Climate Change and Livestock Fertility

Climate change is a global threat to livestock sector to so many species and ecosystem in different parts of the world. Climate change, heat stress, and nutritional stress are the major intriguing factors responsible for reduced fertility in farm animals in tropical countries. Heat and nutritional stresses affect the reproductive performance by decreasing the expression of estrous behavior, altering ovarian follicular development and hormonal profiles, compromising oocyte competence, and inhibiting embryonic development in livestock. Climate is changed by greenhouse gases that released into atmosphere through man-made activities. Livestock contribute 18% of the production of greenhouse gases itself and causes climate change including heat stress, which has direct and indirect impact on fertility of the animals as well as reduce milk production. Adaptation to climate change and lowering its negative effect by alteration of animal micro-environment using different essential technologies are the main mitigation strategies to recover heat stress damage in this respect.

Loss of PDK1 Induces Meiotic Defects in Oocytes From Diabetic Mice

Maternal diabetes has been shown to impair oocyte quality; however, the underlying mechanisms remain unclear. Here, using a streptozotocin (STZ)-induced diabetic mouse model, we first detected and reduced expression of pyruvate dehydrogenase kinase 1 (PDK1) in diabetic oocytes, accompanying with the lowered phosphorylation of serine residue 232 on α subunit of the pyruvate dehydrogenase (PDH) complex (Ser232-PDHE1α). Importantly, forced expression of PDK1 not only elevated the phosphorylation level of Ser232-PDHE1α, but also partly prevented the spindle disorganization and chromosome misalignment in oocytes from diabetic mice, with no beneficial effects on metabolic dysfunction. Moreover, a phospho-mimetic S232D-PDHE1α mutant is also capable of ameliorating the maternal diabetes-associated meiotic defects. In sum, our data indicate that PDK1-controlled Ser232-PDHE1α phosphorylation pathway mediates the effects of diabetic environment on oocyte competence.

Effect of Ethanol on Parthenogenetic Activation and α-Tocopherol Supplementation during In Vitro Maturation on Developmental Competence of Summer-Collected Bovine Oocytes

The use of α-tocopherol during in vitro maturation (IVM) is an alternative to minimize the adverse effects of heat stress on oocyte competence. However, α-tocopherol is diluted in ethanol, which can induce oocyte parthenogenetic activation (PA). This study aimed to evaluate the role of ethanol concentration on PA and the effect of α-tocopherol supplementation during IVM on the developmental competence and the expression of key genes in blastocysts derived from summer-collected oocytes. All in vitro embryo production was conducted at 5% O2, 5% CO2 at 38.5 °C. Experiment 1: oocytes were cultured with or without 0.05% ethanol. As positive PA control matured oocytes were subjected to 3% or 7% ethanol for 7 min. Oocytes from all groups were placed in fertilization medium (22 h) and culture medium (9 days). Ethanol at 0.05% during IVM did not induce oocyte PA, however, 3% and 7% ethanol were effective parthenogenetic inductors. Experiment 2: oocytes were cultured in maturation medium supplemented with 0, 50, 100 and 200 μM α-tocopherol, diluted in 0.05% ethanol. After in vitro fertilization and embryo culture, we assessed blastocyst apoptotic index and the transcription of a panel of genes. The results showed that supplementation with 100 μM α-tocopherol reduced apoptotic index and increased the expression of SOD2. In conclusion, 100 μM α-tocopherol, diluted in 0.05% ethanol, can be used during IVM to embryonic quality.

Cumulus Cell DNA Damage as an Index of Human Oocyte Competence

The determination of oocyte quality is crucial for achieving effective syngamy post-sperm injection and embryonic development. Cumulus cells (CCs) have been proposed as biomarkers of oocyte quality because of their close bio-dynamic relationship with the oocyte. To determine the quality of the oocyte, CCs were sampled during oocyte preparation for ICSI to determine a CC DNA fragmentation index (CCDFI) of each individual oocyte using a variant of the chromatin dispersion test. One hundred and thirty oocytes were selected and studied from two Spanish fertility clinics, 90 of which were fertilized and developed to embryos. Significant differences were found between the CCDFI of unfertilized and fertilized oocytes (p < .001) and between the CCDFI of embryos that were discarded and those that developed suitable for transfer or cryopreservation (p < .001). Oocyte quality was negatively correlated with CCDFI (Spearman’s rho = − 0.45; p < .001). Receiver operator characteristics curves (ROC) suggested that a cut-off value of 24% CCDFI was able to discriminate the capacity of the gametes to result in syngamy with a sensitivity and specificity of 75.6% and 65%, respectively. This cut-off supports the application of CCDFI as potential index for the evaluation of the reproductive potential of oocytes prior to fertilization.

Increased Systemic Antioxidant Power Ameliorates the Aging-Related Reduction in Oocyte Competence in Mice

Ovarian aging is associated with elevated oxidative stress and diminished oocyte developmental competence. We aimed to determine the impact of systemic antioxidant treatment in aged mice. Female outbred CF-1 mice were aged for 9 months prior to an 8-week 45 mg Euterpe oleracea (açaí) daily supplement. The açaí treatment induced a threefold increase in serum antioxidant power (FRAP) compared to both young and aged mice (p < 0.0001). Compared to young mice, aged mice had fewer oocytes and reduced blastocyst development (p < 0.0001); açaí did not affect the oocyte numbers, but improved blastocyst formation (p < 0.05). Additionally, açaí alleviated the aging-related decrease in implantation potential (p < 0.01). The aged mice showed evidence of elevated ovarian ER stress (increased whole-ovary PDIA4 expression, granulosa cell and oocyte GRP78 expression, and oocyte PDIA4 protein), reduced oocyte mitochondrial quality (higher PRKN activation and mitochondrial DNA oxidative damage), and dysregulated uterine glandular epithelium. Antioxidant intervention was sufficient to lessen these effects of ovarian aging, likely in part by the upregulation of NRF2. We conclude that açaí treatment is a promising strategy to improve ER and mitochondrial function in the ovaries, thereby ameliorating the decreased oocyte competence that occurs with ovarian aging.

Human adipose-derived stromal cells transplantation prolongs reproductive lifespan on mouse models of mild and severe premature ovarian insufficiency

Background Although recent studies have investigated the ability of Mesenchymal Stromal Cells (MSCs) to alleviate short-term ovarian damage in animal models of chemotherapy-induced Premature Ovarian Insufficiency (POI), no data are available on reproductive lifespan recovery, especially in a severe POI condition. For this reason, we investigated the potential of MSCs isolated from human adipose tissue (hASCs), since they are easy to harvest and abundant, in ameliorating the length and performance of reproductive life in both mild and severe chemotherapy-induced murine POI models. Methods Mild and severe POI models were established by intraperitoneally administering a light (12 mg/kg busulfan + 120 mg/kg cyclophosphamide) or heavy (30 mg/kg busulfan + 120 mg/kg cyclophosphamide) dose of chemotherapy, respectively, in CD1 mice. In both cases, a week later, 1 × 106 hASCs were transplanted systemically through the tail vein. After four additional weeks, some females were sacrificed to collect ovaries for morphological evaluation. H&E staining was performed to assess stroma alteration and to count follicle numbers; immunofluorescence staining for αSMA was used to analyse vascularization. Of the remaining females, some were mated after superovulation to collect 2-cell embryos in order to evaluate their pre-implantation developmental capacity in vitro, while others were naturally mated to monitor litters and reproductive lifespan length. F1 litters’ weight, ovaries and reproductive lifespan were also analysed. Results hASC transplantation alleviated ovarian weight loss and size decrease and reduced alterations on ovarian stroma and vasculature, concurrently preventing the progressive follicle stockpile depletion caused by chemotherapy. These effects were associated with the preservation of the oocyte competence to develop into blastocyst in vitro and, more interestingly, with a significant decrease of chemotherapy-induced POI features, like shortness of reproductive lifespan, reduced number of litters and longer time to plug (the latter only presented in the severe POI model). Conclusion Human ASC transplantation was able to significantly reduce all the alterations induced by the chemotherapeutic treatment, while improving oocyte quality and prolonging reproductive functions, thus counteracting infertility. These results, strengthened by the use of an outbred model, support the potential applications of hASCs in women with POI, nowadays mainly induced by anticancer therapies. Graphic abstract

266 Effects of Inflammatory States on Ovarian Biology and Oocyte Competence

Female fertility is dependent on estradiol concentrations which regulate a multitude of ovarian functions including follicle development and oocyte maturation leading to ovulation of a viable oocyte. Estradiol biosynthesis is regulated by coordinated actions of follicle-stimulating hormone and intra-ovarian control mechanisms including the co-transcription factor beta-catenin. Beta-catenin is a multi-faceted protein recognized for its role in granulosa cell steroid production and is shown to be modulated by lipopolysaccharide (LPS), the endotoxin responsible for stimulation of the immune system in infections caused by Gram-negative bacteria. Beef heifers treated with subacute concentrations of LPS during a synchronized follicular wave demonstrate a decline in serum estradiol concentrations 50 h after CIDR withdrawal, corresponding with dominant follicle maturation and preceding ovulation. The endotoxin exposure also resulted in increased LPS concentration and E2:P4 ratios in follicular fluid suggesting that low dose LPS modulates the intra-follicular hormonal milieu. Additionally, in a granulosa cell line, LPS treatment decreased mRNA expression of aromatase and beta-catenin. These data indicate that LPS alters E2 synthesis by modulating beta-catenin and subsequent steroidogenic enzyme expression. To further explore the contribution of naturally occurring LPS exposure on follicular steroid production and developing oocytes, bovine ovary pairs were collected from local abattoirs. Oocytes were aspirated from small follicles and matured in vitro to evaluate meiotic events related to nuclear maturation and spindle morphology. Small follicles from ovarian pairs were separated by the detectable LPS concentrations into high and low LPS groups. Oocytes matured from low LPS follicles demonstrated an increase in the percent of abnormal maturation events. Data indicate that induced or naturally occurring low doses of LPS can alter circulating and follicular estradiol concentrations impairing oocyte maturation. Perturbation to local ovarian signaling cascades from subclinical inflammatory disease may be an unappreciated factor altering fertility and leading to decreased cow retention.

Follicle Rescue From Prepubertal Ovaries After Recent Treatment With Cyclophosphamide—An Experimental Culture System Using Mice to Achieve Mature Oocytes for Fertility Preservation

Ovarian tissue cryopreservation is the only feasible method for fertility preservation in prepubertal girls that will undergo gonadotoxic chemotherapy. To date, the only clinical use of cryopreserved tissue is by a later tissue retransplantation to the patient. Clinical challenges in fertility preservation of very young patients with cancer include time constraints that do not allow to retrieve the tissue for cryopreservation before starting chemotherapy and the preclusion of future ovarian tissue transplantation due to the risk of reintroduction of malignant cells in patients with systemic diseases. To overcome these two challenges, we investigated using an experimental model the feasibility of retrieving secondary follicles from ovaries of prepubertal mice after cyclophosphamide (CPA) treatment in increasing doses of 50, 75, and 100 mg/kg. The follicles were thereafter cultured and matured in vitro. The main outcomes included the efficiency of the method in terms of obtained matured oocytes and the safety of these potentially fertility preservative procedures in terms of analyses of oocyte competence regarding normality of the spindle and chromosome configurations. Our findings demonstrated that it was feasible to isolate and culture secondary follicles and to obtain mature oocytes from prepubertal mice ovaries recently treated with CPA. The efficiency of this method was highly demonstrated in the 100 mg/kg CPA group, with near 90% follicle survival rate after 12 days’ culture, similarly to control. Around 80% of the follicles met the criteria to put into maturation, and more than 40% of them achieved metaphase II, with normal spindle and chromosome configurations observed. Suboptimal results were obtained in the 50 and 75 mg/kg CPA groups. These paradoxical findings towards CPA dose might probably reflect a more difficult selection of damaged growing follicles from ovaries recently treated with lower doses of CPA and a hampered ability to identify and discard those with reduced viability for the culture.

In vitro Maturation of Buffalo Oocytes and Expression of Selected Biomarker Candidate Genes

Background: Number of genes expressed during in vitro maturation (IVM) of which selected genes can be used as the potential biomarkers of oocyte competence. Hence, this study was planned to evaluate selected gene expression of GDF9, HAS2, SPRY, ARHGAP22, COL18A1 and GPC4 in IVM and immature cumulus oocyte complexes (COCs). Methods: The COCs were recovered from slaughter origin ovaries of buffaloes. Of which first three grade COCs were randomly allotted in immature (IMT; n=217) and IVM group (n=272). IVM of COCs was performed under drops of BO-maturation media in CO2 incubator at 39.0°C for 24 hours. The expression of genes was evaluated using qPCR and the relative expression of each gene was calculated using the ΔΔCt method with efficiency correction. The logarithmic transformation of fold change of each candidate genes in the IVM group was computed against the IMT group using the Ct values. Result: The expression obtained for IVM group of earlier reported up-regulated genes (GDF9, HAS2, SPRY1) was higher (upto 10xfold) compared to the IMT group. Relatively lower expression was observed for the rest of the three candidate genes (ARHGAP22, COL18A1, GPC4) in the bovine transcripts of oocyte which were earlier also reported as down regulated.