Lunar Cycle Influences Reproductive Performance of Crossbred Brahman Cows Under Tropical Conditions.

Evidence has accumulated over the years indicating that the moon influences some aspects of the reproductive activity in animals and humans. However, little is known about the influence of the lunar cycle on the reproductive performance of cows under tropical conditions, where the environment strongly affects reproduction. This retrospective study was conducted with the aim of assessing the influence of the lunar cycle on some reproductive traits of tropical crossbred cows managed in a pasture-based system. Data from 5869 reproductive records from two commercial farms localized in the Maracaibo Lake Basin of Zulia State, Venezuela, were analyzed. Variables studied were first service conception rate, calving frequency, first postpartum estrous frequency, and pregnancy frequency. In addition to the lunar cycle, the effects of farm, season, and predominant breed were also considered. Data were analyzed using logistic regression and general linear model from SAS. First service conception was affected by lunar phases and predominant breed, but not by farm or season. For frequencies of calving, first postpartum estrus, and pregnancy, there was no main effect of farm, season, and predominant breed, whereas the effect of lunar phases was highly significant. First service conception was significantly greater in waning than in crescent phase of the lunar cycle. Frequencies of calving, first estrus, and pregnancy were highly correlated and showed greater figures around full moon and new moon. In conclusion, lunar cycle influenced first service conception, attaining greater values in the waning phase of the moon cycle. Frequencies of calving, first postpartum estrus, and pregnancy in crossbred cows showed a clear bimodal rhythm, whose greatest values coincided with new moon and full moon.

Efecto de la Dosis de eCG sobre las Características Foliculares y Luteales, momento y Tasa de Ovulación de Novillas Mestizas Cebú Sincronizadas con Progestágeno Intravaginal / Effect of the eCG Dose on Follicular and Luteal Characteristics, Ovulation Time and Ovulation Rate of Crossbred Zebu Heifers Synchronized with an Intravaginal Progestagen Device

Para evaluar el efecto de una dosis reducida de la gonadotropina coriónica equina (eCG) sobre el crecimiento folicular, las características luteales, el momento de la ovulación y la tasa de ovulación en novillas mestizas cebú sincronizadas con un progestágeno intravaginal, se seleccionaron 41 novillas cíclicas. Para la sincronización se utilizó una esponja intravaginal (EI) impregnada con 250 mg de medroxi-acetato-progesterona (MAP), bajo el protocolo siguiente: Día 0: Inserción de la EI más 2 mg de benzoato de estradiol (BE) im y 25 mg de PGF2a im; Día 6: 12,5 mg de PGF2a, 21 novillas recibieron 400 UI de eCG im (T1; n=21) y 20 recibieron 200 UI (T2; n=20); Día 8: retiro de EI; Día 9: 0,5 mg de BE im. La evaluación ultrasonográfica se realizó entre los días 6 y 8, cada 24 h, para determinar la tasa de crecimiento folicular (TCF). A partir del día 8 hasta la ovulación, se evaluaron cada 8 h para determinar el diámetro del folículo ovulatorio (DFO), el momento de la ovulación (MO), el diámetro y área del cuerpo lúteo (DCL y ACL), así como la tasa de ovulación (TO). Las variables TCF, DFO, DCL y ACL se analizaron con el PROC GLM, mientras que el MO mediante el procedimiento NPAR1WAY y la TO mediantec2, utilizando el PROC FREQ del SAS. No hubo diferencias en las variables TCF, DFO y ACL (1,44±0,27 vs.1,72±0,27 mm/d; 12,61±0,47 vs.13,85±0,47 mm y 33,9±2,1 vs. 30,3±2,4 mm² para T1 y T2, respectivamente). Las ovulaciones ocurrieron en promedio 61,67±1,8 y 62,73±1,92 h, respectivamente después de retirado el dispositivo para T1 y T2. El DCL fue mayor (P<0,05) en T1 que en T2 (21,7±1,44 vs.16,9±1,56 mm). Se encontraron mayores porcentajes para la TO (P=0,06) en el grupo T1 que en T2 (95,25 vs. 75%). En conclusión, la dosis de 200 UI de eCG fue suficiente para inducir un adecuado crecimiento folicular y no afectar el momento de la ovulación y la dosis de 400 UI de eCG mejoró las características del cuerpo lúteo y permitió obtener un mayor número de ovulaciones en novillas mestizas cebú sincronizadas con un progestágeno intravaginal.

The effect of a reduced dose of eCG on follicular growth, luteal characteristics, ovulation time (OT) and ovulation rate (OR) was evaluated in crossbred Zebu heifers synchronized with an intravaginal progestagen device. A total of 41 cyclic heifers was selected and synchronized with an intravaginal sponge (IE) impregnated with 250 mg of medroxy-progesterone-acetate (MPA), using the following protocol. Day 0: insertion of IE plus 2 mg of estradiol benzoate (EB) im and 25 mg of PGF2a im. Day 6: 12.5 mg de PGF2a, 21 heifers received 400 IU eCG im (T1; n = 21) and 20 received 200 IU (T2; n = 20). Day 8: IE withdrawal. Day 9: 0.5 mg of BE im. Heifers were timed-artificial inseminated (TAI) between 52 and 56 h after IE withdrawal. Ultrasound evaluation was performed between d 6 until d 8, every 24 h, to determine the rate of follicular growth (FGR). Then, from d 8 until ovulation, heifers were evaluated every 8 h to determine diameter of the ovulatory follicle (DOF), OT, and diameter (CLD) and area (CLA) of the corpus luteum. The OR was also assessed. The variables FGR, DOF, CLD and CLA were analyzed using PROC GLM, while OT was analyzed using the NPAR1WAY procedure and OR using Chi-square with PROC FREQ from SAS. No differences were found in FGR, DOF, and CLA (1.44±0.27 vs. 1.72±0.27 mm/d; 12.61±0.47 vs. 13.85±0.47 mm and 33.9±2.1 vs. 30.3±2.4 mm² for T1 and T2, respectively). Ovulations occurred at 61.67±1.8 h and 62.73±1.92 h, respectively, after IE withdrawal for T1 y T2, respectively. The CLD in T1 was higher (P<0.05) than T2 (21.7±1.44 vs. 16.9±1.56 mm). Higher OR (P=0.06) were found in T1 than in T2 (95.25 vs. 75.0%). In conclusion, the dose of 200 IU of eCG was sufficient to induce an suitable follicular growth without affecting OT and dosing with 400 IU of eCG improved the corpus luteum characteristics and allowed to obtain a greater number of ovulations in crossbred Zebu heifers synchronized with an intravaginal progestagen.

Factors affecting porcine sperm mediated gene transfer.

"Sperm mediated gene transfer" (SMGT) is based on the ability of sperm cells to bind exogenous DNA. The main objective of this study was to improve the production of transgenic pigs by SMGT. Taking into account that there is a lack of repeatability in studies of SMGT and that the mechanism of binding and internalization of exogenous DNA is a question that has not been solved, different factors involved in the production of transgenic animals by SMGT method were evaluated. Here we set out to: (1) evaluate the sperm capacity to bind exogenous DNA after DMSO treatment; (2) determine the location of the transgene-spermatozoa interaction; and (3) evaluate the efficiency of production of transgenic piglets by deep intrauterine artificial insemination (AI) with sperm incubated with DNA. The percentage of DNA binding was higher than 30% after 2h of co-culture, but it was not affected by sperm treatment with DMSO (0.3% or 3%). The integrity of the sperm plasma membrane plays a critical role in DNA interaction, and altered plasma membranes facilitate interactions with exogenous DNA. DNA bound mainly to spermatozoa with reduced viability. DNA molecules were found to be mainly associated to the post-acrosomal region (61.9%). After deep intrauterine AI a total of 29 piglets were obtained, but none of them integrated the transgene. In conclusion, although it has been confirmed that DNA can associate with boar spermatozoa, the efficiency of producing transgenic pigs by AI was not confirmed by the present experiments, mainly due to a reduced DNA binding to functional spermatozoa.

Production of transgenic piglets using ICSI-sperm-mediated gene transfer in combination with recombinase RecA.

Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) protein-coated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA-DNA complexes at 5 microg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT-ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase.

Transgénesis mediada por espermatozoides en la especie porcina: efecto de la presencia de ADN exógeno sobre la calidad seminal y evaluación de la producción in vivo de embriones transgénicos / Sperm mediated gene transfer in pigs: Effect of exogenous DNA presence in seminal quality and evaluation of in vivo transgenic embryo production

Una alternativa biotecnológica para la producción de animales transgénicos es la transgénesis mediada por espermatozoides (SMGT), basada en la habilidad de las células espermáticas de unir e interiorizar ADN exógeno. En este estudio se plantearon dos objetivos principales: 1) evaluar el efecto de la presencia de ADN exógeno sobre la calidad seminal y 2) evaluar la eficiencia en la producción de embriones transgénicos porcinos in vivo mediante inseminación intrauterina quirúrgica con espermatozoides incubados con el transgén. Para alcanzar estos objetivos, los espermatozoides (libres de plasma seminal) fueron incubados en presencia del transgén EGFP (proteína verde fluorescente) durante 2 h a 16°C (en una relación de 10(8) cels/mL y 5 µg ADN/mL). Se valoró la motilidad, motilidad progresiva e integridad de membrana de los espermatozoides incubados, en presencia o ausencia del transgén. Los resultados mostraron que la presencia del ADN no afectó a ninguno de los parámetros seminales estudiados. Con muestras seminales incubadas con el transgén se llevaron a cabo inseminaciones intrauterinas mediante laparotomía en 4 hembras prepúberes. A los 6-7 días tras la inseminación se recolectaron los embriones, para evaluar la tasa de fecundación y la expresión de la proteína verde fluorescente EGFP en los mismos. El número medio de cuerpos lúteos por cerda fue de 10,50 ± 2,90 con una tasa de recolección de 11,90%. Se recolectó un total de 5 embriones presentando todos ellos un estado normal de desarrollo y una alta calidad (dos de ellos presentaban más de 400 células por embrión). Cuando fue analizada la expresión mediante microscopia de fluorescencia, ninguno de ellos expresaba la proteína EGFP. Este resultado, bajo las condiciones experimentales del presente estudio, podría indicar que el transgén se una en su mayoría a espermatozoides con baja viabilidad por lo que la probabilidad de que los espermatozoides vivos unan el ADN y sean capaces de fecundar en comparación con espermatozoides sin el transgén es realmente baja.

An alternative technology for producing transgenic animals is sperm mediated gene transfer (SMGT), based on the ability of sperm cells to bind and internalize exogenous DNA. The aims of this work were: 1) evaluate the effects of the presence of exogenous DNA in the seminal quality and 2) evaluate the efficiency of transgenic embryos production by surgery artificial insemination using sperm mediated gene transfer technique. Fresh spermatozoa (removed seminal plasma) were incubated with DNA (EGFP) at 16°C for 2 h (10(8) cells/mL and 5 µg DNA/mL). At first motility, progressive motility and viability were evaluated in spermatozoa incubated with or without exogenous DNA. The results showed that the presence of the DNA did not affect any of the studied sperm parameters (motility, progressive motility and viability). On the other hand insemination was carried out in 4 gilts by laparotomy in utero tubaric junction. Six-seven days after insemination blastocysts were collected and fertilization rate and transgene expression were determined. The mean number of corpora lutea/gilt was 10.50 ± 2.90 with a mean recovery rate of 11.90%. After the surgery, a total of 5 embryos were recovered and the percentage of normally developed embryos was 100% and with a high quality (two of them had more than 400 cells). When the blastocysts recovered after 6-7 days post-insemination were analyzed by fluorescence microscopy, it was revealed that none of them expressed protein EGFP. These results show the possibility that exogenous DNA bound to the sperm with low viability and only a low percentage to viable sperm, so the probability that live sperm bind the DNA and are able to fertilize in comparison with live sperm without the transgene are really low.

Calves born under tropical conditions after direct transfer of cryopreserved in vitro produced embryos

En el presente estudio se comparó el desempeño de 56 novillas doble propósito que resultaron preñadas luego de la transferencia directa de embriones producidos in vitro cultivados en un medio suplementado con suero o en uno químicamente definido. No se observaron diferencias en las tasas de aborto (30,43% vs 24,24%), distocias (52,17% vs 51,52%) y parto normal (17,39% vs 24,24%) entre las novillas que recibieron embriones cultivados en el medio suplemento con suero y las que recibieron embriones cultivados en el medio químicamente definido. El sexo de la cría afectó significativamente el porcentaje de distocias, 83,33% para machos y 50% para hembras, (P<0,05). El peso al nacimiento de los becerros tampoco se vio afectado (p>0,05) por la suplementación sérica durante el cultivo (46,86 ± 2,04 kg, para los becerros derivados de los embriones cultivados en el medio suplementado con suero y 46,28 ± 1,42 kg, para los derivados de los embriones cultivados en el medio químicamente definido) ni por el sexo de la cría (machos 47,20 ± 1,50 kg y hembras, 45,45 ± 1,84 kg). El peso de los becerros que nacieron muertos o que murieron luego del nacimiento fue significativamente (P< 0,05) mayor (51,92 ± 1,76 kg) al de los becerros que sobrevivieron (43,88 ± 1,22 kg). La sobrevivencia perinatal no se vió afectada ni la suplementación sérica durante el cultivo embrionario, ni por el sexo de los becerros o el nacimiento de un parto distócico. En conclusión, la presencia de suero en el medio de cultivo no afectó el desempeño de las novillas doble propósito que resultaron preñadas luego de la transferencia de embriones producidos in vitro. En este estudio se observó la presencia de becerros con síndrome del recién nacido gigante evidenciado por un alto peso al nacimiento y una alta tasa de abortos y distocias.

Determinación del protocolo de criopreservación de semen canino: reporte preliminar / Determination of the protocol for the criopreservation of the canine semen: preliminary report

Se recolectaron muestras de semen a 7 machos caninos de varias razas, a las cuales se les practicó un espermatograma completo. Aquellas muestras seminales clasificadas como óptimas fueron utilizadas para ser sometidas al proceso de criopreservación. Se procedió a congelar el semen utilizando un diluente a base de TRIS, ácido crítico, glucosa, penicilina, estreptomicina, yema de huevo, agua destilada y glicerol. El semen fue congelado en vapores de nitrógeno líquido a 10 centímetros por encima de su nivel. El semen fue sometido a pruebas de evaluación considerándose los parámetros de volumen, color, pH, motilidad, concentración y morfología. Se realizaron pruebas de resistencia térmica en placas calentadas para el semen fresco, diluido, glicerinado y descongelado, por períodos de tiempo de 15, 30, 45, 60 y 90 minutos, obteniéndoseresultados de motilidad de hasta 30% al cabo de 90 min. Se pudo observar la versatilidad y sensillez del uso de este dilutor y de la técnica de congelación en vapores de nitrógeno líquido para la criopreservación del semen canino.