Background: In vitro assisted reproductive techniques in horses have experienced considerable improvement in the last years. Although the results of in vitro fertilization are still very poor in horses, intracytoplasmatic sperm injection (ICSI) has been proved to be an efficient and successful technique to produce in vitro embryos and is become very popular in the equine industry 1. The big advantage of the ICSI technique is that embryos can be obtained from poor quality semen or very low sperm numbers which would be insufficient for a normal insemination 1,2. An additional advantage of ICSI, is that multiple oocytes can be retrieved from the ovaries and therefore multiple oocytes of the same mare can be fertilized 1,3,4. Before ICSI can take place, the oocytes have to mature to metaphase II in vitro. Equine oocytes meiotic competence depends upon many follicular and technical factors 5. Some laboratories have a strict selection of oocytes before maturation, for example in cattle only the compact cumulus oocytes complex oocytes (Cp oocytes) are selected to undergo ICSI procedure. Cp oocytes can originate from mature follicles, or mainly from immature follicles. In the horse most of those Cp oocyte are not competent enough for in vitro maturation to metaphase II. Therefore, a similar selection of equine oocytes would eliminate the majority of meiotic competent oocytes in the mare. Moreover, expanded cumulus oocyte complex oocytes (Ex oocytes) have a significant higher prevalence to maturate into metaphase I and metaphase II than Cp oocytes 5,6. Ex oocytes also have a significant lower prevalence of degeneration than CP oocytes and require less time to prepare the germinal vesicle breakdown 6,7. Maturation of equine oocytes is a complex process, involving several nuclear and cytoplasmic developmental changes, and is essential in order to become viable, fertilizable and competent for further development. Several different types of media and incubation periods have been evaluated for the maturation of equine oocytes 8. However, maturation to metaphase II have been disappointing in most of the media 9. Not only technical factors have an influence on the meiotic competence of equine oocytes. In most of mammalian species, reproductive success decreases with maternal age. Aging induces multifactorial changes in the reproductive system which in turn affect oocyte competence probably through altering their mitochondria functionality 10,11. Maternal aging has been associated with increased susceptibility to mitochondrial damage and loss in equine oocytes during in vitro maturation 10,11. Other underlying age dependent causes of de-creased reproductive success have been proposed. It was shown that oocytes from old women (40-45 years) showed more abnormalities in chromosome placement in metaphase plates if compared with oocytes from young women (20-25 years). Spindle abnormalities may be caused by changing in regulatory factors and/or altered the timing of the phases of meiosis resulting in microtubule irregularities and unusual chromosome placement 12. Similarly to women, also in the equine species chromosomal abnormalities in the oocytes have been proposed as a cause of early embryonic loss, however no information is currently available on the incidence of these abnormalities in horses 13. Moreover the majority of the studies published so far are on mice and there is no information on the effect of age on equine oocyte quality. The aim of the current study is to focus on the effect of maternal age, initial cumulus appearance and maturation medium on the quality of equine oocytes from slaughterhouse material, based on oocyte morphology and phase of replication. Material and Methods: All the ovaries were obtained from 161 slaughtered mares aging 2 to 26 years in the months of November-December 2014 and June-December 2015. The ovaries were divided in two groups; ovaries from young mares (age up to 14 years) and ovaries of old mares (more than 15 years of age). In the 1st experiment, oocytes (n=170) recovered were washed 4 times in H-SOF. After the washing steps, the oocytes were matured in vitro to reach metaphase II. Two different in vitro maturation (IVM) media were used. The first medium (“SR” medium) contained Dulbecco’s Modified Eagle Medium (DMEM) 3,6 ml (Gibco, The Netherlands), Serum Replacement 400 μl (Gibco, The Netherlands), Human Epidermal Growth Factor (EGF) 4 μl (Peprotech, USA), Cysteine+Cysteamine 40 μl ( Sigma, USA), Lactate solution 40 μl (Sigma, USA), Insuline, Transferin, Selenite (ITS) 4 μl (VWR International BV, the Netherlands), Follicle Stimulating Hormone (FSH) 4 μl (Sigma, USA). The second medium (“FCS”medium) contained instead of 400 μl SR, 400 μl of Fetal Calf Serum (FCS) (Gibco, the Netherlands). All oocytes were incubated for 24 to 36 hours at 38,5oC with 5% CO2 and 5% O2 in 5 ml tubes containing 500 μl IVM medium and a maximum of 25 oocytes per tube. We divided them in 4 groups, depending of the age and media: Serum Replacement - young oocytes, Fetal Calf Serum - young oocytes, Serum Replacement - old oocytes, Fetal Calf Serum - old oocytes. After maturation, oocytes were denuded from the cumulus cells, fixed and stained. All oocytes were assessed for morphology and nuclear maturation under the microscope. In the 2nd experiment, oocytes (n=815) recovered were matured only in FCS medium at the same previous conditions. They were divided in 4 groups depending of the age and initial cumulus appearance: COCs old – compact, COCs old – expanded, COCs young – compact and COCs young – expanded. After denudation, each oocyte from each group was directly assessed for maturation stage (GV, MI, MII) using the Micromanipulator (Eppendorf TransferMan® NK2). Results: Oocytes matured in FCS appeared to have a more expanded cumulus oocyte complex than the oocyte matured in SR. Also in the denudation process, the oocytes out of FCS media were more easier to denudate than those out of SR media. Although no significant difference in oocyte morphology and DNA maturation between the two age group was observed, the percentage of oocytes that matured to metaphase II, was higher in the old group 84,6%) compared to the young group (67,7%). Lastly, independently of the age, the percentage of oocytes that matured to metaphase II was higher in the expanded group (48,2%) compared to the compact group (25,1%). Discussion and Conclusion: In the current study, based on the cumulus appearance, the morphology and the phase of replication neither the maternal age nor the media had an effect on the developmental abilities and quality of equine oocyte from slaughterhouse material. Although we saw a difference in the expansion of the cumulus-oocytes-complex between the two different media, we did not record any significant difference in oocyte quality or developmental ability between the two media used. Moreover, as already described in literature and in accordance with it, meiotic competence of horse oocytes is dependent upon initial cumulus configuration, with a higher rate of maturation for the expanded oocytes compared to the compact ones. Therefore we concluded that both of our media could be used to mature equine oocytes to metaphase II. However, it is possible that with the current methods we underestimated the effect of maternal age on oocyte developmental capacity; in fact a study evaluating the spindle morphology and alignment or the centromeres cohesion could possibly have revealed more subtly effects of aging on the spindle assembly abilities. Therefore further research is needed to better understand the effect of advanced maternal age on oocyte quality in mares. References 1. Hinrichs K. Update on equine ICSI and cloning. Theriogenology 2005;64(3):535-41. 2. Stout T. Equine embryo transfer: Review of developing potential. Equine Vet J 2006;38(5): 467-78. 3. Hinrichs K. Assisted reproduction techniques in the horse. Reproduction, Fertility and Development 2012;25(1):80-93. 4. Hinrichs K. Application of assisted reproductive technologies (ART) to clinical practice. Proc am assoc equine pract; 2010. 5. Hinrichs K. The equine oocyte: Factors affecting meiotic and developmental competence. Mol Reprod Dev 2010;77(8):651-61. 6. Hinrichs K. In vitro production of equine em-bryos: State of the art. Reproduction in Do-mestic Animals 2010;45(s2):3-8. 7. Hinrichs K, Williams KA. Relationships among oocyte-cumulus morphology, follicular atresia, initial chromatin configuration, and oocyte meiotic competence in the horse. Biol Reprod 1997 Aug;57(2):377-84. 8. Carneiro G, Lorenzo P, Pimentel C, Pegoraro L, Bertolini M, Ball B, Anderson G, Liu I. Influence of insulin-like growth factor-I and its in-teraction with gonadotropins, estradiol, and fetal calf serum on in vitro maturation and parthenogenic development in equine oocytes. Biol Reprod 2001 Sep;65(3):899-905. 9. Tremoleda J, Schoevers E, Stout T, Colenbrander B, Bevers M. Organisation of the cytoskeleton during in vitro maturation of horse oocytes. Mol Reprod Dev 2001;60(2):260-9. 10. Rambags B, van Boxtel D, Tharasanit T, Lenstra J, Colenbrander B, Stout T. Advancing maternal age predisposes to mitochondrial damage and loss during maturation of equine oocytes in vitro. Theriogenology 2014;81(7):959-65. 11. Altermatt J, Suh T, Stokes J, Carnevale E. Effects of age and equine follicle- stimulating hor-mone (eFSH) on collection and viability of eq-uine oocytes assessed by morphology and developmental competency after intracyto-plasmic sperm injection (ICSI). Reproduction, Fertility and Development 2009;21(4):615-23. 12. Battaglia DE, Goodwin P, Klein NA, Soules MR. Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum Reprod 1996 Oct; 11(10):2217-22. 13.Rambags B, Krijtenburg P, Van Drie H, Lazzari G, Galli C, Pearson P, Colenbrander B, Stout T. Numerical chromosomal abnormalities in eq-uine embryos produced in vivo and in vitro. Mol Reprod Dev 2005;72(1):77-87.

Effetti dell’età e dei media di maturazione sulla qualità e capacità di sviluppo di oociti prelevati da ovaie di cavalle dopo macellazione

RIZZO, MARILENA
2017-02-20

Abstract

Background: In vitro assisted reproductive techniques in horses have experienced considerable improvement in the last years. Although the results of in vitro fertilization are still very poor in horses, intracytoplasmatic sperm injection (ICSI) has been proved to be an efficient and successful technique to produce in vitro embryos and is become very popular in the equine industry 1. The big advantage of the ICSI technique is that embryos can be obtained from poor quality semen or very low sperm numbers which would be insufficient for a normal insemination 1,2. An additional advantage of ICSI, is that multiple oocytes can be retrieved from the ovaries and therefore multiple oocytes of the same mare can be fertilized 1,3,4. Before ICSI can take place, the oocytes have to mature to metaphase II in vitro. Equine oocytes meiotic competence depends upon many follicular and technical factors 5. Some laboratories have a strict selection of oocytes before maturation, for example in cattle only the compact cumulus oocytes complex oocytes (Cp oocytes) are selected to undergo ICSI procedure. Cp oocytes can originate from mature follicles, or mainly from immature follicles. In the horse most of those Cp oocyte are not competent enough for in vitro maturation to metaphase II. Therefore, a similar selection of equine oocytes would eliminate the majority of meiotic competent oocytes in the mare. Moreover, expanded cumulus oocyte complex oocytes (Ex oocytes) have a significant higher prevalence to maturate into metaphase I and metaphase II than Cp oocytes 5,6. Ex oocytes also have a significant lower prevalence of degeneration than CP oocytes and require less time to prepare the germinal vesicle breakdown 6,7. Maturation of equine oocytes is a complex process, involving several nuclear and cytoplasmic developmental changes, and is essential in order to become viable, fertilizable and competent for further development. Several different types of media and incubation periods have been evaluated for the maturation of equine oocytes 8. However, maturation to metaphase II have been disappointing in most of the media 9. Not only technical factors have an influence on the meiotic competence of equine oocytes. In most of mammalian species, reproductive success decreases with maternal age. Aging induces multifactorial changes in the reproductive system which in turn affect oocyte competence probably through altering their mitochondria functionality 10,11. Maternal aging has been associated with increased susceptibility to mitochondrial damage and loss in equine oocytes during in vitro maturation 10,11. Other underlying age dependent causes of de-creased reproductive success have been proposed. It was shown that oocytes from old women (40-45 years) showed more abnormalities in chromosome placement in metaphase plates if compared with oocytes from young women (20-25 years). Spindle abnormalities may be caused by changing in regulatory factors and/or altered the timing of the phases of meiosis resulting in microtubule irregularities and unusual chromosome placement 12. Similarly to women, also in the equine species chromosomal abnormalities in the oocytes have been proposed as a cause of early embryonic loss, however no information is currently available on the incidence of these abnormalities in horses 13. Moreover the majority of the studies published so far are on mice and there is no information on the effect of age on equine oocyte quality. The aim of the current study is to focus on the effect of maternal age, initial cumulus appearance and maturation medium on the quality of equine oocytes from slaughterhouse material, based on oocyte morphology and phase of replication. Material and Methods: All the ovaries were obtained from 161 slaughtered mares aging 2 to 26 years in the months of November-December 2014 and June-December 2015. The ovaries were divided in two groups; ovaries from young mares (age up to 14 years) and ovaries of old mares (more than 15 years of age). In the 1st experiment, oocytes (n=170) recovered were washed 4 times in H-SOF. After the washing steps, the oocytes were matured in vitro to reach metaphase II. Two different in vitro maturation (IVM) media were used. The first medium (“SR” medium) contained Dulbecco’s Modified Eagle Medium (DMEM) 3,6 ml (Gibco, The Netherlands), Serum Replacement 400 μl (Gibco, The Netherlands), Human Epidermal Growth Factor (EGF) 4 μl (Peprotech, USA), Cysteine+Cysteamine 40 μl ( Sigma, USA), Lactate solution 40 μl (Sigma, USA), Insuline, Transferin, Selenite (ITS) 4 μl (VWR International BV, the Netherlands), Follicle Stimulating Hormone (FSH) 4 μl (Sigma, USA). The second medium (“FCS”medium) contained instead of 400 μl SR, 400 μl of Fetal Calf Serum (FCS) (Gibco, the Netherlands). All oocytes were incubated for 24 to 36 hours at 38,5oC with 5% CO2 and 5% O2 in 5 ml tubes containing 500 μl IVM medium and a maximum of 25 oocytes per tube. We divided them in 4 groups, depending of the age and media: Serum Replacement - young oocytes, Fetal Calf Serum - young oocytes, Serum Replacement - old oocytes, Fetal Calf Serum - old oocytes. After maturation, oocytes were denuded from the cumulus cells, fixed and stained. All oocytes were assessed for morphology and nuclear maturation under the microscope. In the 2nd experiment, oocytes (n=815) recovered were matured only in FCS medium at the same previous conditions. They were divided in 4 groups depending of the age and initial cumulus appearance: COCs old – compact, COCs old – expanded, COCs young – compact and COCs young – expanded. After denudation, each oocyte from each group was directly assessed for maturation stage (GV, MI, MII) using the Micromanipulator (Eppendorf TransferMan® NK2). Results: Oocytes matured in FCS appeared to have a more expanded cumulus oocyte complex than the oocyte matured in SR. Also in the denudation process, the oocytes out of FCS media were more easier to denudate than those out of SR media. Although no significant difference in oocyte morphology and DNA maturation between the two age group was observed, the percentage of oocytes that matured to metaphase II, was higher in the old group 84,6%) compared to the young group (67,7%). Lastly, independently of the age, the percentage of oocytes that matured to metaphase II was higher in the expanded group (48,2%) compared to the compact group (25,1%). Discussion and Conclusion: In the current study, based on the cumulus appearance, the morphology and the phase of replication neither the maternal age nor the media had an effect on the developmental abilities and quality of equine oocyte from slaughterhouse material. Although we saw a difference in the expansion of the cumulus-oocytes-complex between the two different media, we did not record any significant difference in oocyte quality or developmental ability between the two media used. Moreover, as already described in literature and in accordance with it, meiotic competence of horse oocytes is dependent upon initial cumulus configuration, with a higher rate of maturation for the expanded oocytes compared to the compact ones. Therefore we concluded that both of our media could be used to mature equine oocytes to metaphase II. However, it is possible that with the current methods we underestimated the effect of maternal age on oocyte developmental capacity; in fact a study evaluating the spindle morphology and alignment or the centromeres cohesion could possibly have revealed more subtly effects of aging on the spindle assembly abilities. Therefore further research is needed to better understand the effect of advanced maternal age on oocyte quality in mares. References 1. Hinrichs K. Update on equine ICSI and cloning. Theriogenology 2005;64(3):535-41. 2. Stout T. Equine embryo transfer: Review of developing potential. Equine Vet J 2006;38(5): 467-78. 3. Hinrichs K. Assisted reproduction techniques in the horse. Reproduction, Fertility and Development 2012;25(1):80-93. 4. Hinrichs K. Application of assisted reproductive technologies (ART) to clinical practice. Proc am assoc equine pract; 2010. 5. Hinrichs K. The equine oocyte: Factors affecting meiotic and developmental competence. Mol Reprod Dev 2010;77(8):651-61. 6. Hinrichs K. In vitro production of equine em-bryos: State of the art. Reproduction in Do-mestic Animals 2010;45(s2):3-8. 7. Hinrichs K, Williams KA. Relationships among oocyte-cumulus morphology, follicular atresia, initial chromatin configuration, and oocyte meiotic competence in the horse. Biol Reprod 1997 Aug;57(2):377-84. 8. Carneiro G, Lorenzo P, Pimentel C, Pegoraro L, Bertolini M, Ball B, Anderson G, Liu I. Influence of insulin-like growth factor-I and its in-teraction with gonadotropins, estradiol, and fetal calf serum on in vitro maturation and parthenogenic development in equine oocytes. Biol Reprod 2001 Sep;65(3):899-905. 9. Tremoleda J, Schoevers E, Stout T, Colenbrander B, Bevers M. Organisation of the cytoskeleton during in vitro maturation of horse oocytes. Mol Reprod Dev 2001;60(2):260-9. 10. Rambags B, van Boxtel D, Tharasanit T, Lenstra J, Colenbrander B, Stout T. Advancing maternal age predisposes to mitochondrial damage and loss during maturation of equine oocytes in vitro. Theriogenology 2014;81(7):959-65. 11. Altermatt J, Suh T, Stokes J, Carnevale E. Effects of age and equine follicle- stimulating hor-mone (eFSH) on collection and viability of eq-uine oocytes assessed by morphology and developmental competency after intracyto-plasmic sperm injection (ICSI). Reproduction, Fertility and Development 2009;21(4):615-23. 12. Battaglia DE, Goodwin P, Klein NA, Soules MR. Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum Reprod 1996 Oct; 11(10):2217-22. 13.Rambags B, Krijtenburg P, Van Drie H, Lazzari G, Galli C, Pearson P, Colenbrander B, Stout T. Numerical chromosomal abnormalities in eq-uine embryos produced in vivo and in vitro. Mol Reprod Dev 2005;72(1):77-87.
20-feb-2017
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