Identification of measures predictive of age of puberty onset in gilts.

A potential indicator of female lifetime productivity in swine is age of puberty, when a gilt achieves her first behavioral estrus. Follicular activity, as determined by tertiary follicle development, in prepubertal gilts begins during postnatal day (PND) 75 to 115. The central hypothesis of this study is that gilts demonstrating tertiary follicle development earlier in life, assessed using vulva size as a proxy, achieve puberty earlier in life compared with counterparts of a similar age and weight that lack tertiary follicle development. The objectives of this project were to identify a developmental time point when variation in ovarian development exists and to determine whether a relationship between the age prepubertal ovarian development and the age at onset of puberty exists. To accomplish this, 155 gilts of similar age (± 2 d) were weighed and vulva size measured on PND 75, 85, 95, 105, and 115. Vulva measures, including vulva width (VW), vulva length (VL), and vulva area (VA), were utilized as developmental proxies for follicular activity. At each time point, gilts (n = 10) were sacrificed and ovarian follicular activity recorded. In a subset of gilts (n = 105), estrus detection was conducted daily on PND days 126 to 200. Mean VA on PND 75, 85, 95, 105, and 115 was 596 ± 206, 683 ± 190, 864 ± 212, 1014 ± 228, and 1265 ± 252 mm2, respectively. Of the gilts demonstrating behavioral estrus, 28 were within PND 140 to 160, 36 between PND 161 to 180, 15 between PND 181 to 200, and 26 did not demonstrate estrus behavior within 200 d of age. All gilts euthanized at PND 75 lacked follicular activity as defined by having a minimum of 2 antral follicles per ovary, whereas 60%, 80%, 90%, and 100% demonstrated follicular activity on PND 85, 95, 105, and 115, respectively. Body weight at PND 75 and VW at PND 115 were correlated to age at first estrus (P < 0.05). Of the gilts whose VA was less than 1 SD from the mean on PND 95 (i.e., <652 mm2), 31% and 50% demonstrated their first behavioral estrus by PND 180 and 200, respectively. However, of gilts whose VA was within or greater than 1 SD of the mean (i.e., ≥652 mm2), 66% and 79% exhibited estrus prior to PND 180 and 200, respectively. These data support utilization of VA changes between 95 and 115 d of age as a useful tool to identify replacement gilts prior to puberty for inclusion into the sow herd.

Pharmacologic treatment with CPI-613 and PS48 decreases mitochondrial membrane potential and increases quantity of autolysosomes in porcine fibroblasts.

A metabolic phenomenon known as the Warburg effect has been characterized in certain cancerous cells, embryonic stem cells, and other rapidly proliferative cell types. Previously, our attempts to induce a Warburg-like state pharmaceutically via CPI-613 and PS48 treatment did augment metabolite production and gene expression; however, this treatment demonstrated a Reverse Warburg effect phenotype observed in cancer-associated stroma. In the current study, we inquired whether the mitochondria were affected by the aforementioned pharmaceutical treatment as observed in cancerous stromal fibroblasts. While the pharmaceutical agents decreased mitochondrial membrane potential in porcine fetal fibroblasts, the number and size of mitochondria were similar, as was the overall cell size. Moreover, the fibroblasts that were treated with CPI-613 and PS48 for a week had increased numbers of large autolysosome vesicles. This coincided with increased intensity of LysoTracker staining in treated cells as observed by flow cytometry. Treated fibroblasts thus may utilize changes in metabolism and autophagy to mitigate the damage of treatment with pharmaceutical agents. These findings shed light on how these pharmaceutical agents interact and how treated cells augment metabolism to sustain viability.

Late gestation supplementation of corn dried distiller's grains plus solubles to beef cows fed a low-quality forage: III. effects on mammary gland blood flow, colostrum and milk production, and calf body weights.

Objectives were to investigate the effects of supplementation with corn dried distiller's grains plus solubles (DDGS) to late gestating beef cows on arterial blood flow to the mammary glands during late gestation and early lactation; colostrum and milk production; dystocia and immunity; and calf BW. Cows were fed a control (CON; n = 15; 5.1% CP; 36.2% ADF) diet consisting of 90% corn stover and 10% corn silage on a dry basis offered ad libitum or CON diet with supplementation of DDGS (0.30% of BW; SUP n = 12). Mammary gland blood flow was assessed on day 245 of gestation. At parturition, maternal and calving parameters were assessed; colostrum and jugular blood was sampled; and dams were weighed. Mammary gland blood flow and milk production was measured on day 44 of lactation. Calves were weighed fortnightly for 8 wk and at weaning. Colostrum production tended to be greater in SUP dams than in CON dams (837 vs. 614 ± 95 g, P = 0.10). Calves of SUP dams were heavier at birth and 24 h (0 h, 43.2 vs. 39.8 ± 1.0 kg, P = 0.02; 24 h, 44.0 vs. 40.4 ± 1.1 kg, P = 0.02). At birth and 24 h, blood pCO2 was greater in calves born to SUP dams (6.82 vs. 6.00 ± 0.41 kPa, P = 0.04). Serum IgG did not differ (P = 0.21) at 24 h. Ipsilateral mammary gland blood flow of SUP cows was greater than CON cows (2.76 vs. 1.76 ± 0.30 L/min; P = 0.03); however, when summed with contralateral, total blood flow was similar (P = 0.33). Hemodynamic measures on day 44 of lactation were similar (P ≥ 0.32). Milk production tended to be increased (13.5 vs. 10.2 ± 1.2 kg/d, P = 0.07) in SUP vs. CON cows. Despite similar BW through 56 d, calves from SUP cows were heavier (P = 0.04) at weaning (309.7 vs. 292.0 ± 6.0 kg). In conclusion, we accept our hypothesis that DDGS supplementation during gestation influenced mammary blood flow, milk production and calf weights. These findings implicate maternal nutrition's leverage on both nutrient and passive immunity delivery to the calf early in life as well as potential advantages on long-term performance.

Improvement of in vitro and early in utero porcine clone development after somatic donor cells are cultured under hypoxia.

Genetically engineered pigs serve as excellent biomedical and agricultural models. To date, the most reliable way to generate genetically engineered pigs is via somatic cell nuclear transfer (SCNT), however, the efficiency of cloning in pigs is low (1-3%). Somatic cells such as fibroblasts frequently used in nuclear transfer utilize the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation for efficient energy production. The metabolism of somatic cells contrasts with cells within the early embryo, which predominately use glycolysis. We hypothesized that fibroblast cells could become blastomere-like if mitochondrial oxidative phosphorylation was inhibited by hypoxia and that this would result in improved in vitro embryonic development after SCNT. In a previous study, we demonstrated that fibroblasts cultured under hypoxic conditions had changes in gene expression consistent with increased glycolytic/gluconeogenic metabolism. The goal of this pilot study was to determine if subsequent in vitro embryo development is impacted by cloning porcine embryonic fibroblasts cultured in hypoxia. Here we demonstrate that in vitro measures such as early cleavage, blastocyst development, and blastocyst cell number are improved (4.4%, 5.5%, and 17.6 cells, respectively) when donor cells are cultured in hypoxia before nuclear transfer. Survival probability was increased in clones from hypoxic cultured donors compared to controls (8.5 vs. 4.0 ± 0.2). These results suggest that the clones from donor cells cultured in hypoxia are more developmentally competent and this may be due to improved nuclear reprogramming during somatic cell nuclear transfer.

Porcine Fetal-Derived Fibroblasts Alter Gene Expression and Mitochondria to Compensate for Hypoxic Stress During Culture.

The Warburg effect is characterized by decreased mitochondrial oxidative phosphorylation and increased glycolytic flux in adequate oxygen. The preimplantation embryo has been described to have characteristics of the Warburg effect, including similar changes in gene expression and mitochondria, which are more rudimentary in appearance. We hypothesized hypoxia would facilitate anaerobic glycolysis in fibroblasts thereby promoting gene expression and media metabolite production reflecting the Warburg effect hallmarks in early embryos. Additionally, we speculated that hypoxia would induce a rudimentary small mitochondrial phenotype observed in several cell types evidenced to demonstrate the Warburg effect. While many have examined the role hypoxia plays in pathological conditions, few studies have investigated changes in primary cells which could be used in somatic cell nuclear transfer. We found that cells grown in 1.25% O2 had normal cell viability and more, but smaller mitochondria. Several hypoxia-inducible genes were identified, including seven genes for glycolytic enzymes. In conditioned media from hypoxic cells, the quantities of gluconolactone, cytosine, and uric acid were decreased indicating higher consumption than control cells. These results indicate that fibroblasts alter gene expression and mitochondria to compensate for hypoxic stress and maintain viability. Furthermore, the metabolic changes observed, making them more similar to preimplantation embryos, could be facilitating nuclear reprogramming making these cells more amendable to future use in somatic cell nuclear transfer.

Pharmacologic treatment of donor cells induced to have a Warburg effect-like metabolism does not alter embryonic development in vitro or survival during early gestation when used in somatic cell nuclear transfer in pigs.

Somatic cell nuclear transfer is a valuable technique for the generation of genetically engineered animals, however, the efficiency of cloning in mammalian species is low (1-3%). Differentiated somatic cells commonly used in nuclear transfer utilize the tricarboxylic acid cycle and cellular respiration for energy production. Comparatively the metabolism of somatic cells contrasts that of the cells within the early embryos which predominately use glycolysis. Early embryos (prior to implantation) are evidenced to exhibit characteristics of a Warburg Effect (WE)-like metabolism. We hypothesized that pharmacologically driven fibroblast cells can become more blastomere-like and result in improved in vitro embryonic development after SCNT. The goals were to determine if subsequent in vitro embryo development is impacted by (1) cloning pharmacologically treated donor cells pushed to have a WE-like metabolism or (2) culturing non-treated donor clones with pharmaceuticals used to push a WE-like metabolism. Additionally, we investigated early gestational survival of the donor-treated clone embryos. Here we demonstrate that in vitro development of clones is not hindered by pharmacologically treating either the donor cells or the embryos themselves with CPI, PS48, or the combination of these drugs. Furthermore, these experiments demonstrate that early embryos (or at least in vitro produced embryos) have a low proportion of mitochondria which have high membrane potential and treatment with these pharmaceuticals does not further alter the mitochondrial function in early embryos. Lastly, we show that survival in early gestation was not different between clones from pharmacologically induced WE-like donor cells and controls.

Pharmacologic Reprogramming Designed to Induce a Warburg Effect in Porcine Fetal Fibroblasts Alters Gene Expression and Quantities of Metabolites from Conditioned Media Without Increased Cell Proliferation.

The Warburg effect is a metabolic phenomenon characterized by increased glycolytic activity, decreased mitochondrial oxidative phosphorylation, and the production of lactate. This metabolic phenotype is characterized in rapidly proliferative cell types such as cancerous cells and embryonic stem cells. We hypothesized that a Warburg-like metabolism could be achieved in other cell types by treatment with pharmacological agents, which might, in turn, facilitate nuclear reprogramming. The aim of this study was to treat fibroblasts with CPI-613 and PS48 to induce a Warburg-like metabolic state. We demonstrate that treatment with both drugs altered the expression of 69 genes and changed the level of 21 metabolites in conditioned culture media, but did not induce higher proliferation compared to the control treatment. These results support a role for the reverse Warburg effect, whereby cancer cells induce cancer-associated fibroblast cells in the surrounding stroma to exhibit the metabolically characterized Warburg effect. Cancer-associated fibroblasts then produce and secrete metabolites such as pyruvate to supply the cancerous cells, thereby supporting tumor growth and metastasis. While anticipating an increase in the production of lactate and increased cellular proliferation, both hallmarks of the Warburg effect, we instead observed increased secretion of pyruvate without changes in proliferation.

Some assembly required: evolutionary and systems perspectives on the mammalian reproductive system.

In this review, we discuss the way that insights from evolutionary theory and systems biology shed light on form and function in mammalian reproductive systems. In the first part of the review, we contrast the rapid evolution seen in some reproductive genes with the generally conservative nature of development. We discuss directional selection and coevolution as potential drivers of rapid evolution in sperm and egg proteins. Such rapid change is very different from the highly conservative nature of later embryo development. However, it is not unique, as some regions of the sex chromosomes also show elevated rates of evolutionary change. To explain these contradictory trends, we argue that it is not reproductive functions per se that induce rapid evolution. Rather, it is the fact that biotic interactions, such as speciation events and sexual conflict, have no evolutionary endpoint and hence can drive continuous evolutionary changes. Returning to the question of sex chromosome evolution, we discuss the way that recent advances in evolutionary genomics and systems biology and, in particular, the development of a theory of gene balance provide a better understanding of the evolutionary patterns seen on these chromosomes. We end the review with a discussion of a surprising and incompletely understood phenomenon observed in early embryos: namely the Warburg effect, whereby glucose is fermented to lactate and alanine rather than respired to carbon dioxide. We argue that evolutionary insights, from both yeasts and tumor cells, help to explain the Warburg effect, and that new metabolic modeling approaches are useful in assessing the potential sources of the effect.