José Carlos Ferreira-Silva

Use of retinoids during oocyte maturation diminishes apoptosis in caprine embryos

Exposure of caprine oocytes and embryos to retinoids enhances embryonic development, but the mechanisms governing this phenomenon have not been characterised. The aim of the present study was to evaluate if the incidence of apoptosis is affected by the addition of retinyl acetate (RAc) and 9-cis-retinoic acid (RA) during in vitro maturation (IVM) of caprine oocytes. Embryonic development was recorded on days 3 and 8 post-fertilisation, and apoptosis was measured by caspase activity and DNA fragmentation (TUNEL assay). Control zygotes had lower capacity to cleave and reach the blastocyst stage (24.45 ± 2.32 and 5.32 ± 0.81, respectively) than those of RAc- (29.96 ± 1.62 and 7.94 ± 0.93, respectively) and RA-treated groups (30.12 ± 1.51 and 7.36 ± 1.02, respectively). Oocytes and blastocysts positive for TUNEL assay were more frequent, respectively, in the controls (8.20 ± 0.78, 8.70 ± 1.05) than in RAc (5.60 ± 0.52, 4.80 ± 0.51) and RA (6.40 ± 0.69, 5.40 ± 0.69). Caspase activity did not differ between control oocytes (7.20 ± 0.91), RAc (6.60 ± 0.68) and RA (7.30 ± 0.67), but it was reduced in RAc- (5.05 ± 0.62) and RA-treated blastocysts (5.75 ± 0.22) compared to controls (8.35 ± 0.71). These results indicate that the addition of retinoids during IVM increases the developmental potential of goat embryos with a concomitant reduction in apoptosis rates.

Incidence of apoptosis after retinoids and insulin-like growth factor-I (IGF-I) supplementation during goat in vitro embryo production.

The addition of growth factors and vitamins enhances goat embryonic development in vitro. However, few attempts have been reported trying to identify supplementation regimens for oocyte maturation or embryo culture with additive properties. The present report was aimed to evaluate if retinoids [0.3 μM retinyl acetate (RAc) and 0.5 μM 9-cis-retinoic acid (RA)] supplementation during goat oocyte maturation and retinoids and/or 50 ng mL-1 IGF-I during embryo culture synergically enhanced embryonic development while diminishing the incidence of apoptosis. All combinations of RAc and RA treatment produced blastocysts with similar efficiencies, while IGF-I enhanced embryos yields irrespectively of retinoid addition. Moreover, retinoids and IGF-I supplementation showed similar caspase activity or DNA fragmentation indexes in blastocysts. In conclusion, supplementation with retinoids and IGF-I during goat embryo culture enhances blastocysts development without synergic reduction of apoptosis.

Conceptus loss in Santa Inês ewes carrying twin pregnancies by natural mating or embryo transfer

Commercial application of reproductive biotechnologies such as multiple ovulation and embryo transfer depends on its overall efficiency. Sheep embryo transfer is gradually gaining wider adoption, but pregnancy rates after embryo transfer remain lower than those derived from natural mating for most breeds. The work was aimed to evaluate embryonic and fetal losses in Santa Inês ewes carrying twin pregnancies by natural mating or embryo transfer. Ewes were subjected to synchronized natural mating by ram effect or used as recipients for embryo transfer. Ewes diagnosed as carrying twin pregnancies at day 25 were used in the experiment (n = 42). Conceptus viability was monitored by ultrasonography on days 30, 35, 40, 45, 50, and 55 after conception. Conceptus loss was similar (P > 0.05) within natural mating 11/42 (26.19%) and embryo transfer 14/42 (33.34%). However, overall embryonic loss (80.0%) was greater (P < 0.05) than fetal loss (20.0%), with no difference within groups The results allow the conclusion that conceptus loss after embryo transfer is similar to natural mating and occurs predominantly during the embryonic stages.

Freezing of Stallion Semen: In Vitro Evaluation of Motility and Acrosin Activity in Sperm Cells Cryopreserved Using Different Semen Extenders

The work described here aimed to verify the efficiency of different extenders for cryopreservation of equine semen using sperm motility and acrosin activity as spermatic parameters. The semen was fractioned into two equal parts and resuspended in an 11% lactose solution in a 1:1 proportion, where it remained for 20 minutes at room temperature. The semen was centrifuged at 600 g for 10 minutes, and after the second centrifugation, each pellet received the freezing extender (Merck or Zorlesco) and was loaded into 4 mL straws. Each straw was placed in liquid nitrogen vapor steam for 15 minutes and further immersion in liquid nitrogen at -196°C for long-term storage. After thawing, semen samples were initially evaluated for sperm motility, both total and progressive, and acrosin activity. Moreover, semen was incubated at 37°C and further assessed at 60 and 120 minutes in a thermoresistance test (TRT) for sperm motility and acrosin activity. Immediately after thawing, both progressive and total motility, and acrosin activity were lower (p < 0.05) in thawed semen than in fresh semen. During the TRT, total sperm motility and acrosin activity after 60 minutes were lower (p < 0.05) than those obtained after thawing. Similarly, total sperm motility and acrosin activity were lower (p < 0.05) after 120 minutes than at 60 minutes of the TRT. The analysis of motility and acrosin activity allowed the conclusion that both extenders have a similar capacity to preserve the integrity of sperm cells subject to freezing and thawing.

Role of Male effect on Reproductive Efficiency of Nulliparous Santa Inês and Morada Nova ewes raised in Different Regions

Background: The male effect is an attractive strategy to increase herd production by concentrating mating events and deliveries and further allowing the adoption of genetic improvement programs. It holds similar efficiency to those chemically based estrous synchronization methods, but has the advantage of being a natural method. The work was aimed to evaluate the influence of male effect on estrous induction and synchronization, pregnancy and prolificacy of nulliparous Santa Ines and Morada Nova ewes raised in Semiarid and Zona da Mata regions of Pernambuco state. Materials, Methods & Results: Santa Ines (n = 80) and Morada Nova (n = 80) females, with age from 11 to 12 months, after being evaluated and selected, were identified with plastic ear tags, weighted and maintained isolated from males, during 30 days before experiment onset, without any physical, visual, olfactive and auditive contact. Estrous events were observed twice a day (6:00 and 16:00 h) by trained personnel, during a breeding season of 60 days, and estrous were considered synchronized when detected, within first five days of breeding season. Rams of Santa Ines (n = 2) and Morada Nova (n = 2) breeds were selected based upon reproductive capacity by an andrology exam, and were marked on the externum bone region with a wax and ink (4:1) mixture, and were marked in female lots in order to identify females in estrous. After ten days of breeding season onset, rams were again marked with the same wax and ink mixture, but with a different ink color. Pregnancy diagnosis was performed on day 30 by ultrasonography and confirmed on day 60 after the last mating. The statistical analysis was performed using SAS, version 8. Differences of 5% (P 0.05). The total pregnancy was 42.50% on first, 64.70% on second service and total delivery was 86.20% singletons, 12.06% twins and 3.33% triples with prolificacy of 1.15 ± 0.31. On both Semiarid and Zona da Mata regions, the majority of estrous events occurred between the11th and 15th day of the breeding season for Santa Ines ewes and between 6th and 10th day for Morada Nova ewes. Discussion: The occurrence of estrous, for both breeds, in both regions, were detected throughout the breeding seasons, despite most estrous detections were within the initial fifteen days, which normally happens with cycling pluriparous females, in disagreement with findings in the literature that young females display lower reproductive performance on the first breeding season. However, the sexual inexperience of young females is not equivalent to lack of male receptivity, since then, could not be responsible for late estrous onset in a breeding season of young females. In agreement with this statement, and based on the data described here, it has been described that young ewes display estrous within the initial 18 days of breeding season onset. The estrous dispersion in biostimulation programs is normally due to female cyclicity, a physiological condition that lowers the sensibility to estradiol negative feedback response, but are still responsible to the presence of males.

Induction of Ovulation in Mangalarga Marchador Mares by hCG or GnRH

Background: Induction of ovulation is a key procedure for horse assisted reproduction technologies, such as for artificial insemination (AI) and embryo transfer. The application of hCG remains as the primary ovulation-inducing agent for horse assisted reproduction, but alternatives are in demand to avoid its adverse effects, such as loss of ovulation-inducing efficiency over multiple applications by hCC-antibody production. Despite reports on alternative ovulation-inducing agents, pair-wise comparisons of such agents under similar conditions have been limited. Under such scenario, the work was aimed to determine the efficiency of both hCG and Buserelin at inducing ovulation in Mangalarga Marchador mares raised in the Northeast of Brazil under an AI program. Materials, Methods & Results: Mares were initially selected based on their reproductive performance, the absence of clinical-reproductive alterations and adequate body condition score. Mares in diestrus were randomly distributed in three experimental conditions, received 5 mg of Dinoprost and were monitored daily for estrus detection. After estrus detection, ovaries were monitored by ultrasonography, in 12-h intervals, until the follicle reached 35 mm. At this time-point, ovulation was induced with 0.042 mg of Buserelin endovenously, with 3,000 IU hCG by an intramuscular shot, and control mares received 2 mL of saline solution, also by an intramuscular shot. Both hCG and Buserelin displayed similar efficiencies (P > 0.05) for induction of ovulation and that both agents were effective (P < 0.05) for such purpose, since greater percentages (P < 0.05) of induction on mares treated from those of the control. Moreover, the total number of ovulations in mares treated at the end of the experiment was not different (P > 0.05) from those found in the Control. All ovulations occurred within a 72-h period after treatment. It can be observed that in mares treated with hCG or Buserelin, ovulations occurred both in more mares (P < 0.05) and at earlier time-points than mares from the control. It is also possible to note that pregnancy was not different (P > 0.05) between hCG and Buserelin groups, and that pregnancy of mares treated with ovulation-inducing factors was similar to the control. Discussion: The majority of ovulations in mares occurred within initial 48-h after treatment for both hCG and GnRH, suggesting a similar potential for horse assisted reproduction. Both hCG and Buserelin are two commonly used agents for induction of ovulation in mares. As described here, the majority of ovulations occurred within initial 48-h after treatment, a fact which can be attributed to hCG and GnRH activity, since it can happen in intervals from 36 to 48-h after treatment. Pregnancy rates did not differ among groups. These results are under the working hypothesis that hCG and Buserelin would display similar efficiencies on pregnancy rates. Despite the understanding of hCG activity on induction of ovulation due to its high specificity toward LH receptors and results from a direct effect on diminishing estradiol concentration, increasing LH, and further inducing ovulation within 48-h after treatment. In contrast, Buserelin has a similar functional property but acts upon LH synthesis and its release. In conclusion, ovulation in mares can be induced with both hCG and Buserelin, and both ovulation-inducing agents do not affect pregnancy rates.