A novel culture medium with reduced nutrient concentrations supports the development and viability of mouse embryos

Further refinement of culture media is needed to improve the quality of embryos generated in vitro. Previous results from our laboratory demonstrated that uptake of nutrients by the embryo is significantly less than what is supplied in traditional culture media. Our objective was to determine the impact of reduced nutrient concentrations in culture medium on mouse embryo development, metabolism, and quality as a possible platform for next generation medium formulation. Concentrations of carbohydrates, amino acids, and vitamins could be reduced by 50% with no detrimental effects, but blastocyst development was impaired at 25% of standard nutrient provision (reduced nutrient medium; RN). Addition of pyruvate and L-lactate (+PL) to RN at 50% of standard concentrations restored blastocyst development, hatching, and cell number. In addition, blastocysts produced in RN\u2009+PL contained more ICM cells and ATP than blastocysts cultured in our control (100% nutrient) medium; however, metabolic activity was altered. Similarly, embryos produced in the RN medium with elevated (50% control) concentrations of pyruvate and lactate in the first step medium and EAA and Glu in the second step medium were competent to implant and develop into fetuses at a similar rate as embryos produced in the control medium. This novel approach to culture medium formulation could help define the optimal nutrient requirements of embryos in culture and provide a means of shifting metabolic activity towards the utilization of specific metabolic pathways that may be beneficial for embryo viability

Detection of noncyling cows by heatmount decectors and ultrasound before treatment with progesterone

Our objective was to determine accuracy of identifying anovulatory lactating dairy cows before the application of a timed AI protocol [with or without progesterone supplementation via a controlled internal drug release (CIDR) insert and 2 different timings of AI] by using heatmount detectors and a single ovarian ultrasound examination. At 6 Midwest locations, 1,072 cows were enrolled in a Presynch protocol (2 injections of prostaglandin F2α(PGF2α) 14 days apart) with the second injection administered 14 days before initiating the Ovsynch protocol (injection of gonadotropin releasing hormone (GnRH) 7 days before and 48 hours after PGF2αinjection, with timed AI at 0 or 24 hours after the second GnRH injection). Heatmount detectors were applied to cows at the time of the first Presynch injection, assessed 14 days later at the second Presynch injection and again at initiation of the Ovsynch protocol, and ovaries were examined for presence of a visible corpus luteum (CL) by ultrasound before initiation of treatment. Treatments were assigned to cows based on presence or absence of a visible CL: 1) anovulatory (no CL + CIDR insert for 7 d); 2) anovulatory (no CL + no CIDR); and 3) cycling (CL present). Further, every other cow in the 3 treatments was assigned to be inseminated concurrent with the second GnRH injection of Ovsynch (0 hour) or 24 hours later. Pregnancy was diagnosed at 33 and 61 days after the second GnRH injection. Heatmount detectors and a single ultrasound examination both underestimated proportions of cows classified as anovulatory or having no prior luteal activity compared with those classifications determined by concentrations of progesterone in blood serum. Overall accuracy of heatmount detectors and ultrasound was 71 and 84%, respectively. Application of progesterone to cows without a CL at the time of the first injection of GnRH reduced incidence of ovulation but improved pregnancy rates at day 33 or 61 compared with nontreated cows without a CL at the onset of the Ovsynch protocol. Pregnancy rates and pregnancy survival did not differ for cows having a CL before treatment compared with those not having a CL but treated with progesterone. Pregnancy rates were 1.5-fold greater for cows ovulating in response to the first GnRH injection. Timing of AI at 0 or 24 hours after the second GnRH injection did not alter pregnancy rates, but cows having prior luteal activity before treatment had improved pregnancy rates compared with anovulatory cows. We conclude that identifying anovulatory cows by ultrasound was more accurate than by heatmount detectors. Subsequent treatment of potential anovulatory cows with progesterone failed to improve fertility but had benefit for cows with prior estrous cycles at the onset of the timed AI (TAI) protocol, regardless of luteal status before the final luteolytic injection of PGF2α.; Dairy Day, 2007, Kansas State University, Manhattan, KS, 2007; Dairy Research, 2007 is known as Dairy Day, 200

Alterations in oocyte mitochondrial number and function are related to spindle defects and occur with maternal aging in mice and humans

The objective of this work was to determine the role of mitochondria in the loss of oocyte quality with maternal aging. Our results show that mitochondrial DNA (mtDNA) copy number and function are reduced in eggs from aged mice after both in vivo and in vitro maturation. Higher incidences of spindle abnormalities were observed in old eggs. However, no correlation with egg ATP content was found. In vitro matured eggs from aged mice did not have a normal cortical distribution of active mitochondria and were subject to increased oxidative stress due to higher levels of reactive oxygen species and lower expression of glutamate-cysteine ligase, catalytic subunit (Gclc). Supplementation of antioxidants during in vitro maturation of old eggs mitigated this affect, resulting in increased mtDNA copy number and mitochondrial function, a mitochondria distribution pattern similar to young eggs, and improved chromosomal alignment. Eggs from women of advanced maternal age (AMA) had lower mitochondrial function than eggs from young women, although both age groups displayed a cortical distribution pattern of active mitochondria. In contrast to the mouse, human eggs from AMA women had higher mtDNA copy number than eggs from young women following in vitro maturation. In summary, oocytes of older females are more susceptible to perturbations in mitochondrial number and function, which are associated with increased spindle abnormalities and oxidative stress during in vitro maturation. These results demonstrate that oocyte mitochondria play a critical role in age-related infertility