Animal Reproduction最新文献

The aim of this study was to investigate the effects of modulating reactive oxygen species (ROS) in vitrified bovine in vitro produced (IVP) embryos. In experiment I we compared ROS production in fresh and vitrified-warmed blastocysts. In experiment II we evaluated the effects of antioxidant supplementation (100 μM of 2-mercaptoethanol; BME; 0 h to 2 h during warming) on ROS levels in vitrified-warmed blastocysts, and in experiment III we compared the development of fresh and vitrified-warmed blastocysts in the presence (BME) or absence (Control) of antioxidant (100 μM BME; 0 h to 48 h during warming). Higher ROS production (Fresh: 68.48 ± 7.92 vs Vitrified: 123.53 ± 13.15; P<0.05) and lower cell number was observed in vitrified compared to fresh embryos (Fresh: 123.01 ± 5.67 vs Vitrified: 103.04 ± 4.25; P<0.05). Antioxidant supplementation reduced ROS levels (Vitrified: 38.24 ± 1.27 vs. Vitrified/BME: 33.54 ± 1.08; P<0.05) and increased cell number in treated embryos (Vitrified: 100.65 ± 3.98 vs. Vitrified/BME: 112.95 ± 3.72; P<0.05). No differences were observed in the re-expansion rates of vitrified embryos cultured in the absence and presence of BME at 0, 2, and 4 h after warming (P>0.05). The embryo hatching rate did not differ (P>0.05) among embryos from the fresh, vitrified and vitrified/BME groups. However, the total cell numbers were higher (P<0.05) in vitrified embryos supplemented with BME (143.02 ± 6.97) than in vitrified embryos without BME (113.25 ± 5.09) but similar (P>0.05) to that observed in fresh embryos cultured with (150.54 ± 8.99) and without BME (142.71 ± 13.60). It was concluded that the vitrification and warming processes increased ROS levels in blastocysts and its attenuation with BME antioxidant improved embryonic quality.

More than 90% of spermatozoa of boars in pork producing countries is stored in liquid at 17 °C; however, the quality of these spermatozoa is affected by bacterial breeding and oxidative damage. This study analyzed sperm quality and sperm capacitation after storage to study the effects of the effects of ZnO nanoparticles (ZnO NPs) supplementation on seminal plasma (SP)-free sperm preservation. We investigated the effects of adding 20, 50, 100 and 200 μg/mL of ZnO NPs to a seminal free boar sperm diluent over a 7-day period at 17 °C to assess the changes in non-capacitated/capacitated sperm quality parameters, antioxidant capacity, ATP content and extent of protein tyrosine phosphorylation. The addition of different doses of ZnO NPs to stored sperm did not induce significant effects on the sperm motility and ATP content when compared to the sperm without ZnO NPs treatment. However, the addition of 50, 100, 200 μg/mL ZnO NPs to stored sperm improved total antioxidant capacity (T-AOC) and CuZn-superoxide dismutase (CuZn-SOD) (p < 0.05). ZnO NPs also reduced the malondialdehyde (MDA) content of the preserved sperm (p < 0.05). Moreover, our results indicate that the supplementation of 50 μg/mL ZnO NPs to preserved sperm improved the sperm membrane integrity (p < 0.05). ZnO NPs exerted protective effects on protein tyrosine phosphorylation, especially with regards to membrane proteins. Following incubation and capacitation, sperm exhibited good levels of protein tyrosine phosphorylation and ATP levels with high T-AOC and CuZn-SOD activity and low MDA content. ZnO NPs exerted protective capacity to a preservation extender used for SP-free boar sperm during storage at 17 °C. The optimal concentration of ZnO NPs for preservation extender was 50 μg/mL.

This study aimed to develop a non-surgical method to neutralize reproduction in female dogs. Female Beagle puppies, aged 6 days, were treated with pellets designed to release estradiol benzoate (EB; 1.0 mg) and progesterone (P4; 5.0 mg) over approximately 3 weeks. Their estrous cycles were monitored from 6 to 34 months of age by examining their vulvas daily and measuring their serum P4 levels once a month. Vulvar edema and discharge, followed by a serum P4 level above 5 ng/ml, indicated the potential estrus. Each time a dog showed these signs, breeding was attempted by housing with a proven male Beagle. All the treated dogs displayed cyclic progesterone surges with 5 to 6-month-long anestrous intervals. Surprisingly, none exhibited sexual behaviors, and no mating occurred (i.e., no intromission and copulatory tie), resulting in no pups being born. This phenomenon was further explored in laboratory animals. Neonatal female rats were treated with microspheres containing smaller doses of the same steroids (0.3 mg EB + 3.0 mg P4) at 1 or 2 days old. At 3 months old, the rats were ovariectomized, chemically stimulated to exhibit estrus behaviors using a standard protocol and tested for receptivity to proven male rats. Untreated control rats showed normal receptivity (i.e., lordosis) and allowed males to mate. However, rats treated with EB+P4 did not exhibit lordosis or allow mating. These results indicate that the combined use of estrogen and progesterone could be an effective non-surgical method for inhibiting mating behavior and, consequently, neutralizing female dog reproduction.

Ensuring boar sperm quality before insemination is crucial for maximizing field fertility and efficient pig production. The computer-assisted sperm analysis (CASA) and fluorescence probes combined with flow cytometry (FC) are commonly used techniques for evaluating sperm kinematics and functions, closely related to animal fertility. However, their high cost and complex operations make it challenging to apply them in laboratories or pig breeding farms with limited resources. Here, our aim was to develop a new protocol using a resazurin redox dye to assess boar sperm quality for practical application. We first created simulated semen samples with different levels of sperm quality (0, 20, 40, 60, 80, 100%) at concentrations of 300 and 150 × 10⁶ cells/mL. Subsequently, the simulated semen was used to establish an optimal standard protocol based on the results of the resazurin colorimetric assay. Finally, the condition that showed the strongest correlation between resazurin redox rate and sperm parameters was selected to perform a linear regression test. Two optimal working conditions were identified, involved incubating 10 µL of resazurin reagent with 100 µL of semen for either 20 or 40 min, depending on the sperm concentration of either 300 or 150 × 10⁶ cells/mL. We subsequently conducted a linear regression analysis using data that included the resazurin reaction rate and measurements of sperm parameters. Finally, we obtained two sets of five equations, allowing directly convert the absorbance of the resazurin assay into values for sperm quality parameters. These parameters include total motility, progressive motility, viability, acrosome integrity, and mitochondrial activity. This new protocol is valuable for sperm evaluation because it is cost-effective, time-efficient, and labor-saving.

This study aimed to compare the effects of nandrolone decanoate on the morphology and physiology of ovarian tissues in two experimental models, Zebrafish and rats, after in vitro cultivation. A total of 136 animals were used (Wistar rats, n=36, and Zebrafish, n=100). In both experiments, the animals were divided into two groups (Control and Deca) and were exposed to nandrolone decanoate for seven weeks. At the end of the administrations, the animals were euthanized, and the tissues were collected for morphological and biochemical analyses. Data were expressed as mean ± SEM. Tukey and Shapiro-Wilk tests were used. ANOVA and chi-square tests were applied for group comparisons. Differences were considered significant when P<0.05. The results showed an increase in body weight in Wistar rats, while Zebrafish body weight was decreased. In both experiments, the number of atretic follicles increased throughout the in vitro culture, from day 0 to day 7, in the Control group (CTRLr and CTRLz), while in the DECA group (DECAr and DECAz), atretic follicles were reduced from D0 to D7. The antioxidant environment, represented by increased the thiol content, which was significantly higher on day zero in CTRLz compared to CTRLr. SOD activity increased in Zebrafish (group DECAz), while CAT activity decreased in both models (group DECAr and DECAz). In conclusion, the study demonstrated similarity in ovarian physiology between the models exposed or not exposed to nandrolone decanoate, suggesting that, when convenient, researchers could consider changing the experimental model.

Early puberty is associated with improved long-term reproductive performance. Predicting who will achieve early puberty is limited to intensive, invasive serial blood collections for measurement of reproductive hormones. The vaginal genome during pubertal development has potential as biomarkers of early estrus in the pre-pubertal period. Pre-pubertal gilts (n =13) were followed from d74 ± 3 of age until first estrus or d214 ± 1 of age. Blood, vaginal epithelia, and anogenital distance were collected at five timepoints during reproductive development (d74, d104, d130, d160 and first estrus or end of trial). Total RNA was isolated from vaginal epithelia and relative gene expression of two toll-like receptors (TLR-4 and TLR-5), tacykinin precursor-3 (TAC-3), insulin-like growth factor-1 (IGF-1), and estrogen receptor (ERα)-alpha was quantified by real time RT-PCR, relative to expression of RPLP0. Four gilts exhibited estrus early (< d184), 3 were average (d194 to 195), 3 were late (d203 to 213), and 3 were deemed anestrus. Comparison of expression of each gene relative to d70 was performed using the PCR package in RStudio (version 1.2.5025) and Fisher's exact t-test for TLR-4, TLR-5 and TAC-3, and ANOVA for ER-alpha and IGF-1. Correlation analysis examined the relationship between anogenital distance and age at first estrus. A single blood draw for serum progesterone was obtained 8 days after recorded first estrus or end of trial; the presence of serum progesterone supports the visual identification of standing estrus. Expression of IGF-1 and TAC-3 were up-regulated 9- and 7-fold, respectively at d160 (P < 0.05). Expression of ERα tended to be upregulated 3-fold at d104 (P = 0.08) and expression of TLR-4 and TLR-5 was not detected until first estrus. Anogenital distance was positively correlated to the first estrus. These transcripts associated with reproduction warrant further investigation into use as biomarkers to detect early estrus.

This study was conducted to evaluate manila duck's (Cairina moschata) frozen semen quality after cryopreservation in lactated ringer's egg yolk-astaxanthin (LREY-A) with 5 different concentrations of dimethyl sulfoxide (DMSO). Methodology: Semen was collected from 3 manila ducks (Cairina moschata) using the cloaca massage technique twice a week. Fresh semen was evaluated macro and microscopically then polled and divided into 5 tubes of treatments. Each tube was diluted in DMSO4, DMSO6, DMSO8, DMSO10, and DMSO12. The semen of each treatment was loaded into a 0.25 mL straw and equilibrated at 5 °C for 2 h. Freeze above nitrogen vapor and stored a container of liquid nitrogen at -196 °C, then semen thawed in a water bath at 37 °C for 30 sec. Data were analyzed using One-Way ANOVA Analysis. Results of this showed that post-equilibration sperm motility and sperm viability have differed significantly (P<0.05) for each treatment, with the highest % sperm motility DMSO8 and DMSO6, this is also shown in post-thawing sperm motility and viability which have differed significantly (P<0.05) and the highest % sperm viability were DMSO8 and DMSO6. In conclusion, Frozen semen extender formulation of DMSO8 and DMSO6 which are used in manila duck semen cryopreservation was the best to other treatments to maintain % sperm motility and % sperm viability in post-equilibration and post-thawing. The highest sperm motility recovery rate was in DMSO8. The lowest sperm live and dead abnormality was in DMSO8^. It is concluded that the combination of DMSO8 was the best in maintaining the quality of manila duck frozen semen.

Polyethylenimine (PEI) has been explored as an efficient non-viral system for delivering genes to cells; however, there were no protocols for its use in porcine fetal fibroblasts (PFF). Therefore, we compared different concentrations of FITC-PEI (0.625, 1.25, 2.5, 5, 10, 20, 40, or 80 µg/mL) and incubation times (30 min, 1 h, or 2 h). It was observed that the incubation time did not affect the internalization of the PEI-FITC and that 30 min was sufficient to capture the complex. The concentrations higher than 10 µg/mL could reach many marked PFF (>90%). Then, two PEI concentrations were tested, 10 or 40 µg/mL, combined with an N/P of 2 with the pmhyGENIE-5 for 30 min. The percentage of PFF-GFP positive was similar between the PEI concentrations in the evaluation time points (24 h, 48 h, and 72 h). However, 40 µg/mL caused higher membrane damage rates. Thus, it can be concluded that concentrations between 10 - 80 µg/ml of PEI promote high incorporation rates, even in periods as short as 30 minutes. Furthermore, it can be stated that the transfection condition used in Polyplexes 1 (10 µg/mL of PEI and 37.5 µg/mL of pmhyGENIE-5 for 30 min) efficiently produces genetically edited porcine fetal fibroblasts with low cell damage.

Intracytoplasmic Sperm Injection (ICSI) has increased usage in cases of stallion fertility issues, particularly for older stallions, those with reduced sperm numbers or quality, or stallions that have passed away, and only a limited amount of frozen semen is available. By manipulating the frozen semen through thawing, diluting, and refreezing or by cutting the straw under liquid nitrogen, the supply of semen for ICSI can be extended. While ICSI requires a minimal number of spermatozoa per procedure, it is important to consider sperm quality as a crucial factor affecting fertilization and embryo development. Although it is possible to produce healthy embryos and offspring from low quality sperm samples, it is preferable to process and select morphologically and functionally superior sperm to maximize the chances of successful fertilization and embryo development. Several techniques are available for selecting the spermatozoa for ICSI, such as swim-up, washing, density gradient centrifugation, microfluidic sorting, and some combinations. In this review, we will focus on semen type, handling, recent breakthroughs, stallion effects on ICSI efficiency and the prospects of this technology within the equine industry.

The relationships between members of the groups include behaviors related to affiliation, dispute for dominant positions, parental care, and facing disputes for food and territory. All these activities are under hormone modulation and those of a steroidal nature are heavily involved. Despite this, only few data are available on steroid hormones in free-ranging marmosets of the Callithrix genus, which limits the understanding of the physiological functioning and modulation of the socio-sexual behavior by steroid hormones of this taxon. In this study, we characterized fecal concentrations of progesterone, estrogens, and glucocorticoids of six breeding and non-breeding females from two groups of free-ranging Callithrix penicillata (É. Geoffroy, 1812). The concentration of progesterone was significantly higher in females which gave birth, compared to non-breeding females. The levels of fecal estrogens and glucocorticoids did not differ between breeding and non-breeding females. The data are in agreement with the few studies on steroid values of wild and captive marmosets. This study shows the concentrations of progesterone and glucocorticoids in free-ranging C. penicillata for the first time, and it is the only study reporting the concentration of fecal estrogens in wild marmosets. Overall, the high levels of progesterone associated with pregnancy in free-ranging C. penicillata as well as levels of estrogens and glucocorticoids close to those reported for other species, suggest a conserved pattern of hormonal secretion between Callithrix species that have been studied in captivity.