ABSTRACT This experiment aimed to assess the effect of fetal calf serum (FCS) added to culture medium on day 5 to 7 after in vitro fertilization on production and subsequent cryotolerance of blastocysts from Ecuadorian Creole heifers. Immature cumulus-oocyte complexes (COCs) were collected by ovum pick-up (OPU) from 10 Creole heifers and from ovaries collected at abattoir in eight collection sessions. COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and culture (IVC). On day 1 after IVF, presumptive embryos from each oocyte source (OPU [O] and Abattoir [A]) were allocated randomly in two groups according to whether 2.5% (v/v) FCS was added to culture medium on day 5 after IVF: 1) O-FCS+, 2) O-FCS-, 3) A-FCS+, and 4) A-FCS-. On day 7 after IVF, high quality embryos were vitrified. Cryotolerance of vitrified-warmed embryos was assessed according to blastocele re-expansion at 2 h and subsequent re-expansion and hatching at 24 and 48 h of re-incubation. Blastocysts rate on day 7 did not differ between oocyte source and FCS group. After vitrification/warming process, addition of FCS affected the re-expansion rate only at 2 h, irrespective of the oocyte source (p<0.05). Likewise, blastocysts hatching rate at 48 h of incubation was drastically affected only in OPU derived oocyte (p<0.01). Supplementation of FCS on day 5 after IVF did not improve blastocyst production and adversely affected the cryotolerance of in vitro bovine embryos derived from creole heifers raised on Ecuadorian Andean highlands.

RESUMEN El estudio tuvo como objetivo evaluar el efecto del suero fetal bovino (FCS) agregado al medio de cultivo los días 5 a 7 después de la fertilización in vitro sobre la producción y posterior criotolerancia de blastocistos de vaquillonas criollas ecuatorianas. Se recolectaron complejos cúmulo-ovocitos (COCs) inmaduros mediante aspiración intravaginal guiada por ultrasonido (OPU) de 10 vaquillonas criollas y de ovarios de matadero en ocho sesiones. Los COCs se sometieron a maduración in vitro (MIV), fecundación (FIV) y cultivo (IVC). El día 1 después de la FIV, los presuntos embriones de cada fuente de ovocitos (OPU [O] y Matadero [A]) se asignaron aleatoriamente en dos grupos según si se añadió FCS al 2.5% (v/v) al medio de cultivo el día 5 después de la FIV: 1) O-FCS+, 2) O-FCS-, 3) A-FCS+ y 4) A-FCS-. El día 7 después de la FIV se vitrificaron embriones de alta calidad. La criotolerancia de los embriones vitrificados-calentados se evaluó según la reexpansión del blastocele a las 2 h y la posterior reexpansión y eclosión a las 24 y 48 h de incubación. La tasa de blastocistos el día 7 no difirió entre la fuente de ovocitos y el grupo FCS. Después del proceso de vitrificación/ calentamiento, la adición de FCS solo afectó la tasa de reexpansión a las 2 h, independientemente de la fuente de ovocitos (p<0.05). Asimismo, la tasa de eclosión de los blastocistos a las 48 h de incubación se vio drásticamente afectada únicamente en los ovocitos derivados de OPU (p<0.01). En conclusión, la suplementación de FCS el día 5 después de la FIV no mejoró la producción de blastocistos y afectó negativamente la criotolerancia de embriones bovinos in vitro derivados de vaquillonas criollas criadas en los Andes ecuatorianos.

Abstract The objective of this study was to evaluate the effect of three doses of equine chorionic gonadotropin (eCG) for ovarian superstimulation on ovarian response, follicular development and cumulus-oocyte complexes (COCs) collection in llamas. For this purpose, eighteen multiparous non-lactating adult (4–7 yo) female llamas with an average body condition of 2.8 (BCS 1–5) were submitted to a follicular ablation (FA) to induce a new follicular wave emergence. Two days after FA (Day 0), synchronized llamas were randomly allocated to three treatment groups (n = 6/group) and given 500, 750 and 1000 IU of eCG (Novormon®, Syntex, Buenos Aires, Argentina) per animal respectively to induce ovarian superstimulation. Transrectal ultrasonography were performed on Days 2, 4, and 6; and ovum pick up (OPU) was performed on Day 6. Data was evaluated by one-way analysis of variance (ANOVA), repeated measures ANOVA, and 2-tailed Chi-square. The average size (mm) of follicles was greater (p≤ 0.05) in the 1000 IU group compared to the other groups. There was a greater (p≤ 0.05) number of follicles ≥ 7 mm in the 1000 IU group compared to the 500 IU group. Number of COCs collected on Day 6 and the COC recovery rate were not different among groups. In conclusion, a single dose of 1000 IU of eCG induced the best ovarian response resulting in larger and greater number of follicles at the time of OPU. (eCG cumulusoocyte cumulus oocyte (COCs purpose nonlactating non lactating 4–7 47 4 (4– yo 28 2 8 2. BCS 1–5 15 1 5 (FA emergence 0, 0 , 0) n 6/group 6group 75 100 Novormon®, Novormon (Novormon® Syntex Aires Argentina OPU (OPU oneway one way ANOVA (ANOVA) 2tailed tailed Chisquare. Chisquare Chi square. square Chi-square (mm p≤ p (p 0.05 005 05 50 conclusion 4– (4 1– 10 Novormon® (Novormon (ANOVA 0.0 00 ( 0.

BACKGROUND: Rhinoceros are currently one of the most threatened mammal species globally. Slow population growth, increased poaching and habitat destruction have led to increased conservation efforts for each species. Assisted reproductive technologies (ART) have been implemented in an attempt to aid reproductive outputs for the conservation of these endangered species. Developing species-specific ART programmes for wildlife have been challenging. Temperature control during oocyte recovery is essential for ensuring in vitro success OBJECTIVE: This study is the first to investigate the effect of enema warming prior to trans-rectal ovum pick-up (OPU) on in vitro oocyte maturation in Southern white rhinoceros (Ceratotherium simum simum). METHODS: OPUs were performed on 20 rhinoceros cows from three different game farms in South Africa; oocytes were transported to one of two in vitro fertilisation laboratories for culture. The enema fluid was either warmed to 32 °C or not warmed prior to the OPU. Location of the farm, the different laboratories, ambient temperature, season, aspiration probe temperature, media type and enema temperature were investigated as predictor variables for oocyte maturation success RESULTS: After considering all other potential covariates, warming of the enema fluid was the only independent predictor of in vitro oocyte maturation success during this study CONCLUSION: Oocytes retrieved from rhinoceros cows that received an enema warmed to 32 °C were 2.3 times more likely to mature in vitro compared to oocytes from cows that received an unwarmed enema; the findings can be implemented in other rhinoceros ART programmes and in conservation efforts of other endangered mammalian species

Abstract Currently, gonadotropin products (follicle stimulating hormone, FSH, and luteinizing hormone, LH) used in animal reproduction are produced by extraction and purification from abattoir-derived pituitary glands. This method, relying on animal-derived materials, carries the potential risk of hormone contamination and pathogen transmission. Additionally, chorionic gonadotropins are extracted from the blood of pregnant mares (equine chorionic gonadotropin; eCG) or the urine of pregnant women (human chorionic gonadotropin; hCG). However, recent advancements have introduced recombinant gonadotropins for assisted animal reproduction therapies. The traditional use of FSH for superovulation has limitations, including labor requirements and variability in superovulation response, affecting the success of in vivo (SOV) and in vitro (OPU/IVEP) embryo production. FSH treatment for superstimulation before OPU can promote the growth of a homogenous follicular population and the recovery of competent oocytes suitable for IVEP procedures. At present, a single injection of a preparation of long-acting bovine recombinant FSH (rFSH) produced similar superovulation responses resulting in the production of good-quality in vivo and in vitro embryos. Furthermore, the treatment with eCG at FTAI protocol has demonstrated its efficacy in promoting follicular growth, ovulation, and P/AI, mainly in heifers and anestrous cows. Currently, treatment with recombinant glycoproteins with eCG-like activity (r-eCG) have shown promising results in increasing follicular growth, ovulation, and P/AI in cows submitted to P4/E2 -based protocols. Bovine somatotropin (bST) is a naturally occurring hormone found in cows. Recombinant bovine somatotropin (rbST), produced through genetic engineering techniques, has shown potential in enhancing reproductive outcomes in ruminants. Treatment with rbST has been found to improve P/IA, increase donor embryo production, and enhance P/ET in recipients. The use of recombinant hormones allows to produce non-animal-derived products, offering several advantages in assisted reproductive technologies for ruminants. This advancement opens up new possibilities for improving reproductive efficiency and success rates in the field of animal reproduction. Currently follicle LH abattoirderived abattoir derived glands method animalderived materials transmission Additionally equine human hCG. hCG . hCG) However therapies limitations response SOV (SOV OPU/IVEP OPUIVEP (OPU/IVEP procedures present longacting long acting rFSH (rFSH goodquality good quality embryos Furthermore ovulation PAI P AI eCGlike like reCG r (r-eCG P4E2 PE P4 E2 E P4/E based protocols bST (bST rbST, , (rbST) techniques ruminants PIA IA P/IA PET ET recipients nonanimalderived non P4E (rbST

Abstract The aim of this study was to compare embryo production efficiency in Flemish and Holstein donor females using ovum pick-up and in vitro fertilization (OPU-IVF) or in vivo production (superovulation; SOV) procedures. The study was conducted using a split-plot design, with eight Flemish and eight Holstein non-lactating cycling females. Females were subjected to ten weekly OPU/IVF sessions and/or two SOV/embryo collections sessions at a 63-day interval, for a total of 160 OPU-IVF and 32 SOV sessions. Mean numbers of follicles and corpora lutea, and cumulus-oocyte complex (COC) recovery rates were similar between breeds after the OPU and SOV sessions. However, Flemish donors yielded better quality grade II COCs (301, 41.9%) than Holstein females (609, and 202, 33.1%). Also, cleavage and blastocyst rates, and the total number and the mean number of viable embryos obtained after OPU-IVF were higher in Flemish (49.6% and 11.8%, and 63 and 11.8 per donor, respectively) than in Holstein (32.8% and 7.2%, and 34 and 7.2 per donor, respectively) females. Flemish females were also more efficient in yielding viable embryos after SOV (111, 7.3 per donor) than Holstein (48, 3.3 per donor) females. Overall, Flemish donor females had better responses to OPU-IVF or SOV procedures than Holstein counterparts. Irrespective of the breeds, SOV procedures were more efficient than OPU-IVF in yielding more viable embryos, under the conditions of this study. Both reproductive procedures were useful tools for the genetic conservation of the Flemish cattle breed in Southern Brazil. pickup pick up OPUIVF IVF (OPU-IVF superovulation (superovulation splitplot split plot design nonlactating non lactating andor SOVembryo 63day day interval 16 3 lutea cumulusoocyte cumulus oocyte COC (COC However 301, 301 (301 41.9% 419 41 9 609, 609 (609 202 33.1%. 331 33.1% . 33 1 33.1%) Also 49.6% 496 49 6 (49.6 118 11 8 11.8% 11. respectively 32.8% 328 (32.8 72 7 2 7.2% 7. 111, 111 (111 73 48, 48 (48 3. Overall counterparts Brazil 30 (30 41.9 4 60 (60 20 33.1 49.6 (49. 32.8 (32. (11 (4 (3 41. (6 33. 49. (49 32. (32 (1 (

Abstract In order to carry out a retrospective analysis of successive follicular aspirations in brangus cows in a livestock establishment in the Alto Paraná department, records of 10 Brangus breed cows subjected to seven successive follicular aspirations without prior hormonal stimulation were used. In the same, the quantity and quality of oocytes obtained in the aspirations were evaluated. Regarding the number of aspirated oocytes, in an average interval of 45 days between aspirations, an average of 207 ± 14 oocytes was obtained. Statistical analysis was performed by making a random comparison between aspirations: first aspiration with fourth aspiration, finding no statistically significant differences (p> 0.05); then the 4th aspiration was compared with the 7th aspiration, resulting statistically significant (p <0.05). Regarding the number of viable oocytes per aspiration, an average of 206 ± 30 oocytes and 88 non-viable oocytes were obtained, which comparing the first with the fourth aspiration yield statistically significant results (p <0.05). Regarding oocyte quality, the results show a greater quantity of GIII oocytes, they were statistically analyzed in the following way: comparison between the first (22.4) and the fourth aspiration (20.1) of the quality of GI oocytes, GII, GIII, yielding statistically non-significant results (p> 0.05).

Resumen Con el objetivo de realizar un análisis retrospectivo de las aspiraciones foliculares sucesivas en vacas de la raza brangus en un establecimiento ganadero del departamento de Alto Paraná, fueron utilizados registros de 10 vacas de la raza Brangus sometidas a siete aspiraciones foliculares sucesivas sin previa estimulación hormonal. En el mismo fueron evaluadas la cantidad y calidad de ovocitos obtenidos en las aspiraciones. Con respecto a la cantidad de ovocitos aspirados, en intervalo promedio de 45 días entre aspiraciones, se obtuvo un promedio de 207± 14 ovocitos. Se realizó el análisis estadístico realizando una comparación al azar entre aspiraciones: primera aspiración con la cuarta aspiración, no encontrando diferencias estadísticamente significativa (p>0,05) ; luego se compararon la 4ta aspiración con la 7ma aspiración, resultando estadísticamente significativo (p<0,05). Con respecto a la cantidad de ovocitos viables por aspiraciones se obtuvo en promedio 206 ±30 ovocitos y 88 ovocitos no viables, los cuales comparando la primera con la cuarta aspiración arrojan resultados estadísticamente significativos (p< 0,05). En cuanto a calidad de ovocitos los resultados demuestran mayor cantidad de ovocitos GIII, los mismos fueron analizando estadísticamente de la siguiente manera: comparación entre la primera (22,4) y la cuarta aspiración (20,1) de la calidad de ovocitos GI, GII, GIII, arrojando resultados estadísticamente no significativos (p>0,05).

Resumo Objetivou-se avaliar a infuência do estresse térmico em oócitos utilizados na produção in vitro de embriões (PIV) bovinos da raça Holandesa de alta produtividade no dia da aspiração folicular (OPU; 0), 30, 60 e 90 dias antes da OPU. A partir da temperatura média no dia 0 e aos 30, 60 e 90 dias anteriores, foram classificados nos grupos conforto (CT; até 15°C) e estresse por calor (ET -acima de 15°C). Observou-se infuência negativa em oócitos e embriões viáveis (total e grau I). A submissão ao estresse térmico nos períodos de 30 e 60 dias anteriores à OPU resultou em menor produção de oócitos viáveis (P=0,0028; P=0,0092, respectivamente). Sob estresse, no dia da OPU (ET-OPU), as vacas não apresentaram redução na quantidade de oócitos viáveis (P=0,5497) e não houve infuência da temperatura para o grupo estressado 90 dias antes da OPU (P=0,8287). Para embriões totais, a diferença ocorreu apenas no grupo ET-30 (P=0,0317), onde os grupos ET-OPU, ET-60, ET-90 apresentaram, respectivamente, P=0,1987, P=0,0596 e P=0,4580. Em relação à produção de embriões grau 1, não houve diferença para os grupos ET-OPU (P=0,2291) e ET-90 (P=0,2868), porém houve redução para ET-30 (P=0,0143) e ET- 60 (P=0,0253). Em resumo, o estresse por calor teve impacto negativo quando ocorreu 30 ou 60 dias antes da aspiração folicular. Além disso, 30 dias parece ser o período de maior suscetibilidade e que causa os maiores efeitos deletérios na viabilidade oocitária e na PIV.

Abstract The objective of this study was to evaluate the influence of thermal shock on oocytes used in the production of in vitro embryos (IVP) of high productivity Holstein cows on the day of follicular aspiration (OPU; 0), 30, 60 and 90 days before the OPU. From the mean temperature on day 0 and on the previous 30, 60 and 90 days, they were classifed into comfort group (TC; up to 15°C) and heat stress (HS; above 15°C) groups.Anegative influence was observed on oocytes and viable embryos (total and grade I). The heat stress in the periods of 30 and 60 days prior to OPU resulted in lower production of viable oocytes (P=0.0028; P=0.0092, respectively). Under stress, on the day of OPU (HS-OPU), cows showed no reduction in the amount of viable oocytes (P=0.5497) and there was no influence of temperature for the group stressed 90 days before OPU (P=0.8287). For total embryos, the difference occurred only in the HS-30 group (P=0.0317), where the groups HS-OPU, HS-60, HS-90 presented, respectively, P=0. 1987, P=0.0596 and P=0.4580. Regarding the production of embryos of grade 1, there was no difference for the groups HS-OPU (P=0.2291) and HS-90 (P=0.2868), but there was a reduction for HS-30 (P=0.0143) and HS-60 (P=0.0253). In summary, heat stress had a negative impact when it occurred 30 or 60 days before follicular aspiration. In addition, 30 days seems to be the period of more susceptibility and that causes the greatest deleterious effects on oocyte viability and IVP.

ABSTRACT Background: Under-reporting and recognition of occupational disease (OD) is a secular problem in Spain and other European countries. The burden of occupational disease in Spain project (CEPS) aims to contribute. Objective: To describe the process of identification and recognition of OD in seven Spanish between 2017 and 2020. Material and Methods: In the seven hospitals that participate in the study an occupational pathology unit (OPU) was created. The OPU encouraged the healthcare services to identify possible OD treated in the hospital. The patients confirmed by the OPU were accompanied in the recognition process. Results: 308 cases were referred in this period and 154 OD was confirmed (62%). Of these, 78 have been recognized (65%). The confirmation rate (confirmed/ referred cases) varied according to hospitals (from 25% at the Badalona Serveis Assistencials to 100% at the Hospital Universitario Fundación Jiménez Díaz) and diagnosis groups (from 13% for musculoskeletal disorders to 100% for various pathologies). The recognition rate (confirmed/ recognized cases) varied according to hospitals, diagnosis, sex and age, being higher in women, aged ≥ 55 years and diagnosed with various pathologies (ocular, infectious, mental, among others). Conclusion: OPU contribute significantly in the process of identification and recognition of OD, which the patients might not have obtained otherwise.

RESUMEN. Antecedentes : La infra-notificación y reconocimiento de enfermedad profesional (EP) es un problema secular en España y demás países europeos. El proyecto de Carga de la Enfermedad Profesional en España (CEPS) pretende contribuir a resolver este problema. Objetivo: Describir el proceso de identificación y reconocimiento de EP en siete hospitales españoles entre 2017 y 2019. Material y Métodos: En los siete hospitales que participan en el estudio se creó una unidad de patología laboral (UPL) la cual estimulaba en los servicios asistenciales la identificación de posibles EP atendidas en el hospital. Los pacientes confirmados por la UPL fueron acompañados en el proceso de reconocimiento. Resultados: En este periodo fueron derivados 308 casos, y en 154 (62%) se confirmó la sospecha EP. De éstos, 78 (65%) han sido reconocidos. La tasa de confirmación (casos confirmados/ derivados) varió según hospitales (de un 25% en el Badalona Serveis Assistencials a un 100% en el Hospital Universitario Fundación Jiménez Díaz) y grupos de diagnósticos (de un 13% para trastornos musculoesqueléticos a un 100% para patologías varias). La tasa de reconocimiento (casos confirmados/casos reconocidos) varió según centro, diagnóstico, sexo y edad, siendo más alta en mujeres, con ≥ 55 años y diagnosticados con patologías varias (oculares, infecciosas, mentales, entre otras). Conclusión: Las UPL contribuyen significativamente en el proceso de identificación y reconocimiento de EP, lo que los pacientes posiblemente no hubieran obtenido de otra forma.

RESUMEN. Antecedentes : La infra-notificación y reconocimiento de enfermedad profesional (EP) es un problema secular en España y demás países europeos. El proyecto de Carga de la Enfermedad Profesional en España (CEPS) pretende contribuir a resolver este problema. Objetivo: Describir el proceso de identificación y reconocimiento de EP en siete hospitales españoles entre 2017 y 2019. Material y Métodos: En los siete hospitales que participan en el estudio se creó una unidad de patología laboral (UPL) la cual estimulaba en los servicios asistenciales la identificación de posibles EP atendidas en el hospital. Los pacientes confirmados por la UPL fueron acompañados en el proceso de reconocimiento. Resultados: En este periodo fueron derivados 308 casos, y en 154 (62%) se confirmó la sospecha EP. De éstos, 78 (65%) han sido reconocidos. La tasa de confirmación (casos confirmados/ derivados) varió según hospitales (de un 25% en el Badalona Serveis Assistencials a un 100% en el Hospital Universitario Fundación Jiménez Díaz) y grupos de diagnósticos (de un 13% para trastornos musculoesqueléticos a un 100% para patologías varias). La tasa de reconocimiento (casos confirmados/casos reconocidos) varió según centro, diagnóstico, sexo y edad, siendo más alta en mujeres, con ≥ 55 años y diagnosticados con patologías varias (oculares, infecciosas, mentales, entre otras). Conclusión: Las UPL contribuyen significativamente en el proceso de identificación y reconocimiento de EP, lo que los pacientes posiblemente no hubieran obtenido de otra forma.

ABSTRACT Background: Under-reporting and recognition of occupational disease (OD) is a secular problem in Spain and other European countries. The burden of occupational disease in Spain project (CEPS) aims to contribute. Objective: To describe the process of identification and recognition of OD in seven Spanish between 2017 and 2020. Material and Methods: In the seven hospitals that participate in the study an occupational pathology unit (OPU) was created. The OPU encouraged the healthcare services to identify possible OD treated in the hospital. The patients confirmed by the OPU were accompanied in the recognition process. Results: 308 cases were referred in this period and 154 OD was confirmed (62%). Of these, 78 have been recognized (65%). The confirmation rate (confirmed/ referred cases) varied according to hospitals (from 25% at the Badalona Serveis Assistencials to 100% at the Hospital Universitario Fundación Jiménez Díaz) and diagnosis groups (from 13% for musculoskeletal disorders to 100% for various pathologies). The recognition rate (confirmed/ recognized cases) varied according to hospitals, diagnosis, sex and age, being higher in women, aged ≥ 55 years and diagnosed with various pathologies (ocular, infectious, mental, among others). Conclusion: OPU contribute significantly in the process of identification and recognition of OD, which the patients might not have obtained otherwise.

ABSTRACT This study assessed the morphometric and functional characteristics of oocytes from 10 native cows to the Ecuadorian Andes, collected by transvaginal follicular aspiration (OPU) and oocytes collected post mortem (PM) from abattoir ovaries. For this purpose, 1157 cumulus-oocyte complexes (CCOs) of the OPU group (n=271) and PM group (n=886) were recovered in 10 repetitions per group by follicular aspiration at 90 mm Hg pressure and classified as A, B or C, according to the characteristics of the cytoplasm and cumulus cells. The activity of the enzyme glucose 6 phosphate dehydrogenase (G6PD) and the integrity of the plasma membrane was determined by staining two groups of CCOs with cresyl brilliant blue (BCB +) and trypan blue, respectively. The size (diameter and volume) of the oocytes was evaluated using CaptaVisio® software v. 5.1. The data were analysed by logistic regression and by the SAS general linear model, and the means were compared by the least square method. The results showed that the oocytes of the PM group had a larger diameter (126.0 ± 0.48 vs. 122.7 ± 0.79 µm; p<0.01) and integrity of the plasma membrane (86.0 vs. 76.8%; p<0.05) than the OPU group. However, quality Aand B oocytes did not show significant differences between groups with respect to plasma membrane integrity and BCB + values. The results suggest that, although the fact that the size and viability were more affected in the oocytes aspirated by OPU, those of quality A and B from both sources showed similar values regarding the integrity of the plasma membrane and metabolic condition to continue. maturation.

RESUMEN El estudio analizó las características morfométricas y funcionales de ovocitos de 10 vacas nativas de los Andes ecuatorianos, colectados por aspiración folicular transvaginal (OPU) y de ovocitos colectados post mortem (PM) de ovarios de matadero. Para este propósito, 1157 complejos cúmulo-ovocito (CCOs) del grupo OPU (n=271) y PM (n=886) fueron recuperados en 10 repeticiones por grupo por aspiración folicular a 90 mm Hg de presión y clasificados en calidad A, B o C, de acuerdo con las características del citoplasma y células del cúmulo. Se determinó la actividad de la enzima glucosa 6 fosfato deshidrogenasa (G6PD) y la integridad de la membrana plasmática tiñendo dos grupos de CCOs con azul brillante de cresilo (BCB+) y azul de tripán, respectivamente. Se evaluó el tamaño (diámetro y volumen) de los ovocitos usando un software CaptaVision® (v. 5.1). Los datos se analizaron por regresión logística y por el modelo lineal general del SAS, y las medias se compararon por el método de los mínimos cuadrados. Los resultados mostraron que los ovocitos del grupo PM presentaron mayor diámetro (126.0±0.48 vs 122.7±0.79 µm; p<0.01) e integridad de la membrana plasmática (86.0 vs 76.8%; p<0.05) que el grupo OPU. Sin embargo, los ovocitos de calidad A y B no registraron diferencias significativas entre grupos con respecto a la integridad de la membrana plasmática y valores de BCB+. Los resultados sugieren que, a pesar de que el tamaño y la viabilidad fue más afectada en los ovocitos aspirados por OPU, los de calidad A y B provenientes de ambas fuentes mostraron valores similares con respecto a la integridad de la membrana plasmática y condición metabólica para proseguir la maduración.

ABSTRACT The effect of the addition of melatonin (Mt) on the maturation and / or vitrification of bovine oocytes on cleavage and subsequent embryonic development was evaluated. Oocyte-cumulus cell complexes (COCs) were obtained from creole cows by ovum pick up (OPU) and by follicular aspiration from abattoir ovaries (AO). From the pool of oocytes obtained from both sources, those with homogenous cytoplasm and three or more compact layers of cluster cells were selected. The selected COCs were randomly assigned to five treatments: T1, matured with Mt and vitrified without Mt; T2, matured and vitrified with Mt; T3, matured without Mt and vitrified with Mt; T4 (control) matured and vitrified without Mt; T5, matured without Mt and not vitrified. The concentration of Mt in the maturation and vitrification media was 0.01 µM (10-9 M). The oocytes were matured, vitrified, fertilized and the presumed zygotes cultured until day 7 post-fertilization in vitro. The data were analysed by logistic regression. Regardless of the origin of the oocytes, cleavage rate (C) and embryo production (EP) was similar between treatments. The C in the OPU oocytes was greater in T4, and the EP was similar between treatments. In AO, the results did not vary between treatment in C and EP. In conclusion, Mt reduced the C in oocytes collected by OPU, while it did not affect EP. In the AO oocyte source, the addition of Mt did not affect the C nor the EP.

RESUMEN Se evaluó el efecto de la adición de melatonina (Mt) en el medio de maduración y/o de vitrificación de ovocitos bovinos sobre clivaje y posterior desarrollo embrionario. Los complejos ovocito-células del cúmulo (COCs) fueron obtenidos de vacas criollas mediante la técnica de aspiración transvaginal guiada por ultrasonografía (OPU) y de ovarios de matadero (OM). Del pool de ovocitos obtenidos de ambas fuentes se seleccionaron los que tenían citoplasma homogéneo y tres o más capas compactas de células del cúmulo. Los COCs seleccionados fueron asignados aleatoriamente a cinco tratamientos: T1, madurados con Mt y vitrificados sin Mt; T2, madurados y vitrificados con Mt; T3, madurados sin Mt y vitrificados con Mt; T4 (control) madurados y vitrificados sin Mt; T5, madurados sin Mt y no vitrificados. La concentración de Mt en los medios de maduración y vitrificación fue de 0.01 µM (10-9 M). Los ovocitos fueron madurados, vitrificados, fecundados y los presuntos cigotos cultivados hasta el día 7 pos-fecundación in vitro. Los datos fueron analizados por regresión logística. Independientemente del origen de los ovocitos, el porcentaje de clivaje (PC) y de producción de embriones (PIV) fue similar entre tratamientos. El PC en los ovocitos de OPU fue mayor en T4, y la PIV fue similar entre tratamientos. En los de OM, los resultados no variaron entre tratamiento en PC y PIV. En conclusión, la Mt redujo el PC en ovocitos colectados por OPU, mientras que no afectó la PIV. En los colectados de OM la adición de Mt no afectó el PC ni la PIV.

ABSTRACT The aim of this study was to evaluate the effects of gonadotropin treatment and the interval between ovarian stimulation with FSH/LH and the follicular aspiration session, on the quantity and quality of the cumulus oocyte complexes (COCs) obtained by ovum pick up (OPU)) in Ecuadorian Creole heifers. Four heifers were randomly assigned to four groups (FHS/LH 40h; FSH/LH 48h; control 40h; control 48h). In two groups a dose (0.011 mg, IM) of GnRH was applied on day 0, followed by 500 IU, IM, of FSH/LH on day 2, and the OPU session at 40 (FSH/LH 40 h) or 48 hours (FSH/LH 48h) after the application of gonadotropin. In the other two groups, FSH/LH was replaced by a physiological solution. There was no statistical difference in the recovery rate of COCs between FSH/LH 40h and FSH/LH 48h, but approximately twice as many COCs (p<0.05) were obtained than in those who did not receive ovarian stimulation. In addition, 2.4 times more COCs of qualityAwere achieved in the treatment ofFSH/LH 48hthan in that ofFSH/LH 40h.All (100%) of these structures reacted positively to Brilliant Cresyl Blue (BCB+), which means that they finished the growth and were competent to initiate in vitro maturation. It is concluded that the treatment with FSH/LH 48h allowed to obtain a greater number of COCs competent for in vitro maturation.

RESUMEN El objetivo del estudio fue evaluar los efectos del tratamiento con gonadotropina y el intervalo entre la estimulación ovárica con FSH/LH y la sesión de aspiración folicular, sobre la cantidad y calidad de los complejos cúmulus ovocitos (COCs) obtenidos por ovum pick up (OPU) en vaquillas criollas ecuatorianas. Cuatro novillas fueron asignadas aleatoriamente a cuatro grupos (FHS/LH 40h; FSH/LH 48h; control 40h; control 48h). En dos grupos se aplicó una dosis (0.011 mg, IM) de GnRH el día 0, seguida de 500 UI, IM, de FSH/LH el día 2, y la sesión de OPU a los 40 (FSH/LH 40 h) o 48 horas (FSH/LH 48h) después de la aplicación de la gonadotropina. En los otros dos grupos, la FSH/LH se reemplazó por una solución fisiológica. No hubo diferencia estadística en la tasa de recuperación de los COCs entre FSH/LH 40h y FSH/LH 48h, pero se obtuvo aproximadamente el doble de COCs (p<0.05) que en aquellos que no recibieron estimulación ovárica. Se consiguió 2.4 veces más COCs de calidad A en el tratamiento de FSH/LH 48h que en el de FSH/LH40h.El 100% de estas estructuras reaccionaron positivamente al Azul Brillante de Cresilo (BCB+), lo que significa que terminaron el crecimiento y fueron competentes para iniciar la maduración in vitro. Se concluye que el tratamiento con FSH/LH 48h permitió obtener un mayor número de COCs competentes para la maduración in vitro.

RESUMO A fim de avaliar o efeito de diferentes doses da rbST sobre a dinâmica folicular, a produção e a maturação in vitro de oócitos, 20 vacas Sindi, divididas em três grupos, receberam um dispositivo de progesterona intravaginal, estradiol e PGF2α, além de 2mL de solução salina (grupo controle), 250 (grupo rbST 250) ou 500mg de rbST (grupo rbST 500). Cinco dias depois, realizou-se a ovum pick up, e os complexos cumulus-oócitos (CCOs) recuperados foram selecionados, classificados e maturados in vitro. Os dados de contagem foram comparados pelo procedimento glht (General Linear Hypothesis Test), e os dados em porcentagem foram submetidos ao qui-quadrado, no programa estatístico R, onde as diferenças foram consideradas significativas quando P<0,05. Não houve diferença (P>0,05) entre os grupos quanto à quantidade de folículos e à taxa de maturação. Os grupos rbST 250 e rbST 500 foram significativamente superiores (P<0,05) ao grupo controle em relação ao número de folículos grandes (0,42±0,20 vs. 0). O grupo rbST 500 apresentou maior (P<0,05) porcentagem de oócitos viáveis (91,52%) do que os grupos controle (67,85%) e rbST 250 (53,33%). A rbST aumenta o número de folículos grandes, e 500mg de rbST aumentam a porcentagem de oócitos viáveis em vacas Sindi.

ABSTRACT In order to evaluate the effect of different doses of rbST on the follicular dynamics, production, and in vitro maturation of oocytes, 20 Sindhi cows were divided into three groups, receiving an intravaginal progesterone device, estradiol and PGF2α, and 2mL of solution saline (Control Group), 250 (rbST 250 Group) or 500mg rbST (rbST 500 Group). Five days later, the ovum pick up was performed, and the cumulus-oocyte (CCO) complexes recovered were selected, classified, and matured in vitro. The counting data were compared by the glht (General Linear Hypothesis Test) procedure, and the percentage data were submitted to Qui- square, in the statistical program R, where differences were considered significant when P< 0.05. There was no difference (P> 0.05) between the groups regarding follicle quantity and maturation rate. The rbST 250 and rbST 500 groups were significantly higher (P< 0.05) than the Control group in relation to the number of large follicles (0.42±0.20 versus 0). The rbST 500 group presented higher (P< 0.05) percentage of viable oocytes (91.52%) than the Control (67.85%) and rbST 250 (53.33%) groups. rbST increases the number of large follicles and 500mg rbST increases the percentage of viable oocytes in Sindhi cows.

Abstract The objective of this study was to evaluate the fertility of buffalo semen for in vitro embryo production (IVEP) by comparing the effectiveness of refrigerated versus frozen semen. Three OPU sessions were held at 30-day intervals. For oocyte fertilization three buffalo bulls were used, one per session. At each OPU-IVEP session, one ejaculate was collected and divided into two equal aliquots. Each aliquot was either refrigerated at 5ºC/24 hours or frozen. A TRIS extender containing 10% low density lipoproteins, 0.5% lecithin and 10 mM acetylcysteine was used adding 7% glycerol for freezing. Sperm motility/kinetic was evaluated by CASA and sperm membrane integrity by the hypoosmotic swelling test. The evaluations were performed at 0 h (post final dilution at 37ºC), at 4 and 24 hs post-incubation at 5ºC and post-thaw. At 24 hs incubation and immediately post thaw sperm cells were used for in vitro fertilization of buffalo oocytes equally distributed between both groups. Cleavage rates and embryo development were followed. The embryo/matured and embryo/cultured rates were 25.4 x 14.0% and 29.4 x 18.5% (P<0.05), for chilled and frozen semen, respectively. It is concluded that cooled semen can be used for in vitro embryo production in buffalo and that a better efficiency may be expected for cooled compared to frozen semen.

Abstract Knowledge of follicular wave dynamics obtained through the use of real-time ultrasonography and the development of the means by which follicular wave dynamics can be controlled have provided practical approaches for the in vivo and in vitro production and transfer of embryos in cattle. The elective control of follicular wave emergence and ovulation has had a great impact on the application of on-farm embryo transfer, especially when large groups of donors need to be superstimulated at the same time. Although estradiol and progestins have been used for many years, practitioners in countries where estradiol cannot be used have turned to alternative treatments, such as mechanical follicle ablation or the administration of GnRH for the synchronization of follicle wave emergence. In vitro embryo production also benefits from the synchronization of follicle wave emergence prior to Cumulus Oocyte Complexes (COCs) recovery. As Bos indicus cattle have high antral follicle population, large numbers of oocytes can be obtained by ovum pick-up (OPU) without superstimulation. However, synchronization of follicular wave emergence and superstimulation is necessary to obtain high numbers of COCs by OPU and blastocysts following in vitro fertilization in Bos taurus donors. Finally, embryos can now be transferred in commercial beef or dairy herds using efficacious synchronization and re-synchronization protocols that are easily implemented by farm personnel. These technologies can also be used to resolve reproductive problems such as the reduced fertility observed during summer heat stress and/or in repeat-breeder cows in commercial dairy herds.