo State University, UNESP, Botucatu, SP, Brazil Sa In horses, bone marrow and adipose tissue are the main sources of mesenchymal stem cells (MSCs) for therapeutic use. Despite the high potential to restore damaged tissues, its use in reproductive disorders and testicular degeneration in stallions has not been studied. The aim of the present study was to evaluate the scrotal surface temperature (SST), testicular volume (TV) and sperm motility after intratesticular application of allogenic bone marrow derived MSC from a single donor stallion. Obtained cells were processed, cultured and frozen until 15 days before the intratesticular application. After thawing, the MSCs were plated and expanded to confluence to provide 20 106 cells for each animal. For intratesticular application, 18 adult stallions (3-4 yr old) were divided into two groups: 5mL sterile phosphate saline buffer (Control; n¼9) and 5mL of the cell suspension (MSC; n¼9; 10 106 cells/testicle). The injection procedure was performed in the medial curvature of each testicle.The TV was calculated as length X width X height X 0.53 cm3, the SST was obtained using an infrared thermographer (Flir Systems; oC) and the sperm motility was assessed by computer-assisted sperm analyzer (CASA, HTM IVOS). The parameters were monitored at the moment of application (M0) and one day after application (M1)for TV, SST and sperm motility. After 48h of application (M2), only the SST was measured. Parametric data were examined by ANOVA followed by Tukey’s test and non-parametric data by Kruskal-Wallis followed by Dunn test with a significance level if p< 0.05.No difference was observed in the Control group for SST in M0 (29.9±1.2), M1 (29.4 ± 1.0) and M3 (29.6 ± 1.0); for TV in M0 and M1 (136.1 ± 22.2 vs 150.9 ± 73.1), respectively. However, on MSC, the M1 (29.5±0.78) showed a significant increase of SST when compared to M0 (28.4 ± 0.97) (p< 0.05). The SST showed similar results at M0 and M2 (29.3 ± 0.56). No difference was observed in MSC group, for TV in M0 and M1 (162.9 ± 50.3 vs 140.1 ± 36.5), and for sperm motility at M0 and M1 (78.2 ± 11.8 vs 75.0 ± 17.3). In conclusion, the injection of MSCs into testicles did not affect the TV and sperm motility. The intratesticular application of MSCs increased the SST at 24h, however, after 48h returned to physiological, suggesting that this procedure can be safely used for scientific and/or therapeutic purposes. Studies are in progress to determine the effect of MSCs injection on spermatogenesis.
Key Words: stem cells, inflammatory reaction, testicle
48 Intra-uterine injection of equine umbilical cord mesenchymal stem cells in the mare D. Casarini 1, A. Josson 1, N. Saulnier 2 1 VetAgro Sup, 69280 Marcy L’Etoile, France; 2 Vetbiobank, 69280 Marcy L’Etoile, France
Numerous reviews describe the relationship between fertility and specific pathological changes in the mare’s endometrium, but more studies are needed to show if endometrial pathology can be treated topically. Therefore, we designed a preliminary study whereby 2 reproductive healthy Standardbred mares of 8 and 10 yrs old were used to investigate if an increasing dose of equine umbilical cord mesenchymal stem (eUCMS) cells may have an effect on the mare’s endometrium. The eUCMS cells were donated to us by Vetbiobank (France) after being fully characterised following ISCT (International Society for Cellular Transplantation) recommendations. Reported immunophenotype is CD90+/29+/ 44+/105+/MHC2-/CD45- This type of cells are known to differentiate into mesodermal lineage upon induction. As soon as each mare passed transition, she was examined 2 to 3 times weekly by transrectal ultrasound from April to October. Prior to the study, an endometrial culture, uterine cytology and endometrial biopsy were performed during diestrus followed by 1 ml of PGF2 alpha IM. Both mares received between 5 and 30 million eUCMS cells in 20 ml PBS intra-uterine (IU) during 3 estrous cycles each. All IU infusions of eUCMS cells were done when the mare showed estrus with a minimum of a 38 mm follicle and a grade 1 endometrial edema (grade 1 for minimal and grade 4 for maximal) Twenty four hours after eUCMS cells infusion, a uterine swab for culture and a uterine lavage for cytology were performed using between 0.5 and 1 L Ringers Lactate to search for bacteria and neutrophils. In two of the six cultures one contaminant (Granulicatella adiacens) and one weak growth of Streptococcus equi subspecies zooepidemicus were isolated; all other four cultures remained negative. All six endometrial cytologies remained negative through the entire study. At the end of the study an endometrial biopsy was performed on each mare. The results of these biopsies, a grade 1, remained unchanged. Both mares were subsequently inseminated with 1 billion motile sperm with >90% total motility and 14 days later were diagnosed pregnant. We concluded that IU infusions of eUCMS cells, even at 30 million, did not cause any inflammation or irritation of the endometrium which would interfere with fertility. Further research is needed to investigate if the IU use of eUCMS cells would be of benefit in post breeding endometritis, metritis, pyometra or endometriosis. Key Words: mare, stem cells, uterus
49 Plasma membrane lipid profile from resistant and sensitive spermatozoa of Mangalarga Marchador stallions after cooling at 5 C C. Ramires Neto 1, K.R.A. Belaz 2, Y. F. R. Sancler Silva 1, M.J. Sudano 3, D. Zampieri 2, A. Tata 2, M.N. Eberlin 2, C.P. FreitasDell'aqua 1, E.M.S. Santana 1, F.O. Papa 1, M.A. Alvarenga 1 1 ~o Animal e Radiologia Veterina ria da Departamento de Reproduça ria e Zootecnia, UNESP, Botucatu, Faculdade de Medicina Veterina ~o Paulo, Brazil; 2 Instituto de Química, Universidade Estadual de Sa ~o Paulo, Brazil; 3 Universidade Federal do Campinas, Campinas, Sa Pampa, Bage, Rio Grande do Sul, Brazil Equine cooled semen is routinely used for artificial insemination. However, a large proportion of stallions present sperm cells with poor tolerance to the cooling process. The lipid composition of the sperm plasma membrane has been suggested as a factor affecting sperm to tolerate low temperatures. Therefore, the aim of this study was to evaluate and compare membrane phospholipid profiles from stallions presenting sperm resistant or less tolerant to cooled-storage at 5 C. Two ejaculates from each of 20 Mangalarga Marchador stallions were used. All stallions had high fertility (more than 60% with fresh semen) when using fresh