Reproduction in Domestic Animals最新文献

Livestock sex control is one of the core bioengineering technologies for improving quality and efficiency in modern animal husbandry, holding profound practical significance for sex-limited livestock production systems. Precise identification of fetal sex at the early gestational stage constitutes a key prerequisite for achieving targeted sex regulation. The discovery of cell-free fetal DNA (cffDNA) in the peripheral blood of pregnant cows has paved an important technical avenue for establishing a non-invasive and high-precision diagnostic system for early fetal sex identification. In this study, plasma and serum samples collected from pregnant cows were used as experimental materials, and three protocols-phenol-chloroform extraction, heat-based extraction, and a commercial kit specifically designed for isolating cffDNA from plasma/serum-were employed for cffDNA purification and isolation. Y-chromosome-specific genes (either the TSPY or SRY gene) were selected as molecular markers, and optimised detection systems were established by integrating polymerase chain reaction (PCR), real-time quantitative PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP) techniques. A systematic comparison was conducted to evaluate the efficacy and accuracy of different cffDNA extraction methods combined with various amplification technologies for fetal sex identification in both early and late stages of gestation. The actual calving outcomes were used as the standard for validation. The results demonstrated that the quality of cffDNA templates extracted by the commercial kit method was significantly superior to that obtained by the heat-based and phenol-chloroform methods, with the corresponding sex identification accuracy reaching the highest level. Notably, the LAMP technique exhibited unique advantages in detecting fetal sex in extremely early gestational samples (at 1-2 months of pregnancy). Characterised by its simplicity of operation, rapid reaction kinetics, and elimination of the need for sophisticated instrumentation, LAMP is particularly well-suited for on-site large-scale rapid primary screening of fetal sex in livestock farms. It enables the efficient exclusion of male foetuses within a short timeframe, thereby substantially improving the efficiency of breeding selection. Based on the aforementioned findings, this study proposes a combined detection model of "LAMP-based primary screening plus PCR-based confirmation", which can effectively balance detection efficiency and identification accuracy. The research outcomes provide empirical data and methodological references for constructing a non-invasive, early-stage, and high-precision technical system for fetal sex identification in dairy cows. This holds great value for promoting the implementation of precise early reproductive management in dairy farms and enhancing the economic benefits of the livestock industry.

The influence of dead sperm on their healthy counterparts in bovine semen is not well established. This is particularly relevant to artificial insemination (AI), since semen handling and biotechnological procedures can increase the percentage of dead sperm. This study aimed to evaluate the impact of acrosome-disrupted (sonicated) spermatozoa on the quality of neighbouring untreated viable cells after cryopreservation. Semen samples from 12 healthy Holstein bulls were diluted (80 × 10⁶ sperm/mL) in pre-warmed OptiXcell extender at 38°C. A 6 mL portion of diluted semen underwent sonication, and both sonicated and untreated semen samples were mixed to produce treatment groups (TG) as: TG25%, TG50% and TG75% sonicated sperm. Control (CTRL) was not mixed with sonicated sperm. Progressive sperm motility was assessed during a thermo-resistance test after 30 (on-test) and 120 min (off-test) of incubation at 38°C. Results of delta and relative variation of progressive sperm motility showed a significant decline in the TG75% compared to the CTRL (p = 0.013 and 0.034, respectively). Flow cytometry revealed a gradual decline in percentage of viable acrosome-intact sperm with low membrane fluidity and low intracellular calcium (p < 0.001). A comparable decrease was observed for percentage of viable acrosome-intact sperm with high mitochondrial membrane potential (p < 0.001). Considering these findings, this study suggests that post-sonication leakage of acrosomal/cellular content could compromise the functionality of untreated spermatozoa, highlighting the necessity to conduct further mechanistic investigation to evaluate possible damaging pathways.

The objective of this study was to evaluate the effect of insemination timing with sex-sorted semen on fertility in dairy cows subjected to a timed artificial insemination (TAI) protocol. A total of 611 Holstein cows (46 ± 3 DIM) were enrolled and subjected to a presynchronized Ovsynch protocol (G7G; PGF₂α-2d-GnRH-7d-GnRH-7d-PGF₂α-56 h-GnRH), and randomly allocated to four treatment groups. The control group (CONV-14, n = 154) was inseminated with conventional semen 14 h after the final GnRH, while cows in the sex-sorted semen groups were inseminated at 14 (SS-14, n = 152), 18 (SS-18, n = 153), or 22 h (SS-22, n = 152) after the same treatment. The same bull was used for all inseminations. All cows were examined by ultrasonography to individually evaluate ovarian responses to the protocol and pregnancy status. No significant differences were observed among groups in body condition score, milk yield, cyclicity at the beginning of the protocol, response to the protocol, or follicle size at TAI. Pregnancies per artificial insemination were similar with 50.0% (77/154) in the CONV-14 group, 42.8% (65/152), 48.4% (74/153), and 43.4% (66/152) in the SS-14, SS-18, and SS-22 groups, respectively. No significant difference was observed in embryonic loss rates among groups: 5.2% in CONV-14, 9.2% in SS-14, 4.1% in SS-18, and 13.6% in SS-22, while SS-22 was numerically higher (~7%) than the average of the other SS groups. Overall, conception rates were higher in cows responding to the first GnRH than in non-responders (49.7% vs. 32.3%, p < 0.0005), with a significant difference observed only in the CONV-14 and SS-18 groups (p < 0.005). Estrous expression during TAI was associated with higher conception rates in the CONV-14 group (75.0% vs. 45.4%, p = 0.008), while no such difference was detected in the combined SS groups (51.8% vs. 43.3%). However, the conception rate in the SS-22 group (36.7%) was distinctly lower (p < 0.02) than in other SS groups (53.6% in SS-14, 68.0% in SS-18). In conclusion, contrary to the expectation that advancing insemination closer to ovulation with sex-sorted semen would be advantageous, fixed time insemination at 22 h within the TAI program showed poorer outcomes compared to 18 h, which achieved a relative conception rate of 97% compared with conventional semen. It was also concluded that TAI at 22 h should not be recommended in cows exhibiting estrus on the day of insemination.

The main objective of this study was to assess the effects of donor age on the development of in vitro-derived sheep embryos. Ovaries were obtained after slaughter from cycling Polish Longwool ewes aged 1.5-3 years (Group Y-'young'; n = 14) or 8-9 years (Group O-'old'; n = 16). Cumulus-oocyte complexes were collected and subjected to in vitro maturation (IVM), followed by in vitro fertilisation (IVF) with fresh capacitated ram semen. The resultant embryos were then cultured and monitored with a time-lapse (TL) imaging system for up to 8 days (Group Y, n = 64 and Group O, n = 48). The timing of key developmental stages relative to the moment when the oocytes and sperm were combined and including cleavage divisions as well as morula and blastocyst formation was recorded. Both the cleavage (68.75%) and blastocyst formation (26.6%) rates were significantly higher in younger ewes compared with their older counterparts (50.0% and 10.4%, respectively), with Group Y zygotes showing fewer (p < 0.05) incidences of abnormal cleavage and morphology (fragmentation, direct cleavage or asymmetrical cleavage) compared with Group O (10.9% vs. 33.3%, respectively). The first cleavage division occurred earlier (25:42 ± 3:43 vs. 29:20 ± 6:59 [hours: minutes post-insemination]; mean ± SD; p < 0.05) and the duration of the second cell cycle (time between the first and second mitotic division) was greater for Group Y compared with Group O (11:20 ± 9:51 vs. 4:14 ± 6:40; p < 0.05). No significant differences were observed between the times of the following mitotic divisions or formation of morulae and blastocysts. This study documents the specific differences in embryo morphokinetics between donor ewes varying in age and highlights the usefulness of TL imaging for assessing the influence of maternal ageing on embryogenesis in sheep as a model for different mammalian species including humans.

The present study investigated the relationship between seminal parameters and bull conception rate (BCR) in 72 Murrah bulls using Computer-Assisted Sperm Analysis (CASA). The BCR was calculated by obtaining data of artificial insemination spanned over two decades from buffalo farms of two organised herds of India. The association of seminal parameters and BCR was studied using multiple regression and principal component analysis. The average BCR was 38.95% ± 1.51%, ranging from 22.50% to 80.51%. Total motility (TM), progressive motility (PM), average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), and amplitude of lateral head displacement (ALH) showed significant positive correlations with BCR, whereas straightness (STR) and linearity (LIN) were negatively correlated. Multiple regression (R² = 0.29) identified TM as the most reliable predictor of BCR. Principal component analysis (PCA) extracted three components, PC1 (Sperm velocity and head movement, 54.36% variance), PC2 (Trajectory and beat frequency, 19.56%), and PC3 (Progressive motility and path accuracy, 12.98%), explaining 86.90% of the total variance. Regression using PC scores (R² = 0.24) indicated positive effects of PC1 and PC3, and a negative effect of PC2 on fertility. Overall, sperm velocity and progressive motility were primary fertility determinants, while excessive linearity hindered conception success. Therefore, association of seminal parameters with BCR can be explored for enhancing breeding efficiency of bulls. Future breeding programmes should prioritise sperm velocity and progressive motility traits while avoiding excessive linearity to improve bull fertility and conception success.

The objective of the study was to investigate the vanillic acid's (VA) protective effects, a phenolic compound, on the ram semen after freeze-thaw. Semen was obtained from Ramlıç rams and was diluted with control (0 μg/mL VA) and VA-supplemented Tris-based extenders at concentrations of 1, 10 and 50 μg/mL. The diluted semen was equilibrated for 2 h at +4°C, filled into 0.25 mL straws and frozen in liquid nitrogen vapour. It was then stored in a liquid nitrogen container at -196°C. For analysis, the samples were thawed at 37°C for 30 s in a water bath. There was no difference detected among total and progressive motility as well as velocity parameters (p > 0.05) except for rapid progressive motility (p < 0.05). The findings support the idea that VA has an outstanding effect on reducing DNA damage (p < 0.001). While there was no positive development with regard to total oxidant status (p > 0.05), VA enhanced the antioxidant defences of total antioxidant status (p < 0.05). VA administered at doses of 10 and 50 μg increased total antioxidant status (p < 0.01). Lipid peroxidation was not directly affected by VA application (p > 0.05); otherwise, 10 and 50 μg VA treatments showed a positive effect on viability (p < 0.001). Based on findings, it was concluded that although VA was put in the semen extender, it did not have an ameliorative potency on sperm motility and velocity properties except for specific sub-parameters such as rapid progressive motility. All applied doses reduced DNA damage, and 10 and 50 μg doses supported cellular viability.

In vitro maturation (IVM) is a crucial step in the in vitro embryo production (IVEP) of bovine oocytes, requiring coordinated nuclear and cytoplasmic changes for proper embryonic development. However, oocyte quality is often compromised by oxidative stress (OS), primarily caused by reactive oxygen species (ROS) generated under in vitro conditions. Natural antioxidants have been suggested as a solution for OS by neutralising ROS and restoring cellular homeostasis. Over the past few decades, growing research efforts have been directed toward incorporating antioxidants into culture media to enhance oocyte maturation and, consequently, improve the subsequent developmental potential of embryos. Recent studies highlight the roles of enzymatic antioxidants (e.g., superoxide dismutase, catalase) and non-enzymatic antioxidants (e.g., vitamins C and E) in enhancing embryonic development. Strategic combinations of antioxidants have shown promise in optimising embryo quality by mitigating oxidative stress and enhancing developmental outcomes. Here, we aim to recapitulate recent advances in knowledge regarding the effects of antioxidants on bovine oocyte quality and developmental potential during IVM, and subsequent embryo development, and to discuss their importance in the context of enhancing reproductive success.

The TGF-β/Smad signalling pathway plays a critical regulatory role in mammalian follicular development. Vascular Cell Adhesion Molecule 1 (VCAM1), as a member of the immunoglobulin (Ig) superfamily, is commonly expressed in various cells of the mammalian ovary and affects ovarian development. Previous studies have shown that TGF-β1 is a regulator that down-regulates the expression of VCAM1 in human granulosa cells, but its specific mechanism remains unclear. In this study, we revealed that in porcine follicular granulosa cells, TGF-β1 reduces VCAM1 expression by activating TGF-β receptor type I and through the Smad pathway. This down-regulation can be completely reversed by knockdown of Smad2, but not Smad3, suggesting that Smad2 may exert a non-redundant, specialised function in the regulation of VCAM1 by the TGF-β/Smad signalling pathway. These results enhance the understanding of the regulatory mechanisms of the TGF-β signalling pathway in reproduction and provide a theoretical basis for the regulation of reproductive traits.