L-carnitine has an important role in the control of oxidative stress and lipid β-oxidation during in vitro culture and cryopreservation of ovarian follicles, oocytes and embryos. This substance balances the acetyl-CoA/CoA ratio, maintains glucose metabolism and increases energy production in mitochondria. It also plays a key role in reducing endoplasmic reticulum stress, by transferring palmitate to mitochondria or eliminating it to avoid toxicity. By eliminating reactive oxygen species, L-carnitine increases the percentages of mature oocytes with uniform mitochondrial distribution and improves embryo post-thaw cryotolerance. Therefore, L-carnitine controls lipid β-oxidation and oxidative stress during in vitro culture of ovarian follicles, oocyte maturation, embryonic development and cryopreservation.

We have established trophoblast cell lines, from parthenogenesis-derived buffalo blastocysts. The buffalo trophoblast cells were cultured continuously over 200 days and 21 passages. These cells were observed by phase-contrast microscopy for their morphology and characterized by reverse transcriptase polymerase chain reaction and immunofluorescence against trophoblast-specific markers and cytoskeletal proteins. Trophoblast cells showed positive staining for CDX2, a marker of these cells at both blastocyst and cell line levels. Epithelial morphology of these cells was revealed by positive staining against cytokeratins and tubulin but not against vimentin and dolichos biflorus agglutinin. Gene expression profiles of many important placenta-specific genes were studied in the primary trophectoderm outgrowths, which were collected on days 0, 5, 9, 12 and 15 of culture and trophoblast cell line at passages 12–15. Therefore, the trophoblast cell line derived can potentially be used for in vitro studies on buffalo embryonic development.

Metabolite supplementation during in vitro embryo development improves blastocyst quality, however, our understanding of the incorporation of metabolites during in vitro maturation (IVM) is limited. Two important metabolites, follistatin and choline, have beneficial impacts during in vitro culture; however, effects of supplementation during IVM are unknown. The objective of this study was to investigate combining choline and follistatin during IVM on bovine oocytes and subsequent early embryonic development. We hypothesized that supplementation of choline with follistatin would synergistically improve oocyte quality and subsequent early embryonic development. Small follicles were aspirated from slaughterhouse ovaries to obtain cumulus oocyte complexes for IVM with choline (0, 1.3 or 1.8 mM) and follistatin (0 or 10 ng/mL) supplementation in a 3 × 2 design. A subset of oocytes underwent transcriptomic analysis, the remaining oocytes were used for IVF and in vitro culture (IVC). Transcript abundance of CEPT1 tended to be reduced in oocytes supplemented with 1.8 mM choline and follistatin compared to control oocytes (P = 0.07). Combination of follistatin with 1.8 mM choline supplementation during maturation, tended (P = 0.08) to reduce CPEB4 in oocytes. In the blastocysts, HDCA8, NANOG, SAV1 and SOX2 were increased with choline 1.8 mM supplementation without follistatin (P < 0.05), while HDCA8 and SOX2 were increased when follistatin was incorporated (P < 0.05). The combination of choline and follistatin during oocyte maturation may provide a beneficial impact on early embryonic development. Further research is warranted to investigate the interaction between these two metabolites during early embryonic development and long-term influence on fetal development.

The global transition towards diets high in calories has contributed to 2.1 billion people becoming overweight, or obese, which damages male reproduction and harms offspring. Recently, more and more studies have shown that paternal exposure to stress closely affects the health of offspring in an intergenerational and transgenerational way. SET Domain Containing 2 (SETD2), a key epigenetic gene, is highly conserved among species, is a crucial methyltransferase for converting histone 3 lysine 36 dimethylation (H3K36me2) into histone 3 lysine 36 trimethylation (H3K36me3), and plays an important regulator in the response to stress. In this study, we compared patterns of SETD2 expression and the H3K36me3 pattern in pre-implantation embryos derived from normal or obese mice induced by high diet. The results showed that SETD2 mRNA was significantly higher in the high-fat diet (HFD) group than the control diet (CD) group at the 2-cell, 4-cell, 8-cell, and 16-cell stages, and at the morula and blastocyst stages. The relative levels of H3K36me3 in the HFD group at the 2-cell, 4-cell, 8-cell, 16-cell, morula stage, and blastocyst stage were significantly higher than in the CD group. These results indicated that dietary changes in parental generation (F0) male mice fed a HFD were traceable in SETD2/H3K36me3 in embryos, and that a paternal high-fat diet brings about adverse effects for offspring that might be related to SETD2/H3K36me3, which throws new light on the effect of paternal obesity on offspring from an epigenetic perspective.

One of the prominent peculiarities of nanoparticles (NPs) is their ability to cross biological barriers. Therefore, the development of NPs with different properties has great therapeutic potential in the area of reproduction because the association of drugs, hormones and other compounds with NPs represents an alternative for delivering substances directly at a specific site and for treatment of reproductive problems. Additionally, lipid-based NPs can be taken up by the tissues of patients with ovarian failure, deep endometriosis, testicular dysfunctions, etc., opening up new perspectives for the treatment of these diseases. The development of nanomaterials with specific size, shape, ligand density and charge certainly will contribute to the next generation of therapies to solve fertility problems in humans. Therefore, this review discusses the potential of NPs to treat reproductive disorders, as well as to regulate the levels of the associated hormones. The possible limitations of the clinical use of NPs are also highlighted.

Adult male and female Murrah buffalo fibroblast cells were used as donors for the production of embryos using handmade cloning. Both donor cells and reconstructed embryos were treated with 50 nM trichostatin-A (TSA) and 7.5 nM 5-aza-2′-deoxycytidine (5-aza-dC). The blastocyst rate of both treated male (40.1% ± 2.05) and female (37.0% ± 0.83) embryos was significantly lower than in untreated control males (49.7% ± 3.80) and females (47.2% ± 2.44) but their apoptotic index was lower (male, control: 5.90 ± 0.48; treated: 4.96 ± 0.31): (female, control: 8.11 ± 0.67; treated: 6.65 ± 0.43) and epigenetic status in terms of global acetylation and methylation of histone was significantly improved. The expression level of hypoxanthine-guanine phosphoribosyltransferase (HPRT) was higher (P < 0.05) and that of PGK, G6PD, OCT 4, IFN-tau and CASPASE3 was significantly lower (P < 0.05) in treated male blastocyst than control and the expression levels of DNMT1, IGF1R and BCL-XL were not significantly different between the two groups. In the female embryos, the relative mRNA abundance of OCT4 was significantly higher (P < 0.05), and that of XIST and CASPASE3 was significantly lower (P < 0.05) in the epigenetic modifier-treated group compared with that of the control group, whereas the expression levels of HPRT, PGK, G6PD, DNMT1, IFN-tau, IGF1R and BCL-XL were not significantly different between the two groups. In both embryos, a similar effect of treatment was observed on genes related to growth and development, but the effect on the expression of X-linked genes varied. These results indicate that not all X-linked genes respond to TSA and 5-aza-dC treatment in the same manner.

The study was undertaken to examine the relative abundance (RA) of the major developmental important candidate genes in different grades of immature oocytes (A-grade, B-grade, C-grade and D-grade) and various stages of in vitro-produced embryos (2-cell, 4-cell, 8–16-cell, morula, and blastocyst) of buffalo using RT-qPCR. Results showed that the RA of GLUT1, CX43, HSP70.1 and GDF9 was significantly higher (P < 0.05) in the A-grade of oocytes than the C-grade and D-grade but did not differ significantly from the B-grade of oocytes. Similarly, RA of BMP15 and Survivin were significantly higher (P < 0.05) in A-grade than the other grades of oocytes, however, poly(A) polymerase expression was not significantly different (P > 0.05) among the immature oocytes. The expression of GLUT1 was significantly higher (P < 0.05) in the blastocysts, but the expression of CX43 (P < 0.05; P > 0.05), HSP70.1 (P < 0.05; P > 0.05) and GDF9 (P > 0.05) was higher at the 2-cell stage than the other stages of embryos. Interestingly, the expression levels of poly(A) polymerase (P < 0.05), BMP15 (P < 0.05; P > 0.05) and Survivin (P > 0.05) were higher at the 8–16-cell stage than the other stages of embryos. It is concluded that A-grade of immature oocytes has shown more mRNA abundance for the major developmental important genes; therefore A-grade oocytes may be considered as the most developmentally competent and suitable for handmade cloning research in buffalo.

Many studies have focused on the optimization of the composition of embryo culture medium; however, there are few studies involving the effect of a culture medium changing procedure on the preimplantation development of embryos. In this study, three groups were designed: a non-renewal group, a renewal group and a half-renewal group. The levels of reactive oxygen species (ROS), apoptotic index, blastocyst ratio and blastocyst total cell number were analyzed in each group. The results showed that the ROS level and the apoptotic index of blastocyst in the non-renewal group were significantly higher than in the renewal group and the half-renewal group (P < 0.05). The blastocyst ratio and blastocyst total cell number were significantly higher in the half-renewal group than that in non-renewal group and the renewal group (P < 0.05). These results demonstrated that the procedure of changing the culture medium influenced ROS level, apoptotic index, blastocyst ratio and total cell number of blastocysts. In addition, the result suggested that changing the culture medium may lead to a loss of important regulatory factors for embryos, while not changing the culture medium may lead to the accumulation of toxic substances. Half-renewal can alleviate the defects of both no renewal and renewal, and benefit embryo development. This study will be of high value as a reference for the optimization of embryo culture in vitro, and is very significant for assisted reproduction.

The objective of this study was to investigate any effect of cryopreservation of donated eggs on laboratorial and clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles. This retrospective cohort study included 320 oocyte recipients undergoing 307 vitrified and 119 fresh oocyte recipient ICSI cycles, participating in an egg-sharing donation programme, from 2015 to 2018, in a private university-affiliated in vitro fertilization (IVF) centre. A review of donor and recipient ICSI cycles was charted. A general mixed models fit by restricted maximum likelihood, followed by Bonferroni post hoc test was used to compare the means between fresh and warm oocyte donation groups and investigate the effect of cryopreservation on recipient ICSI outcome. The main outcome measure was blastocyst development rates. Fertilization rate, high-quality embryo rates on days 2 and 3, normal cleavage speed rates on days 2 and 3, and blastocyst development rate were significantly higher for the fresh oocyte donation cycles compared with warmed oocyte donation cycles. In the egg-sharing donation programme, fertilization and embryo developmental competence were reduced when vitrified oocytes from infertile couples were used for ICSI compared with fresh oocytes.

This study was carried out to compare the efficacy of different methods to activate buffalo A + B and C + D quality oocytes parthenogenetically and to study the in vitro developmental competence of oocytes and expression of some important genes at the different developmental stages of parthenotes. The percentage of A + B oocytes (62.16 ± 5.06%, range 53.8–71.3%) was significantly higher (P < 0.001) compared with that of C + D oocytes (37.8 ± 5.00%, range 28.6–46.1%) retrieved from slaughterhouse buffalo ovaries. Among all combinations, ethanol activation followed by culture in research vitro cleave medium gave the highest cleavage and blastocyst yields for both A + B and C + D grade oocytes. Total cell numbers, inner cell mass/trophectoderm ratio and apoptotic index of A + B group blastocysts were significantly different (P < 0.05) from their C + D counterpart. To determine the status of expression patterns of developmentally regulated genes, the expression of cumulus–oocyte complexes, fertilization, developmental competence and apoptotic-related genes were also studied in parthenogenetically produced buffalo embryos at different stages, and indicated that the differential expression patterns of the above genes had a role in early embryonic development.

Numerous factors affect vitrification success and post-thaw development of oocytes after in vitro fertilization. Therefore, elaboration of an optimal methodology ensuring higher cryotolerance of oocytes and subsequent blastocyst yield is still of great interest. This paper describes and evaluates critical factors affecting the success of oocyte vitrification. In particular, an appropriate oocyte stage such as maturation status (germinal vesicle stage, metaphase II stage), presence/absence of cumulus cells before vitrification, and the effect of follicle size, as well as different culture systems and media for in vitro production of embryos, the types and concentrations of cryoprotectants, and cooling and warming rates at vitrification are considered. Special attention is paid to various cryocarriers used for low-volume vitrification, which ensures safe storage of oocytes/embryos in liquid nitrogen and their successful post-thaw recovery. At the end, we focussed on how age of oocyte donors (heifers, cows) influences post-thaw development. This review summarizes results of recently published studies describing different methodologies of cryopreservation and post-thaw oocyte development with the main focus on vitrification of bovine oocytes.