Offspring of pre-determined sex has been obtained in several species with the use of sex-sorted semen by flowcytometry followed by artificial insemination or assisted reproduction techniques. Nevertheless in some species such as pig, sheep and horse efficiency is highly reduced compared to conventional AI due to the high number of sperm required for insemination. To obtain offspring from semen available in very limited amounts and with poor motility, as always occurs with sex-sorted frozen stallion semen, the application of the in vitro assisted reproductive techniques, in particular intracytoplasmic sperm injection has become a real option. The scope of this work was to investigate the use of sex-sorted frozen stallion semen for the production of embryos in vitro following ICSI, in vitro culture, freezing and thawing, and their viability after transfer to recipients to produce offsprings. Ejaculates were collected from 2 Standardbred stallions of proven fertility (A, B). Only the ejaculates containing >60% motile spermatozoa after dilution were used for subsequent assessment. Briefly semen samples were diluted, stained with Hoechst 33342 and food dye and sorted with a MoFlo SX® flow cytometer/sperm sorter. Flow-cytometrically sorted spermatozoa were deflected into polypropylene tubes containing 500 μl of 2 % Tes-Tris-egg yolk buffer, than the collected semen was centrifuged, diluted in freezing medium and frozen. Sex-sorted and control non-sexed frozen semen (two stallions of in vitro proven fertility, C, D) was thawed, centrifuged on a Percoll gradient, washed and diluted 1:1 in PVP before ICSI. Oocytes were collected from ovaries of slaughtered mares at the beginning of the breeding season and matured in vitro. Metaphase II oocytes were injected with sperm, subsequently cultured up to the blastocyst stage and frozen in glycerol. Six embryos were subsequently thawed and non-surgically transferred in naturally cycling synchronous recipient 5 days after spontaneous ovulation. Overall 70, 58, 30 and 15 oocytes were injected with sex-sorted and control frozen semen from stallion A, B, C, D respectively. Mean cleavage rates were 20.31% for sorted sexed semen and 71.11% for control showing a significantly lower cleavage rate for sexed semen. This difference was reflected on the number of blastocyst obtained (4.18% vs 20.00%). Out of 6 frozen-thawed embryos transferred we obtained 4 pregnancies, one was lost at 21 days, a second was pharmacologically aborted at 21 days and two were maintained to go to term (one male and one female are currently in the 8th month of gestation). In conclusion, in this study we demonstrated that sex-sorted sperm can be used for producing blastocysts able to establish pregnancies at high rate when transplanted in recipient mares. However the efficiency of the system is low due to the very low cleavage rate obtained with such sex-sorted frozen thawed semen.
Galli C, Colleoni S, Spinaci M, Duchi R, Merlo B, Tamanini C, et al. (2008). Horse pregnancies established with embryos produced in vitro by ICSI with sex-sorted frozen thawed semen.. s.l : s.n.
Horse pregnancies established with embryos produced in vitro by ICSI with sex-sorted frozen thawed semen.
GALLI, CESARE;SPINACI, MARCELLA;MERLO, BARBARA;TAMANINI, CARLO;MARI, GAETANO
2008
Abstract
Offspring of pre-determined sex has been obtained in several species with the use of sex-sorted semen by flowcytometry followed by artificial insemination or assisted reproduction techniques. Nevertheless in some species such as pig, sheep and horse efficiency is highly reduced compared to conventional AI due to the high number of sperm required for insemination. To obtain offspring from semen available in very limited amounts and with poor motility, as always occurs with sex-sorted frozen stallion semen, the application of the in vitro assisted reproductive techniques, in particular intracytoplasmic sperm injection has become a real option. The scope of this work was to investigate the use of sex-sorted frozen stallion semen for the production of embryos in vitro following ICSI, in vitro culture, freezing and thawing, and their viability after transfer to recipients to produce offsprings. Ejaculates were collected from 2 Standardbred stallions of proven fertility (A, B). Only the ejaculates containing >60% motile spermatozoa after dilution were used for subsequent assessment. Briefly semen samples were diluted, stained with Hoechst 33342 and food dye and sorted with a MoFlo SX® flow cytometer/sperm sorter. Flow-cytometrically sorted spermatozoa were deflected into polypropylene tubes containing 500 μl of 2 % Tes-Tris-egg yolk buffer, than the collected semen was centrifuged, diluted in freezing medium and frozen. Sex-sorted and control non-sexed frozen semen (two stallions of in vitro proven fertility, C, D) was thawed, centrifuged on a Percoll gradient, washed and diluted 1:1 in PVP before ICSI. Oocytes were collected from ovaries of slaughtered mares at the beginning of the breeding season and matured in vitro. Metaphase II oocytes were injected with sperm, subsequently cultured up to the blastocyst stage and frozen in glycerol. Six embryos were subsequently thawed and non-surgically transferred in naturally cycling synchronous recipient 5 days after spontaneous ovulation. Overall 70, 58, 30 and 15 oocytes were injected with sex-sorted and control frozen semen from stallion A, B, C, D respectively. Mean cleavage rates were 20.31% for sorted sexed semen and 71.11% for control showing a significantly lower cleavage rate for sexed semen. This difference was reflected on the number of blastocyst obtained (4.18% vs 20.00%). Out of 6 frozen-thawed embryos transferred we obtained 4 pregnancies, one was lost at 21 days, a second was pharmacologically aborted at 21 days and two were maintained to go to term (one male and one female are currently in the 8th month of gestation). In conclusion, in this study we demonstrated that sex-sorted sperm can be used for producing blastocysts able to establish pregnancies at high rate when transplanted in recipient mares. However the efficiency of the system is low due to the very low cleavage rate obtained with such sex-sorted frozen thawed semen.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.