Pregnancy rates obtained in jennies after artificial insemination (AI) using cryopreserved jack semen are very poor in comparison to fresh or cooled semen. It has been hypothesized that the presence of permeable cryoprotectants agents in the freezing extender, and in particular glycerol, may be responsible for causing severe endometritis which result in lower pregnancy rates (Vidament et al. Animal Reproduction Science. 2009; 112:22-35). The aim of this study was to compare uterine inflammatory response of jennies inseminated with vitrified semen in the absence of permeable agents (V) and conventionally frozen semen with glycerol (CF). Spermatozoa from two Amiata donkeys were subjected to vitrification and conventional freezing. Samples were centrifuged, 400×g/7min for vitrification and 600×g/10min for conventional freezing; and resuspended with i) a skimmed milk-egg yolk extender Gent (Minitübe, Germany) supplemented with sucrose at 0.1 molar until a concentration of 300 x 106 spermatozoa/mL (V) or ii) a milk based extender INRA-96 (IMV, L’Aigle, France) with 2.2% glycerol and 2% egg-yolk until 166 x 106 spermatozoa/mL (CF). All samples were slowly cooled to 4ºC. For vitrification, sperm solution was packed in 0.25 mL straws, which were horizontally inserted into 0.5 mL straws, sealed and directly plunged into liquid nitrogen. Conventional freezing was performed following a standard procedure using an automated freezer (Mini-Digitcool, IMV Technologies) at −60°C/min curve from 4 to −140°C. Ten jennies (two cycles/jenny) were inseminated in the uterine body either with vitrified (10 cycles) and frozen semen (10 cycles) after ovulation induction with 0.4 mg/sc buserelin acetate (Suprefact, Sanofi spa, Milano, Italia; 1mg/mL). The uterine inflammatory response was evaluated six hours after AI by uterine lavage with 1 L of Lactated Ringer's Solution and cytology. Endometrial cytology smears were prepared onto glass microscope slides, air-dried and stained with Diff-Quick. A minimum of 200 cells were counted and the percentage of polymorphonuclear neutrophils (PMN, %) was calculated. Results were compared between cryopreservation methods using the Student′s t test and expressed as mean ± standard deviation. No differences (P>0.05) were found between methods regarding inflammatory uterine response: V (67.8% ± 23.5) vs. CF (63.7% ± 27.9). Therefore, the presence of glycerol did not increase the inflammatory response in terms of polymorphonuclear neutrophils production after insemination. In this sense, whether the acute endometritis after AI in jennies with frozen-thawed jack semen is the cause of the poor results remained unclear. Further research is needed including a larger number of animals and cycles employed. This study was supported by project AGL2013-42726-R.
Comparison of uterine inflammatory response of jennies after artificial insemination with vitrified or frozen-thawed donkey sperm
Rota, A.;Panzani, D.;Fanelli, D.;Tesi, M.;Camillo, F.;Monaco, D.;
2020-01-01
Abstract
Pregnancy rates obtained in jennies after artificial insemination (AI) using cryopreserved jack semen are very poor in comparison to fresh or cooled semen. It has been hypothesized that the presence of permeable cryoprotectants agents in the freezing extender, and in particular glycerol, may be responsible for causing severe endometritis which result in lower pregnancy rates (Vidament et al. Animal Reproduction Science. 2009; 112:22-35). The aim of this study was to compare uterine inflammatory response of jennies inseminated with vitrified semen in the absence of permeable agents (V) and conventionally frozen semen with glycerol (CF). Spermatozoa from two Amiata donkeys were subjected to vitrification and conventional freezing. Samples were centrifuged, 400×g/7min for vitrification and 600×g/10min for conventional freezing; and resuspended with i) a skimmed milk-egg yolk extender Gent (Minitübe, Germany) supplemented with sucrose at 0.1 molar until a concentration of 300 x 106 spermatozoa/mL (V) or ii) a milk based extender INRA-96 (IMV, L’Aigle, France) with 2.2% glycerol and 2% egg-yolk until 166 x 106 spermatozoa/mL (CF). All samples were slowly cooled to 4ºC. For vitrification, sperm solution was packed in 0.25 mL straws, which were horizontally inserted into 0.5 mL straws, sealed and directly plunged into liquid nitrogen. Conventional freezing was performed following a standard procedure using an automated freezer (Mini-Digitcool, IMV Technologies) at −60°C/min curve from 4 to −140°C. Ten jennies (two cycles/jenny) were inseminated in the uterine body either with vitrified (10 cycles) and frozen semen (10 cycles) after ovulation induction with 0.4 mg/sc buserelin acetate (Suprefact, Sanofi spa, Milano, Italia; 1mg/mL). The uterine inflammatory response was evaluated six hours after AI by uterine lavage with 1 L of Lactated Ringer's Solution and cytology. Endometrial cytology smears were prepared onto glass microscope slides, air-dried and stained with Diff-Quick. A minimum of 200 cells were counted and the percentage of polymorphonuclear neutrophils (PMN, %) was calculated. Results were compared between cryopreservation methods using the Student′s t test and expressed as mean ± standard deviation. No differences (P>0.05) were found between methods regarding inflammatory uterine response: V (67.8% ± 23.5) vs. CF (63.7% ± 27.9). Therefore, the presence of glycerol did not increase the inflammatory response in terms of polymorphonuclear neutrophils production after insemination. In this sense, whether the acute endometritis after AI in jennies with frozen-thawed jack semen is the cause of the poor results remained unclear. Further research is needed including a larger number of animals and cycles employed. This study was supported by project AGL2013-42726-R.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
