Reproduction in Domestic Animals最新文献

Sex-sorted sperm and preimplantation sex determination are indispensable tools for improving reproductive efficiency and herd management in livestock production. The present study aimed to identify the most effective 'X' and 'Y' specific genes for sexing sorted bovine sperm and embryos. We selected five Y-specific (SRY, HSFY, TSPY, ZFY and OFD1Y) and four X-specific (PLCXD1, SHROOM 2, PLP and MAOA) genes and evaluated their specificity and sensitivity by PCR and qPCR analyses in X-sorted sperm, Y-sorted sperm, mixed sperm and female blood. HSFY and TSPY showed more pronounced and specific amplification in Y-sorted sperm DNA, making them robust Y-specific markers, while MAOA and SHROOM 2 were more specific to X-sorted sperm DNA and identified as effective X-specific markers. These results were further validated in buffalo IVF embryos, confirming their effectiveness in embryo sexing. The findings of this study can be applied in duplex or multiplex PCR assays, which minimise the risk of amplification failure and false negatives, providing a rapid and reliable toolkit for bovine sperm and embryo sexing. Implementing such a molecular approach can significantly benefit livestock management by improving breeding outcomes, reducing the costs and inefficiencies associated with undesired sex ratios.

In the mouse, spermatozoa are highly resistant to DNA damage, even when frozen without cryoprotectants, and can produce offspring when subsequently used for ICSI (intracytoplasmic sperm injection). It is not known whether the same applies to other mammals as well. For example, in the horse, even conventional sperm freezing is still very problematic and frequently leads to sperm immobility. It has, however, never been tested whether sperm immobility also mirrors sperm head DNA damage, and if so, to what extent. In our study, we evaluated the damage to DNA in horse frozen and thawed motile and immotile spermatozoa after their injection into ovulated mouse oocytes. In both groups, injected horse spermatozoa activated the mouse oocytes. This was followed by the extrusion of the second polar body (2 PB) and the formation of maternal pronuclei (Mo-fPN-mouse female pronucleus); in parallel, the horse sperm heads rapidly decondensed in the murine cytoplasm and formed paternal pronuclei (Ho-mPN-horse male pronucleus), which were larger than the female ones. With the exception of one stallion tested, DNA damage has been detected in almost all Ho-mPNs originating from immotile spermatozoa. DNA in motile (even sporadically) spermatozoa was mostly undamaged. Moreover, when the xenogeneic zygotes cleave to the two-cell stage, the incidence of micronuclei in blastomeres mirrors the extent of DNA damage in paternal pronuclei. In conclusion, and contrary to the mouse, where sperm DNA is very resistant to damage, we do not recommend the use of immotile horse spermatozoa for ICSI. On the other hand, even the sporadically motile mouse spermatozoa have no damaged DNA and can thus be used for intragenic ICSI.

The objective of this study was to evaluate the effects of exogenous GnRH administration at the beginning of estrus synchronisation in mares during the spring transitional period. Estrus was synchronised using a progesterone releasing intravaginal device (PRID). The PRID was left in the vagina for 10 days, followed by an injection of 0.4 mg of cloprostenol at PRID removal. The GnRH group (n = 32) was subjected to intramuscular administration of 100 μg of the GnRH agonist triptorelin at PRID insertion, while the control group (n = 32) received 1 mL of sterile physiological saline solution. Ovulation was induced by an intramuscular injection of 3000 IU of human chorionic gonadotropin until the dominant follicle reached a diameter of 35 mm. The mares were examined and insemination was performed. Subsequently, insemination was carried out every 12 h until ovulation. Transrectal palpation and ultrasound were carried out 15 days after ovulation to confirm the presence or absence of an embryonic vesicle. The days of ovulation induction and insemination in the control group were more dispersed than in the GnRH group. Compared with the control group, the time of ovulation induction and insemination in the GnRH group were accelerated and concentrated. In summary, GnRH given at the beginning of the estrus synchronisation program significantly increased synchronisation of ovulation in mares; however, it did not increase pregnancy rates.

In vitro reproductive biotechnologies show promise for fertility preservation but still face challenges, including oxidative stress from high oxygen tension, which impairs cell viability and development. Antioxidants have been widely explored to attenuate oxidative stress during culture. Among them, anethole, a plant-derived phenylpropanoid, stands out for its promising properties. This review explores the mechanisms and applications of anethole in reproductive physiology and its potential to enhance in vitro reproductive systems. Findings indicate that anethole modulates key pathways and may improve outcomes in in vitro follicle culture, oocyte in vitro maturation and in vitro embryo culture. These insights support future research and the strategic inclusion of anethole in reproductive biotechnology protocols.

Follicle-stimulating hormone (FSH) plays an important role in regulating reproductive events, particularly follicular development and oocyte competence acquisition. Some studies using FSH protocols in zebu cattle were performed, but data regarding its application in the Red Sindhi breed are scarce and therefore warranted. In this context, this study aimed to assess the FSH administration regimen-multiple doses (T1-m) and single dose (T2-s) and coasting period (54 h vs. 102 h) in oocyte developmental competence in Sindhi females. A total of 80 mg of FSH was administered either as a single application (40 mg IM + 40 mg, SC) or compared to multiple applications (30 mg + 30 mg + 20 mg, IM). Animals that did not receive FSH treatment serve as controls (CT). The present data showed that both T1 and T2 applications resulted in a greater number of medium-sized follicles (7.80 vs. 8.57, p < 0.05), oocyte recovery (9.76 vs. 9.81), when compared to control (5.20; 6.30, respectively). Animals from T2 also had a greater number of aspirated follicles (12.52 vs. 8.70, p < 0.05), viable oocytes (7.33 vs. 4.45, p < 0.05) and blastocyst rates (43.22% vs. 29.11%, p < 0.05) than control animals. Our results showed that a reduced dose of FSH both single and multiple applications enhance oocyte developmental competence. Moreover, a single application of FSH combined with a longer coasting period offers practical advantages, making this approach more attractive for Sindhi breeding programmes.

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.