Abstract Logistic and economical limitations are often the causes of dog owners not accurately monitoring the estrous cycle and the optimal insemination time. The aim of this study was to evaluate in vivo early embryonic development in bitches, after the analysis of sequential vaginal cytologies associated to single progesterone measurement and single laparoscopic insemination with high quality semen (fresh and with high spermatozoa concentration) or low-quality semen (frozen/thawed and with low spermatozoa concentration) at 48 h post- ovulation time predicted on a single progesterone measurement. Ten bitches were inseminated with 250 x 106 fresh spermatozoa (80% motility), and ten with 80 x 106 frozen/thawed spermatozoa (60% motility) in the cranial part of each uterine horn. Seven days later, ovariohysterectomy was performed and the oviducts and uterine horns and body were flushed to recover embryos and unfertilized oocytes. In 80% of the bitches inseminated with fresh and 50% of bitches inseminated with frozen/thawed semen, embryos at 2 to 8 cells stage were recovered mostly from the, oviducts. This study indicates that pregnancies can be obtained with a single laparoscopic intrauterine insemination after single serum progesterone measurement, although with a low number of embryos. This result should be taken into account in case economic or logistic restrictions that affect the possibility of owners to plan an accurate monitoring of the optimal breeding time using fresh and frozen semen. concentration lowquality frozenthawed thawed 4 post 25 10 (80 motility, motility , 60% 60 (60 horn later oocytes 50 1 (8 6 (6 5 (

ABSTRACT Objective: To assess whether there is an association between the level of progesterone on the day of administration of human chorionic gonadotropin and clinical and laboratory characteristics, in addition to the results of in vitro fertilization of patients with a good prognosis. Methods: A cross-sectional study comprising 103 women who underwent intracytoplasmic sperm injection treatment, between November 2009 and May 2015, aged ≤35 years, with no comorbidities, with fresh embryo transfer. Data were collected from patient medical records. Results: There was a weak positive correlation between the level of progesterone on the day of human chorionic gonadotropin and the number of follicles larger than 14mm (ß=0.02, p=0.001), retrieved oocytes (ß=0.01, p=0.01) and oocytes in metaphase II (MII) (ß=0.02, p=0.02); that is, the increase in progesterone level has a slight association with increased values of these variables. Body mass index was inversely correlated with progesterone level on the day of human chorionic gonadotropin (ß=-0.01, p=0.02). No association was found between the level of progesterone on the day of human chorionic gonadotropin and the protocols used for controlled ovarian stimulation, quality of transferred embryos and the pregnancy rate. Conclusion: There is an association between the value of progesterone on the day of human chorionic gonadotropin administration with body mass index, number of follicles larger than 14mm, number of retrieved oocytes and oocytes in metaphase II. Unlike embryo quality and pregnancy rate, which do not have a statistically significant relation with this value in the population studied.

Abstract The assessment of morphology and digital image opacity may provide valuable information on the present embryo quality. Time-lapse imaging has been employed in research to establish a means of monitoring the dynamic nature of preimplantation embryo development. The aim of present study was to use time-lapse imaging for assessing various prospective morphometric and phototextural markers of the developmental potential of in vitro-derived ovine embryos. Oocytes were obtained by scarification of ovaries from nine Polish Longwool ewes. After in vitro maturation (IVM) and fertilization (IVF) of oocytes with fresh ram semen, the development of embryos to the blastocyst stage was monitored and evaluated using Primo Vision time-lapse imaging technology. Commercially available Image-Pro® Plus software was used to measure zona pellucida thickness, embryo diameter, total area of the perivitelline space, cellular grey-scale pixel intensity and cellular pixel heterogeneity. Statistical assessment of all attributes was done at various time points during embryo development (i.e., presumptive zygote stage: t(0); first cleavage detected at t(2) or t(3); and second cleavage detected at t(4) or t(6)). Out of thirty-seven zygotes analyzed in this study, five did not divide, 26 arrested before and six developed to the blastocyst stage. Our present results indicate that most parameters analyzed did not differ among embryos varying in their developmental fate except for the perivitelline space area that was greater (P<0.05) for non-dividing zygotes than future blastocysts at the presumptive zygote stage (4040±1850 vs. 857±262 µm2, respectively; means±SEM). Consequently, the measurement of perivitelline space at t(0) can potentially be used to prognosticate developmental potential of in vitro-produced ovine embryos albeit further confirmational studies are needed.

RESUMO Objetivou-se testar a vitrificação de ovários de camundongos do ICTB/Fiocruz. Inicialmente, fez-se coleta e maturação in vitro dos oócitos de ovários a fresco e vitrificados, bem como avaliação de estruturas no cultivo embrionário, pós-fertilização in vitro. Fêmeas B6D2F1 foram eutanasiadas para remoção dos ovários (n=60) e divididas em três grupos: grupo 1 (n=30 animais) - oócito de ovários vitrificados, maturados e fertilizados in vitro (120 fragmentos); grupo 2 (n=15) (controle 1) - oócitos coletados a fresco, maturados e fertilizados in vitro; e grupo 3 (n=15) (controle 2) - oócitos maturados in vivo e fertilizados in vitro. A técnica foi verificada no desenvolvimento embrionário in vitro, que foi avaliado pelo teste de qui-quadrado (BioStat 5.0). Recuperaram-se 123, 224 e 328 oócitos nos G1, G2 e G3, respectivamente. Observaram-se diferenças significativas nas taxas de clivagem às 24 horas (embriões ≥ 2 células) entre G1 (8%) e G2 (32%) (P<0,1) e G1 e G3 (49%) (P<0,05), mas não entre G2 e G3 (P>0,05). Para blastocistos, às 96 horas, os grupos G1, G2 e G3 apresentaram, respectivamente, 6%, 11% e 46%, diferindo significativamente entre eles (P<0,05). A vitrificação de ovários, a maturação oocitária e a fertilização in vitro são alternativas para a produção de embriões de camundongos in vitro.

ABSTRACT This work aimed test ovarian vitrification of hybrid mouse from ICTB/Fiocruz. Protocol collection and oocyte in vitro maturation from fresh and vitrified ovaries was established and embryos were evaluated after fertilization. B6D2F1 females were euthanized for ovarian removal (n= 60) and divided into 3 groups: G1 (n= 30) - ovaries fragmented (n= 120), vitrified, matured and fertilized; G2 (n= 15) - in vitro fertilization of oocytes matured in vitro from fresh ovaries; G3 (n= 15) - ampulla region oocytes in vitro fertilizated. Viability was verified by thawing, oocyte in vitro maturation and fertilization. In vitro embryo development of each group was evaluated by Chi-square test (BioStat 5.0). 123, 224 and 328 oocytes were recovered from G1, G2 and G3, respectively. Significant differences were observed in cleavage rates at 24 hours (embryos with 2 cells or more) between G1 (8%) and G2 (32%) (P< 0.1) and G1 and G3 (49%) (P< 0.05) but not between G2 and G3 (P> 0.05). Blastocysts at 96 hours presented 6%, 11% and 46%, respectively for G1, G2 and G3, differing significantly (P< 0.05). Ovary vitrification, oocyte in vitro maturation and in vitro fertilization were available for the production of in vitro mouse embryos.

Abstract A somatic embryogenesis protocol was developed from the immature leaves of adult plants of the macaw palm. Leaf explants from different regions of the palm heart were used for callus initiation in a modified Y3 medium, supplemented with 2,4-D or Picloram at 450 μM. Calli were separated from the leaf explants at 6-, 9- and 12-month periods and transferred to a fresh culture medium of the same composition. They were multiplied for up to 120 days. Reduced concentrations of 2,4-D and Picloram were used to differentiate somatic embryos. They were then germinated in a medium without plant growth regulators. Morphological and anatomical analyses were conducted at different stages of the embryogenic process. The best results for callus induction were achieved by Picloram, when explants were maintained for up to 9 months on culture medium (64.9%). The farthest portions of the apical meristem were those that provided the biggest calli formation. The formation of the somatic embryos was observed from the calli multiplication phase. Reduction in concentrations of growth regulators failed to promote the formation of complete plants. Picloram at 450 μM promotes high callogenesis in leaf tissues of macaw palm, with a potential for somatic embryo formation.

ABSTRACT Introduction: Aedes aegypti (L) (Diptera: Culicidae) is a cosmopolitan species and a vector of arboviruses. Variations in the temperature and salinity of the water affect eclosion and survival during the larval stages. Objective: Evaluate the effect of different temperatures and salinities on the eclosion of eggs and the survival of larvae, pupae and adults in laboratory conditions. Methods: Ae. aegypti larvae were collected from artificial reservoirs in a peri-urban area of Puerto Vallarta, Jalisco, Mexico, and maintained until the adult stage. The eggs obtained were subjected to eight temperatures (15, 17, 20, 25, 27, 30, 32 and 35 °C). Fifteen eggs were placed in quintuplicate and eclosion was evaluated for 96 h. One hundred eggs were placed with water adjusted to 0.3, 2, 5, 10, 15, 18 and 22 psu and eclosion was evaluated until 96 h. Additionally, stage IV larvae were used in quintuplicate, subjecting them to the same salinities and evaluating survival until 48 h. The effect of salinity on oviposition by females was determined by introducing containers with the same salinity into the entomological cages. Results: 100 % eclosion was recorded at 24 and 48 h, whereas no eclosion occurred at a temperature of 35 °C. Salinities of 22 and 18 psu caused 100 % mortality at 24 h, whereas 50 % survived at a salinity of 15 psu. At concentrations of 2, 5 and 10 psu 100 % of the larvae survived until the adult stage. Survival of stage IV larvae in treatments 2, 5 and 10 was 100%, whereas in 15, 18 and 22 psu it fell to 50, 80 and 100 %, respectively (p˂ 0.05). The different salinities did not affect oviposition significantly. Eclosion only occurred at concentrations of 0.3, 2, 5 and 10 psu. Oviposited eggs at concentrations of 15, 18 and 22 psu did not eclose until they were transferred to fresh water, where eclosion percentages ranged between 80 % and 90 %. Conclusions: Ae. aegypti embryos have great plasticity to endure drastic changes in temperature and salinity. Effective control of their populations should include inspection of ponds and reservoirs containing brackish water of up to 18 psu.

RESUMEN Introducción: Aedes aegypti (L) (Diptera: Culicidae), es una especie cosmopolita y vector de arbovirosis. Las variaciones de la temperatura y salinidad del agua influyen en la eclosión y supervivencia de fases larvales. Objetivo: Evaluar el efecto de diferentes temperaturas y salinidades en la eclosión de huevos y la supervivencia de larvas, pupas y adultos bajo condiciones de laboratorio. Métodos: Se colectaron larvas de Ae. aegypti, de reservorios artificiales en la zona periurbana de Puerto Vallarta, Jalisco, México, y se mantuvieron hasta la fase adulta. Los huevos obtenidos se sometieron a ocho temperaturas (15, 17, 20, 25, 27, 30, 32 y 35 °C). Se colocaron 15 huevos por quintuplicado y se evaluó la eclosión durante 96 h. Se colocaron 100 huevos con agua ajustada a 0.3, 2, 5, 10, 15,18 y 22 ups y se evaluó la eclosión hasta las 96 h. Adicionalmente se utilizaron larvas del estadio IV, por quintuplicado, sometiéndose a las mismas salinidades y se evaluó la supervivencia hasta las 48 h. El efecto de la salinidad en la ovoposición de las hembras se llevó a cabo introduciendo recipientes con las mismas concentraciones salinas, dentro en las jaulas entomológicas. Resultados: Se registró el 100 % de eclosión a las 24 y 48 h; la temperatura de 35° C no registró eclosión. Las salinidades de 22 y 18 ups, provocaron mortalidad del 100 % a las 24 h. En la salinidad de 15 ups, sobrevivió el 50 %. Las concentraciones de 2, 5 y 10 ups demostraron 100 % de supervivencia hasta la fase de adulto. La supervivencia de larvas del estadio IV en los tratamientos 2, 5 y 10 fue del 100 % y en 15,18 y 22 ups disminuyó a 50, 80 y 100 %, respectivamente (p˂ 0,05). Las diferentes concentraciones salinas no afectaron significativamente la ovoposición. La eclosión solo se presentó en las concentraciones de 0,3; 2; 5 y 10 ups. Los huevos ovopositados en concentraciones de 15, 18 y 22 ups no eclosionaron hasta que fueron transferidos a agua dulce con porcentajes de eclosión de entre el 80 y 90 %. Conclusiones: Los embriones de Ae. aegypti poseen una amplia plasticidad para soportar cambios drásticos de temperatura y salinidad. El control efectivo de sus poblaciones debe incluir la revisión de charcas o reservorios que contengan aguas salobres hasta 18 ups.

ABSTRACT Recipient cow is the key to success in embryo transfer (ET) programs in cattle. Therefore, it is necessary to know the factors that influence on its reproductive potential. It is considered that multiparous cows have a better response to the ET program than nulliparous cows. The objective of this study was to evaluate the effect of parity on the reproductive efficiency of multiparous and nulliparous cows in a fresh ET program. Twenty-six recipient cows of Suizo Americano breed were divided into two experimental groups (n = 13/each) according to their parity (multiparous and nulliparous). Donor cows underwent a synchronization and superovulation program. Subsequently, the recipient cows were treated with fresh ET. The percentage of cows in estrus was higher on day 1 in multiparous cows than in nulliparous cows (P <0.05). On the other hand, gestation percentage, estrus time, embryo stage and pregnancy percentage per uterine horn were not significantly affected among the experimental groups (P> 0.05). In conclusion, parity did not affect the reproductive response of multiparous and nulliparous recipient cows subjected to a fresh ET program under tropical conditions.

RESUMEN La vaca receptora es la clave del éxito en programas de transferencia de embriones (TE) en el ganado bovino. Por lo tanto, es necesario conocer los factores que influyen en su potencial reproductivo. Se considera que las vacas multíparas presentan una mejor respuesta al programa de TE que las vacas nulíparas. El objetivo del presente estudio fue evaluar el efecto de la paridad en la eficiencia reproductiva de vacas multíparas y nulíparas en un programa de TE en fresco. Veintiséis vacas receptoras de raza Suizo Americano se dividieron en dos grupos experimentales (n = 13/c.u.) de acuerdo con su paridad (multíparas y nulíparas). Las vacas donadoras se sometieron a un programa de sincronización y superovulación, y posteriormente se aplicó la TE en fresco a las vacas receptoras. El porcentaje de vacas en estro fue mayor en el día 1 en las vacas multíparas que en las nulíparas (P<0.05). Por su parte, el porcentaje de gestación, la hora del estro, el estadio del embrión y el porcentaje de gestación por cuerno uterino no se afectaron significativamente entre los grupos experimentales (P>0.05). En conclusión, la paridad no afectó la respuesta reproductiva de las vacas receptoras multíparas y nulíparas sometidas a un programa de TE en fresco en condiciones tropicales.

Abstract This study aimed to evaluate the effect of two Embryo Manipulation Solutions (EMS and EMS supplemented) in maintenance of the viability of embryos, initially using structures derived from mice (first phase). Next, the efficiency of these solutions in routines of bovine embryo transfer was evaluated (second stage). Mice embryos were used in the stages of early blastocyst, and compact morula grades I and II. These embryos were initially randomly distributed and maintained for four hours in three solutions: Modified phosphate buffered saline (PBS; Control); EMS (treatment 1), and EMS supplemented (treatment 2). Subsequently, they were cultured in TCM 199 medium and evaluated in terms of total number of cells, morphometric characteristics, ultra structural aspects, detection of cell apoptosis, and quantification of Hsp70.3 gene expression. In the second phase, these same solutions were tested in the transfer of quality I and II bovine embryos (excellent and good). These embryos were transferred fresh to 58 recipients. The results showed that the total number of cells in embryos expanded blastocyst (ExB), the number of apoptotic cells, the cell, nuclear, nucleolar diameter and the nucleus/nucleolus ratio was similar among the treatments. The pregnancy rate shown on second phase was also similar. However, the EMS supplemented expressed more Hsp70.3 than EMS. The expression of Hsp70.3 was also greater for embryos in EMS than that of EMS supplemented. The McII embryos, EMS and EMS supplemented samples also expressed more Hsp70.3 compared to control embryos. In conclusion, the tested solutions can be used in routine embryo transfer techniques, replacing modified PBS solution as an effective media in maintaining embryo viability.

Resumen Antecedentes: La criopreservación se caracteriza por el mantenimiento de la viabilidad celular a bajas temperaturas, resultando en reducido metabolismo y actividad enzimática intracelular, lo que permite la preservación del material genético por períodos de tiempo indefinidos. Los embriones sometidos a ésta técnica sufren daños morfológicos y funcionales considerables, dando como resultado una sobrevivencia y tasas de desarrollo reducidas. Objetivo: Evaluar la tasa de preñez a partir de embriones Nelore (Bos indicus) producidos in vitro (IVP) después de la vitrificación bajo condiciones de campo. Métodos: Embriones IVP en los estadios de blastocisto (Bl) y blastocisto expandido (Bx) se transfirieron en fresco (n= 137) o después de la vitrificación (n= 127). Resultados: La tasa de preñez a los 35 d fue menor para los embriones transferidos en fresco en fase Bl (41,6) en relación con los Bx (60,9) (p<0,05). Después de la vitrificación, las tasas de preñez a los 35 d fueron similares entre Bl (38,0) y Bx (47,6) (p>0,05). Las pérdidas de preñez a los 60 d fueron similares (p>0,05) tanto para embriones en fresco en Bl (3,1) y Bx (4,8) como para los vitrificados (Bl: 1,9 y Bx: 4,7). Las tasas de nacimiento fueron similares entre los grupos (p>0,05). Conclusión: Las tasas de preñez y nacimiento de embriones IVP vitrificados de Nelore (Bos indicus) bajo condiciones de campo son semejantes a las de embriones en fresco.

Abstract Background: Cryopreservation preserves cellular viability under low temperatures, resulting in diminished intracellular enzymatic activity and reduced cellular metabolism that ultimately allows preserving genetic material for indefinite periods of time. Embryos submitted to cryopreservation suffer from considerable morphological and functional damage, resulting in reduced survival and development rates. Objective: To evaluate pregnancy and delivery rates of in vitro-produced (IVP) Nellore (Bos indicus) embryos after vitrification under field conditions. Methods: The IVP embryos at blastocyst (Bl) and expanded blastocyst (Bx) were transferred fresh (n= 137) or after vitrification (n= 127). Results: Pregnancy rates at 35 d for fresh embryos were lower in Bl (41.6) than in Bx (60.9) (p<0.05). After vitrification, pregnancy rates were similar at 35 d between Bl (38.0) and Bx (47.6) (p>0.05). Pregnancy loss at 60 d were similar (p>0.05) for both fresh (Bl: 3.1 and Bx: 4.8) and vitrified embryos (Bl: 1.9 and Bx: 4.7). Delivery rates were similar between groups (p>0.05). Conclusion: Both pregnancy and delivery rates of Bos indicus IVP embryos vitrified under field conditions are indistinguishable from fresh embryos.

Resumo Antecedentes: A criopreservação é caracterizada pela manutenção da viabilidade celular em baixas temperaturas, resultando em atividade enzimática intracelular e metabolismo celular reduzido, que permite a preservação do material genético por períodos indefinidos de tempo. Embriões submetidos à criopreservação sofrem danos morfológicos e funcionais consideráveis, resultando em sobrevivência reduzida e menores taxas de desenvolvimento. Objetivo: Avaliar a taxa de prenhez a partir de embriões Nelore (Bos indicus) produzidos in vitro (IVP) após a vitrificação sob condições de campo. Métodos: Embriões IVP nos estádios de blastocisto (Bl) e blastocisto expandido (Bx) foram transferidos a fresco (n= 137) ou depois da vitrificação (n= 127). Resultados: A taxa de prenhez aos 35 d foi menor para os embriões transferidos a fresco na fase de Bl (41,6), em relação aos Bx (60,9) (p<0,05). Apos a vitrificação, as taxas de prenhez foram semelhantes aos 35 d entre Bl (38,0) e Bx (47,6) (p>0,05). As perdas de prenhez aos 60 d foram semelhantes (p>0,05) tanto para embriões a fresco nos estádios de Bl (3,1) e Bx (4,8), e vitrificados em Bl (1,9) e Bx (4,7). As taxas de nascimentos foram semelhantes entre os grupos (p>0,05). Conclusão: As taxas de prenhez e nascimentos dos embriões IVP vitrificados de Nelore (Bos indicus) sob condições de campo é semelhante àquela dos embriões a fresco.

ABSTRACT Layer hybrids are more superior in the egg yield than pure breeds. But this superiority causes some problems in the bones during the production period. There are a lot of differences among genotypes in the production period that have been examined extensively, however, the differences throughout incubation period aren’t known yet. Thus, the objective of this study was to evaluate the differences in femur properties of pure breed and layer hybrid embryos during the incubation period. A total of 354 fresh hatching eggs were used from one hybrid (Lohman White) and two pure breeds (Denizli & Gerze). The eggs were incubated. Hatching eggs from each genotype at the beginning of embryonic days (E) 19 and E21 were examined. At these embryonic ages, 12 eggs were selected from each genotype. The eggs were opened and embryos were sacrificed by cervical dislocation. Both femurs were dissected from each embryo and then structure, mineral composition and mechanical properties of femur were determined. The genotype affected the length, width and Zn level in the femur of embryos. Weight, length, width, load of yield and ultimate, ash and all examined minerals increased with embryonic development. These results showed that some femur properties of embryos were influenced by the genotype and layer hybrid embryos are beginning to be advantageous in terms of Zn level in the femur at E21. This study leads to better understanding femur development during the incubation period in layer hybrid and pure breeds.

RESUMO Este estudo testou a eficiência de protocolo alternativo de criopreservação de embriões caprinos de diferentes qualidades morfológicas. Foram utilizados 58 embriões, coletados entre o sexto e o sétimo dia do ciclo estral (n=29/grupo). Embriões congelados passaram por solução 1,5M etilenoglicol por 10min e foram aspirados durante esse tempo para parte central de palheta 0,25mL, separada por bolhas de ar de colunas contendo PBS 0,4% BSA e 20% SFB. As palhetas foram congeladas em máquina de congelação de 20ºC a -6ºC, com taxa de resfriamento de 3ºC/min, estabilização por 15min (seeding após 5min), -6ºC a -32ºC a 0,6ºC/min, e imersas em nitrogênio líquido. Os embriões foram descongelados por 30s a 37ºC, em água. Embriões frescos foram mantidos em solução de manutenção (37ºC). Embriões frescos e congelados/descongelados foram transferidos para 30 receptoras no sétimo dia do ciclo estral. A taxa de partos e a de crias nascidas (respectivamente) foram similares (P>0,05) para receptoras recebendo embriões frescos (66,7% ou 10/15; 55,2% ou 16/29) ou congelados/descongelados (60,0% ou 9/15; 51,7% ou 15/29). O protocolo de criopreservação de embriões utilizado no presente estudo resultou em índices de eficiência semelhantes aos de embriões frescos.

ABSTRACT The efficiency of an alternative freezing protocol for goat embryos of different morphology and quality was tested. Fifty-eight embryos on Day 6-7 stage were transferred as fresh or after freeze-thawing (n=29/group). For freezing, embryos were placed into 1.5M ethylene-glycol solution for 10min. During this time, they were loaded in the central part of 0.25mL straw, separated by air bubble from columns containing PBS/BSA 0.4% plus 20% BFS. Straws were then frozen using a freezing machine from 20ºC to -6ºC at a cooling rate of 3ºC/min, stabilization for 15min (seeding after 5min), from -6 C to -32ºC at 0.6 C/min,and plunged into liquid nitrogen. Frozen embryos were thawed for 30s at 37ºC in a water bath. Embryos subjected to fresh transfer were maintained in holding medium (37ºC). Fresh and frozen-thawed embryos were transferred at day 7 post-estrus to 30 recipients. Kidding and kid born rates were similar (P> 0.05), respectively, for recipients receiving fresh (66.7% or 10/15; 55.2% or 16/29) or frozen-thawed (60% or 9/15; 51.7% or 15/29) embryos. The cryopreservation of goat embryos using slow-freezing protocol and 1.5MEG resulted in similar efficiency rates of fresh embryos.

Abstract OBJECTIVE: To know the reproductive results in an embryo transfer program on day 4 and compare them with transfers in D3 and D5 MATERIALS AND METHODS: A prospective analysis comprised a period from March 2015 to July 2017, with 306 couples undergoing an IVF / ICSI procedure and who met the inclusion criteria (patients of all ages, cycles captured and transferred in fresh and patients with all the factors of infertility) and exclusion (cases of ovodonation, cycles in which there were no embryos to be transferred and cycles in which embryo transfer was difficult). The population was divided into 3 groups: Group A with transferred on day 3; Group B with transferred on day 4) and Group C with transferred on day 5 (TE D5). The luteal phase support for the 3 groups was with progesterone vaginally for 14 days. Pregnancy rates per transfer day were evaluated with the number of clinical pregnancies regarding the number of transfers made and comparing the results with a chi-square test considering significant differences with p < 0.05. RESULTS: We analyze 306 couples. Group A with 205 patients transferred on day 3 (TE D3); Group B with 62 patients transferred on day 4 (TE D4) and Group C with 39 patients transferred on day 5 (TE D5). A significant difference (p = 0.035) was found between the pregnancy rate on day 3 and 4 for patients over 40 years of age (11.4% and 31.6% respectively), however, an increase tendency was observed at all ages. in the pregnancy rate when the embryo transfer takes place on day 4. CONCLUSIONS: The embryo transfer on day 4 of development can be considered as one more option, to present better results with respect to the transfers made on day 3, and a very viable alternative in transfers made in D5 with very similar pregnancy rate and in this even greater study.

Resumen OBJETIVO: Conocer las repercusiones en la reproducción de la aplicación de un programa de trasferencia embrionaria en día 4 y compararlas con trasferencias en día 3 y día 5. MATERIALES Y MÉTODOS: Estudio prospectivo efectuado en parejas a quienes se aplicó un procedimiento de FIV-ICSI entre marzo de 2015 y julio de 2017. Criterios de inclusión: pacientes de todas las edades, ciclos capturados y trasferidos en fresco y con todos los factores de infertilidad. Criterios de exclusión: casos de ovodonación, ciclos en donde no hubiera embriones a trasferir y ciclos en donde la trasferencia embrionaria fue difícil. Para fines de estudio la población se dividió en tres grupos: grupo A con trasferencia en día 3; grupo B con trasferencia en día 4 y grupo C con trasferencia en día 5. El soporte de fase lútea para los tres grupos fue con progesterona por vía vaginal durante 14 días. Se evaluaron las tasas de embarazo por día de tras-ferencia con el número de embarazos clínicos respecto del número de trasferencias efectuadas y los resultados se compararon con c2; se consideró diferencia significativa el valor de p < 0.05. RESULTADOS: Se estudiaron 306 parejas. Grupo A: 205 pacientes trasferidas en día 3; grupo B: 62 pacientes trasferidas en día 4; grupo C: 39 pacientes trasferidas en día 5. Se encontró una diferencia significativa (p = 0.035) entre la tasa de embarazo en día 3 y 4 para pacientes mayores de 40 años (11.4 y 31.6%, respectivamente). En todas las edades se observó una tendencia al incremento en la tasa de embarazo cuando la trasferencia embrionaria se llevó a cabo en día 4. CONCLUSIONES: La trasferencia embrionaria en día 4 de desarrollo puede considerarse una opción más. En este ensayo hubo mejores resultados con respecto a las trasferencias efectuadas en día 3. Es una alternativa viable en trasferencias efectuadas en día 5 con tasas de embarazo muy similares y, en este estudio, incluso mayor.

Abstract OBJECTIVE To evaluate the reproductive effects when recombinant luteinizing hormone is added and to compare two stimulation schemes by number of aspirated oocytes, mature oocytes, fertilization and implantation rates, live newborn and number of vitrified embryos. MATERIALS AND METHODS Longitudinal, observational and retrospective study carried out in patients of the Hisparep Reproduction Clinic of the Spanish Hospital with diagnosis of infertility, over 35 years old, who received a controlled ovarian hyperstimulation cycle with IVF-ICSI during the period 2014-2016. The statistical analysis was carried out with the Student t test for independent samples. The studies were analyzed with the IBM SPSS statistical package, version 22. RESULTS We analyzed 201 women with infertility, over 35 years of age. Group 1 (n = 101) of IVF-ICSI received stimulation with recombinant follicle-stimulating hormone and recombinant luteinizing hormone 2: 1 with menotropins (Pergoveris® and Merapur®) from the second day of the cycle. Group 2 (n = 100) received recombinant follicle stimulating hormone and menotropins (Gonal F® and Merapur®); in both schemes, GnRH antagonist was used from day 7 of the cycle. The average number of aspirated oocytes was 7.5 in Group 1 and 9.1 in Group 2 (p = 0.058). Mean mature oocytes were 6.2 in Group 1 vs 7.4 in Group 2 (p = 0.085). The fertilization rate in group 1 was 57 vs. 67% in Group 2 (p = 0.045). In Group 1 the implantation rate per embryo transferred fresh was 24.1 vs 10.3% in Group 2 (p = 0.40), the live newborn rate was 30% in Group 1 vs 20.6% in Group 2. The mean number of vitrified embryos in Group 1 was 1.47 vs 1.38 in Group 2. CONCLUSIONS The probable advantage of supplementation with recombinant follicle-stimulating hormone during ovarian stimulation in women over 35 years of age is of interest and its evaluation is required in subsequent studies.

Resumen OBJETIVO Evaluar los resultados en ciclos de FIV-ICSI de dos protocolos de estimulación ovárica en mujeres mayores de 35 años e investigar si agregar hormona luteinizante recombinante a FSH-r en un protocolo de estimulación mejora la respuesta ovárica y, en consecuencia, las tasas de embarazo en este grupo poblacional. MATERIALES Y MÉTODOS Estudio longitudinal, observacional y retrospectivo efectuado en pacientes de la Clínica de Reproducción Hisparep del Hospital Español con diagnóstico de infertilidad, mayores de 35 años, que recibieron un ciclo de hiperestimulación ovárica controlada con FIV-ICSI durante el periodo 2014-2016. El análisis estadístico se efectuó con la prueba de t de Student para muestras independientes. Los estudios se analizaron con el paquete estadístico SPSS IBM, versión 22. RESULTADOS Se analizaron 201 mujeres con infertilidad, mayores de 35 años. El grupo 1 (n = 101) de FIV-ICSI recibió estimulación con hormona folículo estimulante recombinante y hormona luteinizante recombinante 2:1 con menotropinas (Pergoveris® y Merapur®) a partir del segundo día del ciclo. El grupo 2 (n = 100) recibió hormona folículo estimulante recombinante y menotropinas (Gonal F® y Merapur®); en ambos esquemas se utilizó antagonista de GnRH a partir del día 7 del ciclo. La media de ovocitos aspirados fue de 7.5 en el grupo 1 y 9.1 en el grupo 2 (p = 0.058). La media de ovocitos maduros fue 6.2 en el Grupo 1 vs 7.4 en el grupo 2 (p = 0.085). La tasa de fecundación en el grupo 1 fue de 57 vs 67% en el grupo 2 (p = 0.045). En el grupo 1 la tasa de implantación por embrión transferido en fresco fue 24.1 vs 10.3% en el Grupo 2 (p = 0.40), la tasa de recién nacido vivo fue de 30% en el Grupo 1 vs 20.6% en el Grupo 2. La media de embriones vitrificados en el Grupo 1 fue 1.47 vs 1.38 en el Grupo 2. CONCLUSIONES La probable ventaja de la complementación con hormona folículo estimulante recombinante durante la estimulación ovárica en mujeres mayores de 35 años es de interés y se requiere su evaluación en estudios posteriores.

The objective of the study was to characterize the morphological and physiological traits of the seeds of five Morus alba L. varieties (cubana, tigreada, universidad, universidad mejorada and yu-62), which were harvested at the Pastures and Forages Research Station Indio Hatuey Matanzas, Cuba. A completely randomized design was used, and the type of embryo, seed size and mass, moisture content, allocation of biomass to the seed reserves (embryo-endosperm) and desiccation tolerance index, were determined. Simple classification variance analysis was performed on the data. The results indicated that the seeds had ovate to round shape; while the embryo was developed, of folded type. The fresh mass varied between 1,30 and 1,46 mg, and most of the seed resources were aimed at reserve formation (between 62,3 and 68,1 %). The moisture content varied between 11,5 and 13,2 %, and the desiccation tolerance index was lower than 0,5; value which is in correspondence with that of orthodox seeds. It is concluded that the information obtained on the biology of the M. alba seed is highly useful for the conservation of the germplasm bank of this species and for its sexual propagation.

El objetivo del estudio fue caracterizar los rasgos morfológicos y fisiológicos de las semillas de cinco variedades de Morus alba L. (cubana, tigreada, universidad, universidad mejorada y yu -62) que se cosecharon en la Estación Experimental de Pastos y Forrajes Indio Hatuey Matanzas, Cuba. Se empleó un diseño totalmente aleatorizado, y se determinó el tipo de embrión, las dimensiones y la masa de las semillas, su contenido de humedad, la asignación de biomasa a las reservas seminales (embrión-endospermo) y el índice de tolerancia a la desecación. A los datos se les realizó un análisis de varianza de clasificación simple. Los resultados indicaron que las semillas tenían forma de ovada a circular; mientras que el embrión era desarrollado, de tipo doblado. La masa fresca varió entre 1,30 y 1,46 mg, y la mayor parte de sus recursos se destinaron a la formación de reservas (entre 62,3 y 68,1 %). El contenido de humedad varió entre 11,5 y 13,2 %, y el índice de tolerancia a la desecación fue menor que 0,5; valor que se corresponde con el de semillas ortodoxas. Se concluye que la información obtenida sobre la biología de la semilla de M. alba resulta de gran utilidad para la conservación del banco de germoplasma de esta especie y para su propagación por vía sexual.