Reproduction in Domestic Animals最新文献

In this study, we evaluated sheep sperm quality after using Tris-citrate-fructose-based extender with and without egg yolk, a Tris-citrate without fructose and with egg yolk and the commercial extender Biladyl®, preserving diluted semen at 15 and 23°C for different times (4, 24, 48 and 72 h). The results showed that the diluents with fructose and egg yolk gave the best results of seminal quality. Moreover, the production of ROS was higher for the temperature of 23°C compared to the temperature of 15°C (control). In addition, VCL and the percentage of spermatozoa with intact acrosome decreased with temperatures of 23°C. Finally, a drastic decrease in sperm quality was observed after 24 hours of preservation for most of the parameters evaluated.

A protocol for conventional in vitro fertilization (IVF) in horses using fresh semen has been described, using a prolonged incubation in FERT-TALP medium (22 h) at 38.2°C in the presence of penicillamine, hypotaurine and epinephrine (PHE). Our work aimed to develop a protocol that maintains quality parameters in frozen-thawed equine spermatozoa incubated for 22 h in the presence of PHE using different media (FERT-TALP and INRA96) and incubation temperatures (30 and 38.2°C). Twelve frozen ejaculates from four stallions were thawed and then incubated in either FERT-TALP or INRA96 with PHE at 30 or 38.2°C for 22 h. Following incubation, total motility (TM), progressive motility (PM), viability and acrosome integrity were evaluated. The results showed that TM was significantly higher (p < .001) at 30°C in both media, while PM was higher for INRA96 at 30°C compared to 38°C (p < .05). Moreover, INRA96 at 30°C exhibited higher sperm viability and acrosome integrity (p < .001) compared to the other experimental groups. These preliminary results suggest that incubating thawed equine spermatozoa at 30°C with PHE in INRA96 successfully maintains motility, viability and acrosome integrity in equine spermatozoa, indicating its potential use for conventional equine IVF.

Goat production is affected by reproductive seasonality. In vitro embryo production (IVEP) could overcome this effect. This study aimed to evaluate the impact of the season of semen collection/freezing on IVEP of prepubertal goat oocytes and on sperm quality and functionality concerning capacitation. Semen from six fertile bucks was collected, pooled and cryopreserved in spring and autumn and used for IVEP of oocytes recovered during the breeding season. Oocytes were IVM in TCM-199 with hormones, EGF and cysteamine; fertilized and cultured in BO-IVF and BO-IVC media (IVF Bioscience, UK). Semen samples were assessed at 0 and 3 h after culture in capacitating (BO-IVF, CAP) and non-capacitating conditions for sperm plasma membrane and acrosome integrity, mitochondrial membrane potential (MMP), intracellular calcium and plasma membrane lipid disorder. Blastocyst production was higher with spring sperm compared to autumn (12.0% vs. 2.1%, respectively; p < .05). After CAP, acrosome reaction and intracellular calcium were higher (p < .05) in spring than autumn sperm. No differences were found in other sperm parameters. In conclusion, seasonal variations in the IVEP of prepubertal goats could be linked to differences in sperm ability to undergo in vitro capacitation.

Premature acrosomal exocytosis in cryopreserved semen is one of the reasons attributed to low fertility among livestock. In the present study, we attempted to enhance the cryopreserved semen quality by selective removal of acrosome-reacted spermatozoa using FITC-PNA conjugated iron magnetic nanoparticles (MNPs). Further, the effect of nano purification on other sperm functional attributes was also assessed. Iron MNPs were prepared using co-precipitation method and dextran-coated MNPs were conjugated with FITC-PNA (0.04 mg/mL). A preliminary experiment was conducted to standardise the dose of FITC-PNA conjugated iron MNPs (0.02, 0.05, 0.1, 0.15, 0.2, 0.4 and 0.6 mg). Among the different doses used, 0.6 mg FITC-PNA conjugated iron MNPs significantly (p < 0.05) removed higher acrosomal reacted spermatozoa from the semen, and therefore, this dose was used in further experiments. Cryopreserved semen from Holstein Friesian breeding bulls (n = 6) were thawed and washed using Sperm-TALP to remove residual extender. Washed spermatozoa (2 × 10⁶) were exposed to 0.6 mg of FITC-PNA conjugated iron MNPs for 10 min at 37°C. The nano purified semen was assessed for various vital sperm parameters viz., viability, intracellular calcium, apoptosis, mitochondrial ROS and mitochondrial membrane potential using flow cytometry. We found that nanopurification using FITC-PNA conjugated iron MNPs significantly (p < 0.05) improved the sperm quality. The proportion of viable non-apoptotic spermatozoa with low intracellular calcium levels was significantly (p < 0.05) enriched in nano purified semen. Nano purification did not affect sperm mitochondrial membrane potential and ROS production. In conclusion, these preliminary findings indicate that FITC-PNA coated iron MNPs effectively removed acrosome reacted spermatozoa and significantly improved sperm functional attributes in the purified fraction.

Spermatogonial stem cells (SSCs) maintain spermatogenesis through self-renewal and differentiation. The proliferation of SSCs in culture systems can provide a valuable source of germ cells. Several studies have investigated new reproductive technologies, including the production of transgenic animals and recombinant proteins secreted from milk in goats. While studies in other species exist, research on goat SSC culture remains limited. We investigated the impact of different testosterone concentrations on the survival and colonisation of cocultured goat SSCs with Sertoli cells. Cells were isolated from immature goats using two-step enzymatic digestion and enriched by differential exclusion method. DMEM/F12 culture medium containing 1% antibiotic and 5% FBS, supplemented with GDNF (20 ng/mL), EGF, bFGF and LIF (10 ng/mL), was used with different testosterone concentrations (0, 60, 120 and 240 μg/mL) and cultured for 10 days. SC subpopulations were confirmed using PGP9.5 immunocytochemistry, and the expression of germ cell markers (ID-4, UCHL-1, THY-1, β1-integrin, BCL6B, VASA, PLZF and OCT-4) was evaluated through RT-PCR. Alkaline phosphatase activity provided additional SSC presence. The survival rate of SSCs after isolation and the number and area of colonies on Days 4, 7 and 10 were measured using an inverted microscope. The presence of PGP 9.5 antigens and germ cell markers (ID-4, UCHL-1, THY-1, β1-integrin, BCL6B, VASA, PLZF and OCT-4) was confirmed by immunocytochemistry and RT-PCR, respectively. According to the results, the group with 60 μg/mL testosterone had the highest number and area of colonies. The number of colonies in the 60 μg/mL testosterone group was significantly higher than the control group (p < 0.05), but no significant difference was observed compared to other groups (p ≥ 0.05). This study suggests that a low testosterone concentration (60 μg/mL) is optimal for goat SSC colonisation and viability in coculture with Sertoli cells, potentially leading to advancements in goat reproductive technologies.

Cryopreservation of bovine oocytes and embryos is essential for long-term preservation and widespread distribution of genetic material, particularly in bovine in vitro embryo production, which has witnessed substantial growth in the past decade due to advancements in reproductive biotechnologies. Among current cryopreservation methods, vitrification has emerged as the preferred cryopreservation technique over slow freezing for preserving oocytes and in vitro-produced (IVP) embryos, as it effectively addresses membrane chilling injury and ice crystal formation. Nonetheless, challenges remain and a simple and robust vitrification protocol that guarantees the efficiency and viability after warming has not yet been developed. Furthermore, although slow cooling can easily be adapted for direct transfer, an easier and more practical vitrification protocol for IVP embryos is required to allow the transfer of IVP embryos on farms using in-straw dilution. In addition, the susceptibility of bovine oocytes and embryos to cryoinjuries highlights the need for novel strategies to improve their cryotolerance. This manuscript examines various methodological approaches for increasing the viability of bovine oocytes and IVP embryos during vitrification. Strategies such as modifying lipid content or mitigating oxidative damage have shown promise in improving cryotolerance. Additionally, mathematical modelling of oocyte and embryo membrane permeability has facilitated the rational design of cryopreservation protocols, optimizing the exposure time and concentration of cryoprotectants to reduce cytotoxicity.

Gonadotropin-releasing hormone (GnRH) -supplemented extenders have emerged as a welfare-orientated method to induce ovulation in the artificial insemination (AI) of rabbits. The main factor that limits the bioavailability of GnRH analogue on intravaginal administration is the proteolytic activity of enzymes present in rabbit seminal plasma. The use of GnRH analogues with higher biological potency would allow us to decrease their concentration in the seminal dose without compromising effectiveness. The current study was designed to assess the efficacy of various GnRH analogues concerning their ability to induce ovulation in rabbit AI. The base solution used for experimental extenders contained an aminopeptidase inhibitor. Four experimental groups were used, females from the Control group were induced to ovulate with an intramuscular administration of 1 μg of buserelin, while in the other three groups females received an intravaginal administration of 3.5 μg of buserelin (BUS), deslorelin (DES) or fertirelin (FER) within the seminal dose. Results showed that the ovulation frequency was similar in all groups studied. A concentration of 3.5 μg of the different GnRH analogues tested in this study showed similar potency in inducing ovulation in non-lactating females, yielding comparable results in terms of pregnancy rate at birth and prolificacy.

This study investigated the optimization of assisted reproductive techniques for wild felid conservation, focusing on in vitro procedures using the domestic cat as a model species. The research evaluated the impact of three different in vitro culture media on blastocyst formation. Oocytes and spermatozoa were collected and processed, followed by in vitro fertilization and culture. Results returned a similar blastocyst rate (ANOVA, p > .05), over 16% across all groups. While demonstrating the potential of these techniques, further investigations are warranted to evaluate embryo quality to refine optimal protocols and their applicability in felid conservation efforts.