Evaluation of fertilization capability of frozen-thawed completely immotile spermatozoa collected from a white bengal tiger after interspecific ICSI with bovine oocytes

The objective of this study was to evaluate the fertilization capability of White Bengal Tiger frozen-thawed completely immotile spermatozoa after interspecific intracytoplasmic sperm injection (ICSI) with bovine oocytes. The fertilization status of presumptive zygotes was assessed 18 h after ICSI by immunofluorescence staining and confocal microscopy. The fertilization rate was 34.8% (8/23), as confirmed by the extrusion of two polar bodies, or male and female pronuclei formation. For unfertilized oocytes (65.2%, 15/23), one activated oocyte had an activated spermatozoon but most were unactivated oocytes with unactivated spermatozoa (1/15, 6.7% vs 10/15, 66.7%, respectively, p < 0.05). These results showed that White Bengal Tiger frozen-thawed completely immotile spermatozoa retained the capacity to fertilize bovine oocytes after interspecific ICSI. This is the first report of in vitro produced zygotes using tiger immotile sperm with bovine oocytes by interspecific ICSI technique, which provides an efficient and feasible method for preservation and utilization of endangered feline animals.(AU)

Intracytoplasmic sperm injection after vitrifcation of immature oocytes in follicular fluid increases bovine embryo production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes. Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20°C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitroembryo production in order to assess embryo production efficiency. A second set of experiments using the FF-Vitri solution compared IVF versus ICSI. With basis on cleaved structures, the morula + blastocyst rate obtained in the Fresh Control (43.9%) was similar to FF-Vitri (31.1%). Conversely, the TH-Vitri (15.7%) and the TH:FF-Vitri (20.4%) rates were significantly lower than the Fresh Control. ICSI showed a positive effect in comparison with IVF. The embryo development rate of Vitri-IVF (18.8%) was the lowest, whereas Vitri-ICSI (37.3%) was similar to the Fresh-IVF (43.9%), but lower than the Fresh-ICSI (57.8%).[...]

Intracytoplasmic Sperm Injection after Vitrification of Immature Oocytes in Follicular Fluid Increases Bovine Embryo Production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes.Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitro embryo production in order to assess embryo production efficiency. A second set

Intracytoplasmic sperm injection after vitrifcation of immature oocytes in follicular fluid increases bovine embryo production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes. Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20°C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitroembryo production in order to assess embryo production efficiency. A second set of experiments using the FF-Vitri solution compared IVF versus ICSI. With basis on cleaved structures, the morula + blastocyst rate obtained in the Fresh Control (43.9%) was similar to FF-Vitri (31.1%). Conversely, the TH-Vitri (15.7%) and the TH:FF-Vitri (20.4%) rates were significantly lower than the Fresh Control. ICSI showed a positive effect in comparison with IVF. The embryo development rate of Vitri-IVF (18.8%) was the lowest, whereas Vitri-ICSI (37.3%) was similar to the Fresh-IVF (43.9%), but lower than the Fresh-ICSI (57.8%).[...](AU)

Equine assisted reproduction and embryo technologies

Assisted reproductive techniques in the horse have been only recently become available compared to other domestic species, in particular ruminants. The scarce availability of abattoir ovaries and t he lack of interest from horse breeders and breed associations, and the anatomical and physiological differences have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established especially after the application of ICSI to obtain in vitro fertilization. The parallel improvement of oocyte maturation conditions and embryo culture media has increased the rates of embryo development from in vitro matured and in vitro cultured ICSI embryos from 5-10% in the early studies to up to 26% in the latest under experimental conditions with abattoir derived oocytes. In 2003, the birth of the first cloned foal established the technology of somatic cell nuclear transfer. The largest set of data on non-surgical embryo transfer of in vitro produced embryos, from ICSI of in vitro-matured Ovum Pick Up (OPU) oocytes, and from somatic cell nuclear transfer, has been obtained in our laboratory. In the clinical context, where OPU and ICSI are applied for the treatment of female and or male infertility, the yield of embryos has been lower compared to experimental conditions. In conclusion, the basic procedures have been established for the use of assisted reproduction and somatic cell nuclear transfer to a degree suitable for clinical applications and the results have been replicated in several laboratories around the world.

Equine assisted reproduction and embryo technologies

Assisted reproductive techniques in the horse have been only recently become available compared to other domestic species, in particular ruminants. The scarce availability of abattoir ovaries and t he lack of interest from horse breeders and breed associations, and the anatomical and physiological differences have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established especially after the application of ICSI to obtain in vitro fertilization. The parallel improvement of oocyte maturation conditions and embryo culture media has increased the rates of embryo development from in vitro matured and in vitro cultured ICSI embryos from 5-10% in the early studies to up to 26% in the latest under experimental conditions with abattoir derived oocytes. In 2003, the birth of the first cloned foal established the technology of somatic cell nuclear transfer. The largest set of data on non-surgical embryo transfer of in vitro produced embryos, from ICSI of in vitro-matured Ovum Pick Up (OPU) oocytes, and from somatic cell nuclear transfer, has been obtained in our laboratory. In the clinical context, where OPU and ICSI are applied for the treatment of female and or male infertility, the yield of embryos has been lower compared to experimental conditions. In conclusion, the basic procedures have been established for the use of assisted reproduction and somatic cell nuclear transfer to a degree suitable for clinical applications and the results have been replicated in several laboratories around the world.(AU)

Técnicas de reprodução assistida aplicadas a equinos / Assisted Reproductive Techniques applied in Horses

Comparada com as demais espécies domésticas, as técnicas de reprodução assistida particularmente a produção in vitro tem tido um progresso lento na espécie equina. Com exceção das técnicas de Transferência de Embriões onde o Brasil ocupa lugar de destaque no mundo, estas técnicas estão longe de serem utilizados em protocolos de rotina. Apesar disso houve um avanço, principalmente a partir do desenvolvimento das técnicas de GIFT e ICSI que possibilitaram a utilização de oócitos recuperados de animais post-mortem ou pós-aspiração in vivo, desenvolvimento embrionário e geração de prenhezes. A técnica da clonagem apesar de se encontrar em uma fase inicial já vem sendo utilizada comercialmente por algumas empresas.

Compared with other domestic species, assisted reproductive techniques, particularly in vitro production has had a slow progress in the equine species. With the exception of embryo transfer where Brazil occupies a prominent place in the world, these techniques are far from being used in routine protocols. Despite this, it has been a breakthrough, especially from the development of the techniques such as GIFT and ICSI that allowed the use of retrieved oocytes from post-mortem or after aspiration from in vivo animals, generating embryonic development and pregnancies. The Cloning technique, despite being at early stages it is already a reality, and has being used commercially for some Companies.

Técnicas de reprodução assistida aplicadas a equinos / Assisted Reproductive Techniques applied in Horses

Comparada com as demais espécies domésticas, as técnicas de reprodução assistida particularmente a produção in vitro tem tido um progresso lento na espécie equina. Com exceção das técnicas de Transferência de Embriões onde o Brasil ocupa lugar de destaque no mundo, estas técnicas estão longe de serem utilizados em protocolos de rotina. Apesar disso houve um avanço, principalmente a partir do desenvolvimento das técnicas de GIFT e ICSI que possibilitaram a utilização de oócitos recuperados de animais post-mortem ou pós-aspiração in vivo, desenvolvimento embrionário e geração de prenhezes. A técnica da clonagem apesar de se encontrar em uma fase inicial já vem sendo utilizada comercialmente por algumas empresas.(AU)

Compared with other domestic species, assisted reproductive techniques, particularly in vitro production has had a slow progress in the equine species. With the exception of embryo transfer where Brazil occupies a prominent place in the world, these techniques are far from being used in routine protocols. Despite this, it has been a breakthrough, especially from the development of the techniques such as GIFT and ICSI that allowed the use of retrieved oocytes from post-mortem or after aspiration from in vivo animals, generating embryonic development and pregnancies. The Cloning technique, despite being at early stages it is already a reality, and has being used commercially for some Companies.(AU)

Injeção Intracitoplasmática de Espermatozoide: Métodos de ativação oocitária e desestabilização da membrana espermática

O desenvolvimento de embriões bovinos produzidos por injeção intracitoplasmática de espermatozóides (ICSI) é baixo em relação aos embriões fertilizados. Deficiência na ativação de oócitos, capacitação inadequada de espermatozóides e falta de descondensação de espermatozóides são os principais transtornos que afetam o sucesso de ICSI em bovinos. No presente trabalho, foram testados métodos de ativação com o intuito de estabelecer um protocolo efetivo para a ativação de oócitos bovinos após a ICSI. Para isso, um inibidor específico de CDK1 (RO-3306) e um ativador específico de PKC (OAG) foram utilizados após a incubação com Ionomicina (ION) para avaliar a retomada da meiose em oócitos bovinos. Além disso, a incubação em meio Fert durante 6 h e a eletroporação (El) de espermatozoides previamente a ICSI foram testadas para verificar o efeito sobre a descondensação do espermatozoide e formação do pró-núcleo (PN) masculino. Oócitos bovinos foram incubados, em diferentes concentrações e períodos de exposição aos tratamentos. Foram avaliados conforme a taxa de ativação oocitária (formação de PN), clivagem, desenvolvimento a blastocisto e número de células nos embriões que se desenvolveram a blastocisto. As taxas de PN foram maiores (P0,01) nos grupos ativados com ION+RO (48.5 %) e ION+RO+OAG (65.6 %) comparado com ION (12.3 %) e ION+OAG (9.2 %). Não houve efeito significativo entre as concentrações 5.0, 7.5 e 10.0 M de RO sobre a taxa de ativação. A taxa de ativação foi significativamente maior (P0,01) em oócitos tratados por 240 min (84.6 %) comparado a 60 (53.6 %) e 120 min (60.0%). No entanto, não houve diferença significativa na taxa de ativação quando o tratamento com RO foi iniciado a 0, 30 ou 60 minutos após a incubação com ION. A taxa de clivagem foi inferior nos grupos ION (11.8 %) e ION+OAG (22.8 %) comparada aos grupos ION+RO (70.2 %) e ION+RO+OAG (62.4 %). A taxa de blastocistos também foi maior no grupo ION+RO+OAG (24.1 %), mas não houve diferença estatística entre os grupos ION+RO (19.7 %) e ION+OAG (9.5 %). Não houve desenvolvimento a blastocisto quando os oócitos foram tratamento somente com ION. Não foi detectada diferença estatística entre os tratamentos sobre o número médio de células por blastocistos. No segundo experimento, espermatozoides não tratados (ICSI-Cont) ou tratados por electroporação (ICSI-El) foram microinjetados em oócitos, os quais foram ativados com ION+RO por 240 min e fixados cerca de 15 h após a injeção para determinar a taxa de formação de PNs masculino e feminino (2PN). A maioria dos oócitos apresentaram o PN feminino bem desenvolvido (66.4 %). A formação de 2PN (masculino e feminino) foi maior no grupo ICSI-El (33.3 %) comparado ao grupo ICSI-Cont (9.4 %). Em conclusão, esse estudo demonstrou que a inibição específica da CDK1 após o tratamento com ION promove a ativação de oócitos bovinos. O tratamento de espermatozoides com eletroporação melhora a formação do PN masculino após ICSI, mas a taxa é inferior a formação do PN feminino.

Development of bovine embryos produced by intracytoplasmic sperm injection (ICSI) is low compared to fertilized embryos. Deficient oocyte activation, inappropriate sperm capacitation, and lack of sperm decondensation are thought to be the main constrains affecting ICSI success in cattle. In the present study, activation compounds were tested to establish an effective protocol for activation of bovine oocytes, and then used for oocyte activation after ICSI. The activation approach consisted of exposing in vitro matured oocytes to Ionomycin (ION) following by a specific CDK1 inhibitor (RO-3306), a specific PKC activator (OAG) or both RO+OAG. In the first experiment, the rate of activation (pronuclear (PN) formation), cleavage, development to the blastocyst stage, and number of cells per blastocyst were evaluated after oocyte treatment. The PN rates were higher (P0.01) in the groups activated with ION+RO (48.5 %) and ION+RO+OAG (65.6 %) compared to ION (12.3 %) and ION+OAG (9.2 %). There was no significant effect between the RO concentrations tested (5, 7.5 and 10 M) on oocyte activation. The PN rate was significantly higher (P0.01) when oocytes were exposed to RO for 240 min (84.6 %) compared to 60 (53.6 %) and 120 min (60.0 %). However, there was no difference between groups when treatment with RO started at 0, 30 or 60 min after on ION exposure. Cleavage rate was higher in ION+RO (70.2 %) and ION+RO+OAG (62.4 %) groups compared to ION (11.8 %) and ION+OAG (22.8 %). Blastocyst rate was also higher in the ION+RO+OAG (24.1 %) group, but not statistically different between ION+RO (19.7 %) and ION+OAG (9.5 %) groups. There was no development to the blastocyst stage after treatment with ION alone. The average cell number in blastocysts was not statistically different among treatments. In the second experiment, the effect of activation with ION+RO (10 M for 240 min) was tested after ICSI using control (ICSI-Cont) or treated by electroporation (ICSI-El) sperm. Most oocytes presented a well-developed female PN (66.4%). Male PN formation was higher (P0.05) in the ICSI-El (33.3%) compared to the ICSI-Cont (9.4%) group. In conclusion, this study revealed that the specific inhibition of CDK1 after ION treatment is an effective approach to activate bovine oocytes. Male pronuclear formation after ICSI is increased by sperm electroporation, but is lower than female pronuclear formation. This indicates that deficient sperm decondensation and male PN formation rather than deficient oocyte activation is likely the main problem to develop an effective protocol for bovine ICSI.

Recent advances in micromanipulation and transgenesis in domestic mammals

Background: Intracytoplasmic sperm injection (ICSI) involves mechanical transfer of a single sperm cell into ooplasm. A new application has been recently found for ICSI, the production of transgenic animals. Since the birth of ''Dolly'', the first adult somatic cloned mammal, viable offspring has been produced by nuclear transfer in many species including cattle. The present review briefly summarizes our experience with ICSI and somatic cell nuclear transfer mainly to produce transgenic embryos, as well as for the generation of new micromanipulation technique. Review: We have evaluated different factors that affect SCNT and transgenesis including the chemical activator, the transfection event and the effect of recloning. Also, we included a brief description of the ICSI technique, which we used in five different species, examining its potential to produce transgenic embryos. Finally different strategies to produce transgenic animals were analyzed: ICSI- mediated gen transfer (ICSI-MGT), Injection of cumulus cell and ooplasmic vesicle incubated for 5 min with the transgene or injection of the plasmid alone. All of them were very efficient in exogenous DNA expression at embryo stages but resulted in mosaic embryos. We demonstrated that "ICSI-MGT" assisted by chemical activation is the only treatment of sperm mediated gen transfer capable to generated transgenic embryos in ovine. Besides, after ICSI-MGT, it is possible to obtain enhanced green fluorescent protein (EGFP)-expressing embryos in five diferent species: ovine, porcine, feline, bovine and equine. Our studies also established for the first time that short term transgene co-incubation with somatic cells can produce transgene-expressing mammalian SCNT embryos, and also that parthenogenic, eDNA- expressing embryos can be obtained by injection of vesicles or eDNA alone. Moreover, eDNA- -expressing embryos can be also obtained by cytoplasmic injection of vesicles in IVF zygotes, simplifying the traditional IVF pronuclear injection technique. We tried a further simplification of the technique in bovine oocytes and zygotes, by intracytoplasmically injecting them with eDNA-liposomes complexes. Approximately 70% of the cleaved embryos and 50% of the blastocysts expressed EGFP, when egfp­liposome was injected 16 h post-fertilization. Different approaches were assayed to reverse the mosaicism including a novel technique of gamete cloning. Our first approach consisted of the production of transgenic IVF embryos by vesicle microinjection to generate transgenic blastomeres to be used as donor cells for cloning. A high efficiency in mosaicism reversal and multiplication of transgenic embryos was attaineded. Other technique assayed was the separation of transgenic blastomeres followed by the aggregation of two-cell fused embryos or by the asynchronous younger blastomere successfully multiplied transgenic embryos, and theoretically reduces mosaicism rates in future offspring [15]. This technology can also be used to multiply embryos from animals with high genetic value. We demonstrated that a sperm and oocyte can be efficiently cloned. Green haploid androgenic blastomeres produced with the injection of a single sperm by egfp ICSI-MGT could be used to fertilized oocytes resulting in several homogeneous expressing embryos. This approach shows great potential because it allows for determination of the sex of the sperm nucleus prior to fertilization. It is also possible to clone previously transfected oocytes followed by the reconstruction of biparental bovine embryos to generate homogeneous transgene-expressing embryos. This review summarizes recent experiments in micromanipulation and gene transfer in domestic animals. The objective is not to exhaustedly describe the research done in this field but to present the promising methods recently developed or evaluated in our lab. Conclusion: Significant advancements have been made in the course of the recent years in micromanipulation and transgenesis techniques. In our lab we have been evaluating ICSI and Nuclear transfer mainly to produce transgenic embryos. We used also transgensis to apply or developed new micromanipulation technique in domestic animals linke sperm and oocyte cloning.

Intracytoplasmic sperm injection in the horse: the ultimate blind date

Background: Intracytoplasmic sperm injection (ICSI) has become a useful technology to produce foals when availability of semen is limited or when in-vitro fertilization is desired, as is the need for subfertile mares. However, its application into clinical practice is challenging. The purpose of this review was to discuss some fundamental molecular aspects of oocyte maturation that should be considered when performing ICSI and to report factors of age and subfertility affecting the success of a commercial ICSI program. Review: The molecular synchrony of oocyte maturation included nuclear, epigenetic and cytoplasmic maturation. Oocyte developmental competence was found to be dependent on the ability to remain in meiotic arrest until the initiation of final maturation, requiring the adequate timing involved with follicular maturation prior to ovulation. Studies performed in cattle and humans have demonstrated that in-vivo oocyte maturation results in high pregnancy rates per oocyte fertilized. Therefore, determining precise maturation of the oocyte would be valuable for the timing of ICSI and subsequent embryo development. Reproductive aging in the mare was characterized by a decline in fertility. Using RT-PCR, quantitative and temporal differences were found in mRNA content of key regulatory maturation genes in granulosa and cumulus cells and in oocytes during in vivo maturation in young and old mares. These results suggested premature oocyte maturation in aged mares that potentially could result in subfertility. Consequently, the timing of oocyte retrival after gonadotropin administration should be carefully evaluated when performing ICSI. In a commercial program, equine patients were classified into normal mares (2.5 to 15 years), problem mares (15-23 years that had not been producing embryos or pregnancies) and old mares (>24 years). Old mares were assessed for endocrine, physical and nutritional imbalances. Follicular and oocyte maturation were induced with a dominant follicle >30 mm in diameter after a normal growth and blood flow and uterine edema with a combination of hCG and GnRH. Transvaginal oocyte retrieval was performed 20 hours after administration of gonadotropins. Oocytes were further cultured in vitro for 12 to 20 hours. Frozen semen was used for all sperm injections. Injected oocytes were further cultured in vitro for at least 24 hours. Embryos were then transferred surgically into oviducts of synchronized recipients. Oocyte recovery rate was 94% (523/557 cycles), cycles per month were 3.3, 2 and 1.3 for young, problem and old mares respectively. Cleavage rates were different (p < 0.05) between young (82 %), problem (70 %) and old (52%) mares. Pregnancy rates at day 60 were also different (p < 0.05) for young (68 %), problem (50 %) and old (23%) mares. Number of pregnancies obtained from a single straw of frozen semen ranged from 2 to 12. Reproductive senescence was observed in 10% of old mares. In addition, Cushing's disease and elevated diestrus FSH were observed in 80% of the old mares. Foaling rates were evaluated in 55 pregnancies; 5% were lost in the last trimester and the remaining foals have shown no apparent abnormalities. Conclusion: More studies are needed to further elucidate the mechanisms of oocyte maturation and activation in the horse, as well as more objective methods to determine oocyte maturity and quality. A clinical ICSI program required an understanding of gamete physiology and detailed mare reproductive management. Our clinical data demonstrated that aging affects fertility profoundly in ways that may be difficult to address with current technology; nonetheless, ICSI has provided the equine industry an alternative to produce offspring from valuable mares and stallions that are subfertile.

Human assisted reproduction

Background: Infertility is defined as the failure to conceive after one year of unprotected intercourse, and this time has been lowered to 6 month if the female partner is older than 35 years. Infertile couples are offered to low and high complexity treatments are available according to their cause of infertility. Low complexity treatments comprise timed intercourse, intrauterine insemination with or without controlled ovarian stimulation. High complexity treatments comprise standard in vitro fertilization (IVF) and IVF by intracytoplasmatic sperm injection (ICSI). Additionally to these treatments, infertile patients might be benefited by accessory techniques such as preimplantational genetic screening and diagnosis. These techniques aim to detect the most common human aneuploydies related to abortion and chromosomal syndromes; and to identify embryos carriers of genetic diseases like talassemia, Huntington's disease, fragile-X syndrome among others. Human assisted reproduction is a dynamic branch of Medicine performed by several medical specialists from gynecologists to surgeons, and professional as nurses, biologists and veterinarians. Review: Initially, human infertility was treated by intracervical insemination due to the risk of endometrial insemination and the occurrence of pelvic inflammation, but the development of better insemination techniques and instruments led to the report of a safe intrauterine insemination method in 1974. However, insemination was restricted to oligozoospermia or cervical factors and it was not able to overcome ovarian and tubal infertility factors, neither severe male infertility factor. The range of infertility treatment was tremendously broadened in 1978 with the report of the first IVF baby birth. Since then infertile couples due to tubal factors, ovarian and moderate male infertility factors started to be treated with IVF. Several changes in embryo culture conditions and instruments for IVF were developed and the technology was world wide spread. The first IVF baby was a girl born in 1984 in Brazil. Even though, some ovarian conditions and severe male factor infertility couldn't be treated until 1992. This landmark was the report of the first babies originated by in vitro fertilization with ICSI. In parallel to those scientific and medical evolutions, drugs and new protocols of controlled ovarian stimulation increased the number of oocytes available for fertilization and, consequently, the number of exceeding embryos. These spare oocytes and embryos required the development of cryopreservation techniques for long term storage. Slow rate freezing and vitrification of embryos were reported approximately 25 years ago, but just recently vitrification of oocytes and embryos became the first option for fertility preservation and/or embryo storage in humans. Preimplantational genetic screening and diagnosis were complementary techniques developed during the same period. These tests require precise and meticulous micromanipulation techniques and skills for the obtainment of a single blastomere with intact genetic material for screening of aneuploydies by fluorescent in situ hybridization or the detection of a deleterious allele by single cell PCR. New drugs, protocols, instruments and techniques for Human Assisted Reproduction are still under development in many Universities, infertility clinics and private companies aiming to increase the efficiency of infertility treatments. Conclusion: Human assisted reproduction is constantly evolving with the development of more precise diagnostic tests and with the improvement of clinical approaches to infertility and better conditions in embryology laboratories and this constant evolution is the result of the synergic interaction of clinical infertility specialists with researchers of different backgrounds.

New assisted reproductive technologies applied to the horse industry: successes and limitations

The development of assisted reproduction technologies (ART) in the horse has been slow compared with that in other large domestic animals. Besides artificial insemination and embryo transfer, other technologies based on in vivo and in vitro procedures of embryo production (IVP) have appeared, but the success rates of equine IVP are still far from allowing their use in routine protocols. Intracytoplasmic sperm injection (ICSI) is one of the most promising techniques applicable to the horse industry. With ICSI just one spermatozoon is injected into a mature oocyte, allowing the use of poor quality semen that could not otherwise be used for artificial insemination. Moreover, ICSI, followed by in vitro culture to the blastocyst stage, may be used in cases where multiple oocytes are available (e.g. when oocytes are obtained post-mortem). Those are just some examples to highlight the importance of ICSI in preserving genetic material. Cloning by Nuclear transfer (NT) can also be used for salvaging valuable equine genetics. The cloning process utilizing somatic cells is a powerful instrument for the preservation of animals with a unique genotype. Although recent reports on horse cloning show that it can be performed relatively efficiently, compared with other species, blastocyst production and thus live foal production is still low with this technique.(AU)

New assisted reproductive technologies applied to the horse industry: successes and limitations

The development of assisted reproduction technologies (ART) in the horse has been slow compared with that in other large domestic animals. Besides artificial insemination and embryo transfer, other technologies based on in vivo and in vitro procedures of embryo production (IVP) have appeared, but the success rates of equine IVP are still far from allowing their use in routine protocols. Intracytoplasmic sperm injection (ICSI) is one of the most promising techniques applicable to the horse industry. With ICSI just one spermatozoon is injected into a mature oocyte, allowing the use of poor quality semen that could not otherwise be used for artificial insemination. Moreover, ICSI, followed by in vitro culture to the blastocyst stage, may be used in cases where multiple oocytes are available (e.g. when oocytes are obtained post-mortem). Those are just some examples to highlight the importance of ICSI in preserving genetic material. Cloning by Nuclear transfer (NT) can also be used for salvaging valuable equine genetics. The cloning process utilizing somatic cells is a powerful instrument for the preservation of animals with a unique genotype. Although recent reports on horse cloning show that it can be performed relatively efficiently, compared with other species, blastocyst production and thus live foal production is still low with this technique.

Intracytoplasmic Sperm Injection after Vitrification of Immature Oocytes in Follicular Fluid Increases Bovine Embryo Production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes.Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitro embryo production in order to assess embryo production efficiency. A second set

Intracytoplasmic Sperm Injection after Vitrification of Immature Oocytes in Follicular Fluid Increases Bovine Embryo Production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes.Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitro embryo production in order to assess embryo production efficiency. A second set

Intracytoplasmic Sperm Injection after Vitrification of Immature Oocytes in Follicular Fluid Increases Bovine Embryo Production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes.Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitro embryo production in order to assess embryo production efficiency. A second set

Intracytoplasmic Sperm Injection after Vitrification of Immature Oocytes in Follicular Fluid Increases Bovine Embryo Production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes.Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitro embryo production in order to assess embryo production efficiency. A second set

Intracytoplasmic Sperm Injection after Vitrification of Immature Oocytes in Follicular Fluid Increases Bovine Embryo Production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes.Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitro embryo production in order to assess embryo production efficiency. A second set