Theriogenology最新文献

Heat shock can impair embryo formation, while growth factors, such as colony-stimulating factor 2 (CSF2), modulate embryonic development. This study evaluated the effect of heat shock between days 2.5 and 3, as well as the impact of CSF2 at day 5 on bovine embryos cultured in a serum-free in vitro medium. The focus was on blastocyst development, the number of blastomeres, DNA fragmentation (TUNEL-positive cells), and mitochondrial activity. Heat shock reduced the proportion of cleaved embryos that developed into blastocysts (P = 0.0603). The resultant blastocysts exhibited a reduced number and proportion of TUNEL-positive cells in the trophectoderm (P = 0.0270 and P = 0.0240, respectively) and in the entire embryo (P = 0.0029 and P = 0.0031, respectively). Additionally, mitochondrial activity was lower in blastocysts derived from heat-shocked embryos (P = 0.0150) and further reduced in embryos exposed to both heat shock and CSF2 (P = 0.0415). In conclusion, the exposure of cleaved embryos to heat shock reduced their development to the blastocyst stage. However, the resulting blastocysts showed decreased DNA fragmentation and mitochondrial activity.

Background

The hypothalamic-pituitary-gonadal (HPG) axis is pivotal in regulating reproductive functions, with gonadotropin-releasing hormone (GnRH) acting as a central regulator. Recently, polyamines have been shown to regulate the HPG axis, including GnRH expression and ovarian biology in old and adult rodents. The present study firstly highlights the age-specific variation in the polyamine and their corresponding biosynthetic enzymes in the ovary during aging, and further, the study focuses on the effect of polyamines, putrescine, and agmatine, in young female mice.

Method and result

Immunofluorescence analysis revealed age-related differences in the expression of ornithine decarboxylase 1 (ODC1), spermine (SPM), and spermidine (SPD) in the ovaries, with adult mice exhibiting significantly higher expression levels compared to young and old mice. Likewise, qPCR analysis showed the mRNA levels of Odc1, Spermidine synthase (Srm), and Spermine synthase (Sms) show a significant increase in adult ovaries, which is then followed by a significant decline in old age. Histological examination demonstrated morphological alterations in the ovaries with age, including decreased follicle numbers and increased stromal cells in old mice. Furthermore, treatment with putrescine, a polyamine, in young mice resulted in larger ovaries and increased follicle numbers compared to controls. Additionally, serum levels of gonadotropin-releasing hormone (GnRH) and progesterone (P4) were measured, showing elevated levels in polyamine-treated mice. GnRH mRNA expression also increased significantly. Gene expression analysis revealed upregulation of genes associated with folliculogenesis such as Fshr, Bmp15, Gdf9, Amh, Star, Hsdb3, and Plaur in the ovaries and onset of puberty such as Tac2, and Kiss1, and a decrease in Mkrn3 in the hypothalamus of polyamine-treated mice.

Conclusion

This study investigates the effect of polyamines in young immature female mice, shedding light on their role in upregulating GnRH, and enhancing folliculogenesis. Overall, these findings suggest that polyamines play a crucial role in ovarian aging and HPG axis regulation, offering potential therapeutics to reinstate fertility in reproductively challenged individuals.

Zinc oxide nanoparticles (ZnO-NPs) have garnered significant attention in biological applications due to their known antioxidant properties. However, their potential impact on assisted reproduction techniques remains largely unexplored, particularly in the context of oocyte quality maintenance within in vitro culture systems, where free radicals can exert detrimental effects. This study investigated the effects of incorporating ZnO-NPs to in vitro maturation (IVM) media on the developmental, cryosurvival, and metabolic profiles of bovine embryos. Three concentrations of ZnO-NPs (0, 1.0, and 1.5 μg/mL) were evaluated. We observed, for the first time, that the inclusion of ZnO-NPs at a concentration of 1.0 μg/mL led to a significant increase in the number of embryonic cells (p < 0.05) accompanied by a reduction in reactive oxygen species production (p < 0.05). Notably, ZnO-NPs did not alter embryonic development, cryosurvival rates, or mitochondrial viability. These findings suggested that ZnO-NPs has antioxidant properties and are compatible with bovine oocytes. Consequently, they may serve as promising supplements to the IVM media, potentially enhancing the efficiency of assisted reproduction techniques.

In this study, we aimed to evaluate the efficacy of cryopreserving canine ovarian tissue using vitrification and slow freezing methods while investigating potential differences in cryotolerance based on follicular type and cryopreservation technique. Twenty-eight ovaries were collected from 14 anoestrus bitches of various breeds, aged between 2 and 5 years, and undergoing elective ovariohysterectomy. The ovaries were sectioned into small fragments and randomly assigned to three groups: vitrification, slow freezing, and a control group (fresh tissue). Vitrification was performed using cryotubes containing DAP 213 solution (2M DMSO, 1M acetamide, 3M propylene glycol) in two stages, while slow freezing involved cryotubes with 1.5M DMSO solution inserted into a programmable machine. The effects of cryopreservation were evaluated by histology and immunohistochemistry (cleaved caspase-3), to determine the percentage of cells undergoing apoptosis. Histological examination revealed that the slow freezing group exhibited a significantly higher percentage of intact follicles (45.75 %) compared to those subjected to vitrification (38.17 %; P = 0.01). Immunohistochemical evaluation further indicated that 84.21 % of the follicles in the slow freezing group did not express caspase-3, suggesting the absence of apoptosis. Conversely, vitrified samples exhibited significantly more apoptotic cells compared to other groups (P < 0.001). Furthermore, early antral follicles displayed a higher susceptibility to degeneration regardless of the cryopreservation method employed. Nevertheless, when comparing the cryopreserved groups, early antral follicles showed greater degeneration in slow freezing group, while preantral follicles were the most affected in the vitrification group. In conclusion, slow freezing demonstrated superior preservation of viable follicles compared to vitrification and emerged as the preferred technique for cryopreserving canine ovarian tissue. These findings contribute valuable insights into optimizing cryopreservation methods for canine ovarian tissue, potentially benefiting reproductive technologies and fertility preservation in canines.

Equine endometrosis is a degenerative and predominantly fibrotic condition resulting from progressive and irreversible multifactorial causes that influence the endometrium of mare. Tissue remodeling in the equine endometrium occurs as part of the pathogenesis of endometrosis, a process characterized by a shift in extracellular matrix (ECM) components. The relationship between matrix metalloproteinases and their specific inhibitors is crucial for the remodeling process. Collagen play a significant role in maintaining a healthy uterus and may promote fibrotic processes. The aim of this study was to quantify endometrial collagen deposition using picrosirius 25 red (PSR) staining, and to evaluate gene expression of collagen type 2 (COL-2) and 3 (COL-3), matrix metalloproteinases 1 (MMP-1) and 2 (MMP-2), their tissue inhibitor (TIMP-2), and tumor necrosis factor (TNF-α) in the endometrium of mares with different grades of fibrosis. The samples (n = 34) were classified into three categories based on the frequency and distribution of fibrosis-related changes in the endometrium: Category I (healthy endometrium, n = 12), Category II (moderate fibrosis, n = 12), and Category III (severe fibrosis, n = 10). Collagen quantification demonstrate a substantial proportional increase (P < 0.0001) in collagen deposition across Category I (11.72 ± 1.39 %), Category II (17.76 ± 1.29 %), and Category III (24.15 ± 1.87 %). In transcript evaluations, higher COL-2 expression was found in Category II than in mares classified as Category I or III. MMP-1 showed increased transcript expression in Category II compared to Category III endometrial samples. Higher expression of MMP-2 was detected in Category III than in Category I and II. TIMP-2 showed lower mRNA expression in Category III vs Category I and II. However, TNF-α gene expression was higher in Category II than in Categories I and III. This study demonstrates that endometrial evaluation using PSR can play an important role in routine analyses for the detection and objective quantification of collagen in endometrial tissues. Additionally, this study demonstrated through gene expression analysis that MMP-1 may be linked to physiological endometrial remodeling. In contrast, MMP-2 could be associated with fibrogenesis in the endometrium, which is regulated by the inhibitor TIMP-2. Furthermore, COL-2 and TNF-α could be considered as biological markers involved in the progression endometrosis in mares. As such, the results of this study may contribute to the development of future antifibrotic therapies that aim to delay or even reverse the pathological remodeling of the extracellular matrix in the uterus, in addition to optimizing the diagnosis and prognosis of endometrial fibrosis in mares.

Extracellular vesicles, also known as exosomes, influence numerous cellular functions by regulating different signaling pathways. However, their role in animal reproduction remains understudied. This study aimed to evaluate the effects of porcine follicular fluid-derived exosomes (pff-Exos) on porcine oocyte in vitro maturation and parthenogenetic embryo development. We obtained pff-Exos through mixed-method ultracentrifugation and size-exclusion chromatography. Transmission electron microscopy revealed an increase in the expression of exosome markers in the first four of thirteen fractions. The number of pff-Exo was 2.2 × 10⁶ particles per microliter. The highest maturation rate of porcine oocytes treated with pff-Exo was observed with 1.1 × 10⁷ particles of pff-Exo in the absence of porcine follicular fluid (pFF) culture conditions. Moreover, increased expression of Gdf9 and Bmp15 was observed. The developmental rate was the highest upon treatment with 1.1 × 10⁷ particles of pff-Exo, which increased the total cell number in blastocysts. Embryonic development to the 2-cell stage was similar between the control and pff-Exo groups; however, development to the 4-cell stage and blastocyst was significantly increased in the pff-Exo group (61.6 ± 6.08 % and 29.72 ± 1.41 %, respectively; P < 0.05) compared with that in the control group (42.0 ± 5.19 % and 18.14 ± 1.78 %, respectively). The expression levels of Oct4, Sox2, Bcl2, Elf4, and Gcn5 significantly increased at the pff-Exo 2-cell stage, whereas those of Bax, Hdac1, Hdac6, and Sirt6 decreased. Specifically, the Oct4, Sox2, Elf4, Gcn5, and Hdac6 levels remained stable in pff-Exo 4-cell embryos, whereas those of p53 and Hat1 were reduced and increased, respectively. Treatment with pffExos significantly increased H3K9 and H3K14 acetylation levels. These results demonstrate that pff-Exo affects the in vitro maturation of porcine oocytes and early embryonic development by regulating gene expression.

Vitrification of oocyte has become an important component of assisted reproductive technology and has important implications for animal reproduction and the preservation of biodiversity. However, vitrification adversely affects mitochondrial function and oocyte developmental potential, mainly because of oxidative damage. Rutin is a highly effective antioxidant, but no information is available to the effect of rutin on the mitochondrial function and development in vitrified oocytes. Therefore, we studied the effects of rutin supplementation of vitrification solution on mitochondrial function and developmental competence of ovine germinal vesicle (GV) stage oocytes post vitrification. The results showed that supplementation of vitrification solution with 0.6 mM rutin significantly increased the cleavage rate (71.6 % vs. 59.3 %) and blastocyst rate (18.9 % vs. 6.8 %) compared to GV-stage oocytes in the vitrified group. Then, we analyzed the reactive oxygen species (ROS), glutathione (GSH), mitochondrial activity and membrane potential (ΔΨm), endoplasmic reticulum (ER) Ca²⁺, and annexin V (AV) of vitrified sheep GV-stage oocytes. Vitrified sheep oocytes exhibited increased levels of ROS and Ca²⁺, higher rate of AV-positive oocytes, and decreased mitochondrial activity, GSH and ΔΨm levels. However, rutin supplementation in vitrification solution decreased the levels of ROS, Ca²⁺ and AV-positive oocytes rate, and increased the GSH and ΔΨm levels in vitrified oocytes. Results revealed that rutin restored mitochondrial function, regulated Ca²⁺ homeostasis and decreased apoptosis potentially caused by mitophagy in oocytes. To understand the mechanism of rutin functions in vitrified GV-stage oocytes in sheep, we analyzed the transcriptome and found that rutin mediated oocytes development and mitochondrial function, mainly by affecting oxidative phosphorylation and the mitophagy pathways. In conclusion, supplementing with 0.6 mM rutin in vitrification solution significantly enhanced developmental potential through improving mitochondrial function and decreased apoptosis potentially caused by mitophagy after vitrification of ovine GV-stage oocytes.

The aim was to evaluate the efficiency of two different ultrasonographic systems, Doppler mode vs. Two-dimensional mode (B mode), to identify the pregnancy status of beef cows and heifers on day 21 (D21) after Timed Artificial Insemination (TAI). The experiment was performed on a commercial beef farm in central-west region of Brazil using 1895 Nelore heifers and cows. All females had ovulation synchronized for a TAI that was performed on D0. Twenty-one days after the TAI, all animals had their ovaries evaluated by ultrasound for pregnancy diagnosis based on the size of the corpus luteum (CL). Using B mode ultrasonography, females without a CL or with a CL ≤ 10 mm in diameter were considered nonpregnant, whereas females with a CL > 10 mm in diameter were considered potentially pregnant. After the B mode examination, the Doppler mode was turned on, and the CL was evaluated by the subjective percentage of blood perfusion in the total area of the CL. Using Doppler mode, females were considered nonpregnant if they had no CL or the CL had ≤25 % of the total area with detectable blood perfusion, whereas animals with >25 % blood perfusion in the CL were considered potentially pregnant. The results for each method (potentially pregnant or nonpregnant) were later compared with the gold standard technique, which was a pregnancy diagnosis on D33 after TAI using ultrasound with visualization of an embryonic heartbeat. The accuracy was determined using the 2 × 2 contingency table approach. The area under the curve using the receiver operating characteristic curve for Doppler mode and B mode were 0.929 and 0.902 (P < 0.01), respectively. There were almost no false negatives (designated non-pregnant but later pregnant at D33) with either technique (0.2 % vs. 0.3 %; P = 0.65 for Doppler mode vs. B mode, respectively). False positives (designated pregnant but non-pregnant on D33) were greater for B mode compared to Doppler (19.1 % vs. 14.0 %; P < 0.01). This resulted in Doppler mode having similar high values as B mode for Negative Predictive Value (99.9 vs. 99.6 %; P = 0.85) and Sensitivity (99.8 vs. 99.7 %; P = 0.86) but there were differences in Specificity (86 vs. 80.9 %; P < 0.01), Positive Predictive Value (88 vs. 84.3 %; P < 0.01), and Accuracy (93.0 vs. 90.4 %; P < 0.01). In conclusion, evaluation of CL blood perfusion by Doppler produced greater accuracy in the early identification of nonpregnant heifers and cows on D21 after TAI than measurement of CL diameter with B mode ultrasound; although both had over 90 % accuracy in identifying pregnant and nonpregnant females.

Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage, transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance.

Cryopreservation of spermatogonia could be a useful tool to preserve the genetic resources of fish, which could be further restored via germ cell transplantation. In this study, the protocol for the cryopreservation of the spermatogonia of Asian sea bass (Lates calcarifer), an economically important fishery resource in the Indo-West Pacific, was optimised. The impact of the cryopreservation technique on cell viability and apoptosis, expression of several genes related to immature germ cell markers, transplantability in allogeneic recipients, and global DNA methylation was evaluated. The slow-freezing method was performed for the cryopreservation of immature testis tissue, which contains a high proportion of spermatogonia. The optimal condition that yielded the highest recovery rate of post-thawed spermatogonia included a cryomedium containing Leibovitz's (L-15) medium and 10 % dimethyl sulfoxide, ice equilibration for 60 min before freezing, and subsequent thawing at 4 °C for 8 min. Moreover, a higher number of early and late apoptotic cells was detected in the cryopreserved than in the fresh testes, suggesting that apoptosis could result in reduced viability. The expression levels of dazl decreased in the cryopreserved testes; however, there were no significant differences in the expression levels of nanos2 or nanos3 between the fresh and cryopreserved testes. Although qRT-PCR showed lower vasa expression in cryopreserved testicular cells, in situ hybridisation showed expressed vasa in the cryopreserved testicular cells. Post-thawed spermatogonia could be incorporated into the genital ridge of allogeneic recipients, suggesting that cryopreserved spermatogonia exhibit transplantability characteristics. Compared with fresh testes, significant changes in the proportion of DNA methylation (decreased 5-mC and 5-caC) were observed in cryomedium-free testicular cells, whereas those of the cryopreserved cells were not significantly different. Therefore, the method we developed for the cryopreservation of the spermatogonia of Asian sea bass enabled post-thaw cells to retain several stemness characteristics and maintain their epigenetic stability.