Reproduction in Domestic Animals最新文献

Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) is part of the tissue inhibitors of the metalloproteinases (TIMPs) family. Its primary function is to regulate the activity of matrix metalloproteinases (MMPs) across various tissues, including those of the reproductive system. This study aimed to quantify the natural levels of TIMP-2 in seminal plasma (SP) and sperm membrane (SM) of bulls, explore potential associations between TIMP-2 levels and semen quality parameters, and examine the relationship between TIMP-2 levels and sperm cryoresistance in bulls. Thirty semen samples from Frieswal breeding bulls were categorized into two groups based on their initial progressive motility (IPM): Good (IPM ≥ 70%; n = 21) and Poor (IPM ≤ 40%; n = 9). The samples were evaluated for their quality parameters at the fresh stage, and TIMP-2 levels were measured in SP and SM using a bovine-specific ELISA kit. Following cryopreservation of Good samples (n = 21), post-thaw motility (PTM) was used to further classify samples into Freezeable (PTM ≥ 50%; n = 14) and Non-Freezable (PTM < 50%; n = 7) groups. In frozen-thawed samples, sperm attributes, kinetics, and functional parameters were assessed, and the results were correlated with retrospective TIMP-2 levels of SP/SM. Our study revealed that the quantified levels of TIMP-2 ranged from 100.27 to 535.95 ng/L in SP and from 0 to 115.78 ng/10 million spermatozoa in SM. TIMP-2 levels in both SP and SM were significantly higher in Good ejaculates compared to Poor ejaculates (p < 0.01). Furthermore, total TIMP-2 levels in the SP/SM of semen samples from bulls showed a positive correlation with fresh semen attributes. However, SP/SM TIMP-2 levels in the Freezeable group did not show any significant differences compared to the Non-Freezable group in post-thaw semen quality attributes, kinetic parameters, and functional tests, except for a significant positive correlation (r = 0.530, p < 0.05) between sperm DNA integrity and SP-TIMP-2 levels. In conclusion, the findings suggested that TIMP-2 can be a positive regulator of semen quality at the neat stage. However, when it comes to the resilience of sperm to cryopreservation, the levels of TIMP-2 do not seem to exert any significant influence in breeding bulls.

This study evaluated the effects of pre-synchronisation with injectable progesterone (P4) on the ovarian follicular dynamics of Bos taurus indicus cows in anoestrous treated with a timed artificial insemination (TAI) protocol. Multiparous Nelore females (n = 47) at 30-60 days postpartum were used in this study. 10 days before (D-10) the TAI protocol, antral follicle count (AFC; follicles ≥ 3 mm), ovarian condition and body condition score (BCS; 1-5) were assessed and were randomly allocated into two groups: Pre-sync (n = 25), which underwent pre-synchronisation with 150 mg of injectable P4 intramuscularly (i.m.), and control (n = 22), which received the same volume of NaCL 0.9%. On D0, the ovarian assessment was repeated, and TAI protocol was initiated in all animals, with the insertion of an intravaginal P4 device and administration of 10.5 μg of buserelin acetate (gonadotropin-releasing hormone-GnRH). On D7, the P4 device was removed, and 300 IU of equine chorionic gonadotropin, 150 μg of D-cloprostenol and 1 mg of estradiol cypionate were administered i.m. On the same day (D7), the presence of the corpus luteum (CL) was assessed, the dominant follicle was measured, and the tail was painted to evaluate estrous expression. On D9, the largest follicle was remeasured, and TAI was performed. Animals that were not detected in oestrous at the time of AI were administered 10.5 μg of GnRH i.m. Numerical data were analysed using the Mann-Whitney U test. Binary data were analysed using the Fisher's exact test (5%). BCS, both at the beginning of pre-synchronisation (p = 0.45) and TAI protocol initiation (p = 0.20), and AFC (p = 0.36) did not differ between control and Pre-sync groups. The diameter of the largest follicle was similar between the control and Pre-sync groups on D-10 (p = 0.32), D0 (p = 0.33), D7 (p = 0.29) and D9 (p = 0.22). On D7 of the protocol, the Pre-sync group had a higher percentage of CL visible on transrectal ultrasonography (84.0%; p = 0.02) than the control group (54.5%); however, the expression during oestrous did not differ between groups (p = 0.59). The pregnancy rate was similar (p = 0.64) between groups and was not influenced by the CL rate on D7 (p = 0.48), oestrous expression (p = 0.20) or their interaction (p > 0.1). Pre-synchronisation effectively increased the proportion of cows with CL on D7 without altering the diameter of the largest follicle, oestrous expression or pregnancy rate in anoestrous cows treated with a GnRH/P4-based TAI protocol.

Supplementing freeze diluents with certain antioxidants can maintain the quality of chilled sperm. The present study was an attempt to investigate the effect of Beltsville extender supplementation with the mitochondrial-targeted antioxidant 'Mitoquinol' on the quality parameters and fertility potential of rooster sperm during the cooling process. Semen samples were diluted in Beltsville extender, divided into five groups, and supplemented with 0, 1, 10, 100 and 1000 nM Mitoquinol. Samples were stored at 5°C for up to 50 h and then assayed for sperm motility, viability, mitochondrial function, membrane integrity and malondialdehyde concentration after 0, 25 and 50 h of cooling. To assess reproductive performance, artificial insemination was performed using sperm cooled for 25 h. The results showed no differences between groups at the beginning time. Extender supplementation with 10 and 100 nM Mitoquinol resulted in an improvement in total motility, progressive motility, membrane integrity, mitochondrial function and viability (p ≤ 0.05), as well as a lower malondialdehyde concentration (p ≤ 0.05) in comparison to the other groups during 25 and 50 h storage. Fertility rates were higher when roosters were inseminated with semen samples supplemented with 10 and 100 nM Mitoquinol, compared to the control group. Therefore, supplementing Beltsville extender with Mitoquinol (10 and 100 nM) effective in improving the quality and fertility potential of cooled rooster sperm.

These studies aimed to determine if shearing ewes at the second or last third of gestation modify the uterine and placentome blood flow, placentome size, and maternal progesterone concentration. Pregnant ewes were assigned to four groups of 12 ewes each according to the gestation period: mid-pregnancy sheared (on day 90 of pregnancy) or unshorn group; and late-pregnancy sheared group (on day 121 of pregnancy) or unshorn group. In both experimental periods, using spectral Doppler ultrasonography, placentomes and uterine artery blood flow and placentome size were evaluated 14 days before and 6 days after shearing. An additional measurement was performed 26 days after shearing in mid-pregnant ewes. Serum progesterone concentration was measured before shearing 4, 24, 72 h, and 22 days after shearing. The uterine artery's end-diastolic velocity (EDV) tended to be greater in the sheared than in the non-sheared ewes (p = 0.1). Peak systolic velocity (PSV) and EDV of placentome increased (p = 0.05 and p = 0.008, respectively) on day 26, accompanied by an increase in placentome area (p = 0.035) in mid-pregnant ewes. In late-pregnant ewes, uterine artery and placentome blood flow and size did not vary. Progesterone concentration varied with time but was not affected by shearing. In conclusion, shearing triggered an increase in placentome size and some changes in blood flow only when ewes were sheared during the second third of their pregnancy. Shearing ewes either the second or last third of gestation did not affect uterine artery blood flow and progesterone secretion.

Elevated non-esterified fatty acids (NEFAs), particularly stearic acid (SA), have a deleterious effect on oocyte maturation, leading to developmental damage and reproductive issues. High SA levels disrupt metabolic processes, inducing lipotoxicity that impairs oocyte quality and contributes to reproductive failures through early embryonic losses. This research investigates the lipotoxic effects of SA and assesses the protective potential of 6-Gingerol (6-G) and Astaxanthin (AX) on porcine oocytes during in vitro maturation (IVM). Herein, 6100 cumulus-oocyte complexes (COCs) were exposed to various concentrations of SA (25-250 μM) to elucidate the concentration-dependent effect on oocyte viability, polar body extrusion (PBE) and cumulus cell expansion index (CCEI). However, the efficacy of 6-G (5-15 μM) and AX (2.5 μM) in combination with SA at 150 μM (SA6) concentration was evaluated to mitigate these adverse effects. The results indicated that SA6 substantially reduced oocyte viability, PBE and CCEI, demonstrating its toxic impact on oocyte developmental competence (p < 0.0001). Moreover, treatment with antioxidants such as SA6 + 6-G (10 μM) and SA6 + AX showed a considerable increase in viability and PBE compared to SA6 alone (p < 0.05). These findings demonstrate the importance of lipid metabolism in oocyte health, where dysregulation impairs reproductive capacity. Both 6-G and AX protected against lipotoxicity induced by SA6 while enhancing lipid homeostasis and the anti-oxidative defences necessary for maintaining cellular integrity. This study finds substantial evidence that optimising the microenvironment with specific antioxidants can improve oocyte quality and provide invaluable knowledge in reproductive technologies and fertility treatments.

In mammals, sex determination is governed by the SRY gene on the Y chromosome, redirecting gonadal development from forming ovaries to testes. Mutations or alterations in the SRY gene can significantly affect phenotypic changes and lineage-specific markers. This study aims to elucidate the role of the SRY gene in buffalo embryos using CRISPR-Cas9 technology. We designed a crRNA targeting the HMG domain of the SRY gene using the CRISPOR algorithm. Nucleofection of sgRNA-Cas9 RNPs into buffalo fibroblasts confirmed efficient cleavage at the targeted site. Using this validated guide, we investigated the role of the SRY gene in sexual determination by electroporating CRISPR-Cas9-RNPs into single-stage zygotes of buffalo. Genetic changes in the SRY gene were confirmed through sequencing, revealing mosaic blastocysts with multiple alleles and non-mosaic mutants. Mutations in SRY gene increased the expression of female lineage-specific gene Wnt4 whereas decreased the expression of male specific gene SOX9 in blastocysts, suggesting reprogramming towards female sex determination pathways. Our findings provide insights into buffalo sex differentiation mechanisms and potential applications in reproductive strategies for breeding programmes.

Bovine leptospirosis is a worldwide disease that causes reproductive issues, including early embryonic death, stillbirths and infertility, which result in significant economic losses. Although bovine leptospirosis is well-documented in cows, the role of bulls in harbouring and potentially transmitting genital leptospirosis has been largely neglected. We examined genital and blood samples from 16 slaughtered bulls using microscopic agglutination testing (MAT), polymerase chain reaction (PCR) and sequencing of amplicons. Our results showed that 81.2% of bulls were seroreactive, and 75% were genitally infected. The amplicons displayed an identity greater than 99% with Leptospira interrogans strains from the Sejroe serogroup, specifically serovar Hardjoprajitno. This study demonstrates that bulls can harbour in their genital tract the same leptospires associated with reproductive syndromes in cows from the same geographic region, highlighting the importance of bulls in maintaining and transmitting Sejroe serogroup strains associated with reproductive disease.

Semen cryopreservation can achieve long-term preservation of sperm. Ice crystal damage, as well as oxidative stress, result in mitochondrial dysfunction and a reduction in sperm motility after thawing. However, limited information exists regarding the impact of reactive oxygen species (ROS) and mitochondria on the cryopreservation of ram sperm. The primary objective of this study was to investigate the relationship between ROS and mitochondria concerning sperm quality during the cryopreservation of ram sperm. This investigation assessed sperm motility, kinematic characteristics, membrane integrity, acrosome integrity, mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) levels, expression of mitochondrial respiratory genes (NDUFV2, SDHA, CYC1, and COXIV), ROS levels, malondialdehyde (MDA) content, phosphatidylserine externalisation rate, sperm ultrastructure, mtDNA copy number, expression of apoptosis-related genes (Bax, Caspase-3, and Caspase-8), Cytochrome C, and Caspase-3 content. The results showed the cryopreservation significantly (p < 0.05) decreased motility, kinetic parameters, membrane integrity, acrosome integrity, MMP, ATP, mRNA expression levels of mitochondrial respiratory-related genes, and significantly (p < 0.05) increased ROS levels, MDA content, phosphatidylserine externalisation rate, damage of sperm ultrastructure, mtDNA copy number, mRNA expression levels of apoptosis-related genes, Cytochrome C and Caspase-3 content compared to the fresh semen group. In conclusion, the cryopreservation causes damage to mitochondria, leading to increased ROS and subsequent oxidative stress. This process also initiates mitochondrial dysfunction and interferes with the electron transport chain, ultimately resulting in decreased MMP and ATP production. Furthermore, the liberation of Cytochrome C prompted the increase in Caspase-3 expression and subsequent sperm apoptosis occurred, ultimately leading to a deterioration in sperm quality after thawing.

The aim of this study was to determine the effect of silver nanoparticles (AgNPs) on donkey sperm parameters and ultrastructure. AgNPs were synthesized, purified and resuspended in the extender. Nine frozen-thawed donkey sperm samples were exposed to different concentrations of AgNPs (0, 1.25, 2.5, 5, 12.5, 25 and 50 μg/mL). Sperm parameters: total (TMOT, %) and progressive (PMOT, %) sperm motility, plasma (LIVE, %) and acrosomal membrane integrity (AIS, %), and sperm morphology (MORF, %) were evaluated immediately after AgNPs exposure (T0) and after 2 h of incubation (T2). The interaction beween AgNPs and spermatozoa was visualized by transmission electron microscopy (TEM). At T0, sperm motility and AIS were reduced (p < .05) when using concentrations ≥50 and ≥25 μg/mL, respectively. At T2, sperm motility and LIVE were significantly decreased (p < .05) in concentrations ≥25 and ≥50 μg/mL, respectively. TEM analysis revealed nanoparticle adhesion to the acrosomal region of the plasma membrane. In conclusion, AgNPs at concentrations ≥25 μg/mL impair motility, acrosome and plasma membrane integrity of donkey sperm, which may be mediated by adhesion to the acrosomal region of the sperm surface membrane.

Nanotechnology and its applications have advanced significantly in recent decades, contributing to various fields, including reproduction. This study introduces a novel method to label porcine oocytes with nanoparticles (NPs) bound to oviductin (OVGP1, Ov) for use in Assisted Reproductive Technologies (ARTs). Despite promising developments, concerns about NP toxicity in gametes necessitate thorough investigation. This research aims to assess the impact of functionalized NPs (NPOv) on sperm functionality. Boar sperm were co-incubated with NPOv for 0, 0.5 and 1 h in two media: BTS (semen dilution and conservation) and TALP (sperm capacitation and in vitro fertilization-IVF). Sperm quality parameters (viability, motility and kinematics) showed no significant differences in TALP medium (p > .05). In BTS, although some kinetic parameters were altered, motility, progressive motility and viability remained unaffected (p > .05). Additionally, NPs presence on the zona pellucida (ZP) of oocytes did not affect sperm attachment (p > .05). In conclusion, in vitro exposure of boar sperm to OVGP1-functionalized NPs in IVF medium or attached to the ZP surface of matured oocytes does not impair sperm functionality, including their binding ability to the ZP.