Extracellular vesicles in low volume uterine lavage and serum: novel and promising biomarker for endometritis in Arabian mares.

BACKGROUND: Extracellular vesicles (EVs) are a promising biomarker and play a vital role in cell-cell communication. This study aimed (I) to identify and characterize EVs from low volume uterine lavage (LVL) and serum in mares with endometritis, compared to healthy controls and (II) to measure serum levels of interleukin 6 (IL-6), and prostaglandins (PGF2α and PGE2). Mares were divided into 30 sub-fertile (endometritis) and 20 fertile (controls). Serum and LVL was collected for EV isolation, and determination of serum levels of inflammatory mediators. Characterization and visualization of EVs were done by electron microscopy, dynamic light scattering and flow cytometry. RESULTS: Serial ultracentrifugation of LVL and use of a commercial kit for serum were strategies for EVs isolation. Mares with endometritis released higher amounts of larger size EVs. The EVs from mares with endometritis differentially expressed CD9 and CD63, compared to controls. Mares suffering from endometritis evoked higher levels of inflammatory mediators. CONCLUSIONS: Thus, EVs could be used for a better understanding the regulatory mechanisms associated with developing endometritis in mares.

Assisted reproduction with gametes and embryos: what research is needed and fundable?

Principles for selecting future research projects include interests of investigators, fundability, potential applications, ethical considerations, being able to formulate testable hypotheses and choosing the best models, including selection of the most appropriate species. The following 10 areas of assisted reproduction seem especially appropriate for further research: efficacious capacitation of bovine spermatozoa in vitro; improved in vitro bovine oocyte maturation; decreasing variability and increasing efficacy of bovine superovulation; improved fertility of sexed semen; improving equine IVF; improving cryopreservation of rooster spermatozoa; understanding differences between males in success of sperm cryopreservation and reasons for success in competitive fertilisation; mechanisms of reprogramming somatic cell nuclei after nuclear transfer; regulation of differentiation of ovarian primordial follicles; and means by which spermatozoa maintain fertility during storage in the epididymis. Issues are species specific for several of these topics, in most cases because the biology is species specific.

Beef cattle in the year 2050.

In 2050, beef likely will be produced much as occurs currently, as (1) a by-product of dairying-cull cows and calves not needed as replacements; (2) intensively managed cattle in environments rich in feed resources; or (3) extensively managed cattle grazing land unsuitable for tillage, with calves often moving to richer feed environments. Genetic progress will continue to depend on information such as weaning weights, but in addition, genetic, epigenetic, and phenotypic information will be obtained from blood, hair, semen, and/or biopsies of embryos.Most cattle will be genetically modified for efficient growth, desirable carcass traits, and management traits such as disease resistance. Some strains of cattle will have Y-chromosome-dependent terminal cross traits; sexed semen thus will automatically result in males with terminal cross characteristics and females with maternally desirable traits. In most cases, mother cows will have shorter gestations and smaller frame sizes than currently to decrease nutrient requirements for maintenance. The cow herd may disappear with some intensively managed systems; with sexed semen, each female can replace herself with a female calf and then be fattened for slaughter. The flexibility of being a ruminant will continue to be exploited by using a variety of feedstuffs, some of which are otherwise of little value.

Pregnancy risk in beef and dairy cows after supplementing semen with transforming growth factor beta-1 at the time of artificial insemination.

Our objective was to determine if the addition of a concentrated human recombinant transforming growth factor beta-1 (TGF) to bovine semen at the time of AI would result in increased risk of pregnancy in beef and dairy cows. Suckled beef cows (n = 1,132) in 11 herds across 2 states and lactating dairy cows (n = 2,208) in one organic-certified herd were enrolled. Beef cows received fixed-time AI (FTAI) following a 7 d CO-Synch + controlled internal drug release estrous synchronization protocol. Dairy cows were inseminated following observation of natural estrus expression. Cows received either no treatment as a control (CON) or 10 ng of TGF in 10 µl added through the cut-end of a thawed straw of semen immediately prior to AI. At the time of FTAI of beef cows, the mean ± SD age was 5.0 ± 2.4 yr, BCS was 5.3 ± 0.7, and days postpartum was 78.2 ± 15.5 d. The overall pregnancy risk in beef cows was 55.2% to AI and 90.5% season-long. Pregnancy risk in beef cows was not affected (P = 0.27) by addition of TGF (53.1% vs. 58.1%). Further, there was no difference (P = 0.88) for season-long pregnancy risk in beef cows that received TGF (91.2% vs. 91.5%). At the time of insemination of dairy cows, the mean ± SD lactation was 3.0 ± 1.3 lactations, BCS was 2.9 ± 0.3, days in milk was 115.6 ± 56.6 d, and cows had received 2.4 ± 1.5 inseminations per cow. The overall pregnancy risk to AI in dairy cows was 23.1%. Pregnancy risk to AI for dairy cows was not affected (P = 0.32) by addition of TGF (22.0% vs. 23.8%). In conclusion, pregnancy risk to AI was not affected by addition of TGF to thawed semen immediately prior to AI in beef or dairy cows.

Factors affecting economics of using sexed semen in dairy cattle.

The use of sexed semen in the dairy industry has grown rapidly. However, high costs and low fertility have limited the use of this potentially valuable tool. This study used simulation to evaluate 160,000 combinations of key variables in 3 spheres of influence related to profit feasibility: (1) market (e.g., milk and calf prices), (2) dairy farm management (e.g., conception rates), and (3) technology (e.g., accuracy of sexing). These influential variables were used to determine the most favorable circumstances in which managers or technicians can effect change. Three distinct scenarios were created to model 3 initiatives that a producer might take with sexed semen: (1) using sexed semen on heifers, (2) using sexed semen on heifers and a fraction of the genetically superior cows, and (3) using sexed semen on heifers and a fraction of the genetically superior cows, and breeding all other cows with beef semen. Due to the large number of management, market, and technology combinations, a response surface and interpretive graphs were created to map the scope of influence for the key variables. Technology variables such as the added cost of sexed semen had relatively little effect on profitability, defined as net present value gain per cow, whereas management variables such as conception rate had a significant effect. Milk price had relatively little effect within each scenario, but was important across scenarios. Profitability was very sensitive to the price of dairy heifer calves, relative to beef and dairy bull calves. Scenarios 1 and 2 added about $50 to $75 per cow in net present value, which ranged from $0 to $200 and from $100 to $300, respectively. Scenario 3 usually was not profitable, primarily because fewer excess dairy replacement heifers were available for sale. Dairy heifer price proved to be the most influential variable, regardless of scenario.

Effects of components of semen extenders on the binding of stallion spermatozoa to bovine or equine zonae pellucidae.

The effects of semen extender components on the ability of stallion sperm to bind to the zona pellucida (ZP) and the suitability of using bovine ZP for a ZP-binding assay for stallion sperm were investigated in a series of experiments. In Experiment I, binding of stallion sperm to both bovine and equine ZP was significantly increased when a skim milk-based extender (EZM) was used. In Experiment II, a threefold increase in sperm binding to ZP was observed when sperm were diluted in EZM compared with diluents, which contained no milk (TALP, LAC, and EmCare). In Experiment III, centrifuging the sperm through Percoll did not increase sperm binding to the ZP but did remove any positive effect of EZM on sperm-ZP binding. In Experiment IV, exposure of either sperm or ZP to EZM before co-incubation did not increase sperm binding to ZP. In Experiment V, sperm diluted in TALP containing skim milk, EZM, or INRA96 bound more efficiently to the ZP than sperm diluted in TALP without milk proteins. In Experiment VI, sodium caseinate, native phosphocaseinate, and caseinoglycopeptide increased sperm binding to the ZP. In conclusion, diluents containing milk or milk proteins markedly enhanced the number of sperm bound to both equine and bovine ZP.

Sexing mammalian sperm - Where do we go from here?

The only commercially viable method of sexing mammalian sperm is to use a flow cytometer to measure sperm DNA content via fluorescence of the DNA-bound fluorophore Hoechst 33342, and then sort sperm into three populations, probably X, probably Y, and undetermined. Millions of insemination doses of sexed sperm are produced annually by this procedure. Although accuracy of sexing usually exceeds 90%, this procedure of sexing one sperm at a time has serious limitations, including cost, sort rates, and damage to sperm resulting in lowered fertility, but not abnormalities in offspring. Suggested areas for research include determining how sperm are damaged and where in the process of fertilization and embryonic development the infertility is manifest. Pre and post sorting procedures are done in approximately hourly batches, and these might be changed to continuous procedures. Numerous genetic, physical, and immunological procedures for sexing millions of sperm in parallel have been proposed, but none appears to be suitable for commercialization at this time due to issues of accuracy, repeatability, damage to sperm, and other problems. However, increasing numbers of reports are appearing concerning improvements in these procedures, and it appears inevitable that one or more of them eventually will prove to be efficacious. In developing such procedures, it is critical to monitor sexing accuracy regularly by rapid and inexpensive procedures such as fluorescence in situ hybridization, quantitative PCR, or sort reanalysis by flow cytometry. Furthermore, monitoring fertility of sexed sperm such as in vitro fertilization should be integral to the development process. Intellectual property issues could be substantive.

Translating Basic Research to Animal Agriculture.

Procedures to maintain viability of mammalian gametes and embryos in vitro, including cryopreservation, have been exceedingly valuable for my research over the past 55 years. Keeping sperm viable in vitro enables artificial insemination, which, when combined with selective breeding, often is the most effective approach to making rapid genetic change in a population. Superovulation and embryo transfer constitute a parallel approach for amplifying reproduction of female mammals. More recent developments include sexing of semen, in vitro fertilization, cloning by nuclear transfer, and genetic modification of germline cells, tools that are enabled by artificial insemination and/or embryo transfer for implementation. I have been fortunate in being able to contribute to the development of many of the above techniques, and to use them for research and applications for improving animal agriculture. Others have built on this work to circumvent human infertility, assist reproduction of companion animals, and rescue endangered species. It also has been a privilege to teach, mentor, and be mentored in this area. Resulting worldwide friendships have enriched me personally and professionally.

Evaluation of biological and economic efficiency of the All Heifer, No Cow beef production system using a system dynamics model based on 6 yr of demonstration herd data.

Alternative management strategies with no cows and all heifers may improve biological and economic efficiency of beef production. The All Heifer, No Cow (AHNC) beef production system involves insemination of nulliparous heifers with female sex-selected semen (FSS) to produce primarily female calves that are early weaned at 3 mo of age. Dams are finished on a high concentrate diet and harvested before 30 mo of age. The objectives of this research were to: 1) build a dynamic model of an AHNC beef production system to quantify system biological and economic efficiency; 2) compare effects of utilizing FSS vs. conventional semen on biological and economic efficiency; 3) evaluate what-if scenarios to determine the effects on biological and economic efficiency of changing variables ±5%, 10%, 15%, and 20% from initial observed values; and 4) evaluate the effects on biological and economic efficiency of changing variables ±10% from initial observed values. A model was built over a 21-yr horizon using Stella Architect. Biological parameter values in the model were based on the 6 yr of data collected from the management of an AHNC demonstration herd. In the model animal, total digestible nutrients (TDN) intake, hot carcass weight (HCW), and age at harvest were randomized. Feed, animal, and carcass prices included in the model were based on 10 yr of historical U.S. price data. Key response variables were biological and economic efficiency (mean ± SD). Biological efficiency was defined as the ratio of output (kilograms of HCW produced) to input (lifetime kilograms of feed TDN consumed), and economic efficiency was measured using a benefit-cost ratio (BCR) and unit variable cost (UVC). Over 40 simulation runs, the predicted mean biological efficiency was 0.0714 ± 0.0008. Economic efficiency was 0.95 ± 0.02 and US $445.41 ± 0.06 for BCR and UVC, respectively. Biological and economic efficiency was improved in the conventional semen scenario; biological efficiency was 0.0738 ± 0.0008, and BCR and UVC were 0.99 ± 0.04 and US $407.24 ± 0.006, respectively. Under this parameterization and market conditions, the AHNC beef production system failed to achieve profitability under any scenario that was evaluated. However, this review did not account for the potential increased genetic benefit from a decreased generation interval and the reduction in feed energy in comparison to a conventional cow/calf system.

Effects of prolonging the interval from progestin removal to prostaglandin F2α injection from 16 to 17 d in a long-term estrus synchronization protocol in beef heifers.

To determine effects of delaying the injection of prostaglandin F2α (PGF) and fixed-time artificial insemination (TAI) in the 14-d CIDR-PG protocol, 1,049 Angus heifers at six locations were enrolled in a completely randomized design. Within location heifers were randomly assigned to one of two treatment groups: 1) PG16 (n = 518), heifers received a controlled internal drug release (CIDR) insert on d 0 for 14 d, a 25-mg injection of PGF 16 d after CIDR removal (d 30), and a 100-µg injection of gonadotropin-releasing hormone concurrent with TAI 66 ± 2 h later; or 2) PG17 (n = 531), heifers were treated the same as PG16, however, PGF was administered 17 d after CIDR removal (d 31), and heifers were TAI 66 ± 2 h later. Estrus detection patches were applied to a subset (n = 482) of heifers at the time of PGF administration and were examined for activation at TAI. Dominant follicle diameter was determined via transrectal ultrasonography at PGF administration and TAI in a subset of heifers (n = 116). Transrectal ultrasonography was performed to determine pregnancy rates to TAI (PR/AI) between 30 and 45 d after TAI. Estrus expression prior to TAI differed by treatment where PG17 heifers had greater (P < 0.01) expression of estrus than PG16 heifers (57.8 ± 6.1% vs. 43.4 ± 6.1%, respectively). Nevertheless, dominant follicle diameters at PGF and at TAI were similar (P ≥ 0.59) between PG16 and PG17 heifers. In addition, PR/AI did not differ (P = 0.29) between PG16 and PG17 treatments (50.5 ± 3.2% vs. 45.7 ± 3.1%, respectively). Results of this experiment indicate that delaying the injection of PGF and TAI in the 14-d CIDR-PG protocol increased estrus expression prior to TAI yet did not improve fertility in beef heifers.

Evaluation of performance and carcass traits for a five-cohort All Heifer, No Cow beef production system demonstration herd.

The All Heifer, No Cow (AHNC) beef production system is an alternative to conventional cow/calf production that involves insemination of nulliparous heifers with sexed semen to produce female calves that are early weaned at 3 mo of age. Dams are finished on a high-concentrate diet and harvested before reaching 30 mo of age. Objectives of this research were to document reproductive, feedyard, calf, and carcass performance of an AHNC herd; evaluate effects of carcass maturity on carcass quality; and determine if performance of initial cohorts (i.e., cohorts 1 and 2) differed from sustaining cohorts (i.e., cohorts 3-5). A total of 272 heifers were enrolled in the AHNC system via five annual cohorts. The system was initiated with 51 yearling, Angus-based heifers, and a replicate set (n = 56) was started 12 mo after. Heifers in cohorts 3 (n = 53), 4 (n = 56), and 5 (n = 56) were primarily offspring of prior cohorts (i.e., cohort 3 heifers born to cohort 1 females), but some were purchased to maintain inventory. Angus replacement heifers were purchased in cohorts 3 (n = 26), 4 (n = 26), and 5 (n = 28). Mean (±standard deviation) pregnancy rate at 30 d after fixed-time artificial insemination (AI) with sexed semen was 50.8% ± 9.4%, and 140-d pregnancy rate was 93.0% ± 1.5%. With AHNC, 61.0% ± 6.5% of females replaced themselves with a heifer. During finishing, average daily gain (ADG) was 1.9 ± 0.4 kg • d-1 and dry matter intake (DMI) was 14.9 ± 1.9 kg • d-1. Hot carcass weight (HCW) was 367 ± 35 kg. The USDA grading system classified 20.5% of all carcasses (n = 220) as C maturity (A00 = 100, B00 = 200, etc.), 62.4% ± 29.1% of carcasses as USDA Choice. USDA yield grade (YG) was 2.6 ± 0.7. Based on cohorts 1 and 2, there were no differences (P = 0.96) in Warner-Bratzler shear force values between A and B maturity vs. C maturity carcasses. Across all cohorts, there were no differences in USDA YG, marbling score (MA), and lean maturity between A and B maturity vs. C maturity carcasses; there were differences in age (P < 0.001), bone maturity (P < 0.001), and overall maturity (P <0.001). A comparison of initial vs. sustaining cohorts showed that initial cohorts had lower (P < 0.001) DMI, heavier (P < 0.001) HCW, and more advanced (P < 0.05) bone maturity. However, there were no differences for 30- and 140-d pregnancy rates, ADG, USDA YG, and MA between initial and sustaining cohorts. The AHNC beef production system can effectively produce female calves and quality carcasses for harvest.

The involvement of proline-rich 15 in early conceptus development in sheep.

The ruminant conceptus undergoes a period of elongation that is required for maternal recognition of pregnancy, prior to attaching to the endometrium. The purpose of these studies was to investigate the role of proline-rich 15 (PRR15) in the sheep conceptus by examining mRNA expression, protein localization, and the effect of PRR15 mRNA degradation. Conceptuses were collected on Days 11, 13, 15, 16, 17, 21, and 30 after mating. Quantitative RT-PCR showed expression of PRR15 mRNA corresponded with the process of trophoblast elongation, with peak expression occurring on Days 15 and 16. A recombinant ovine PRR15 was generated and used to create polyclonal antibodies against PRR15. Immunohistochemistry of a Day 15 conceptus indicated that PRR15 was localized predominantly in the nucleus of the trophectoderm and extraembryonic primitive endoderm. To test whether PRR15 was required during early conceptus development, RNA interference was employed. Blastocysts collected on Day 8 after mating were infected with a lentivirus expressing a short-hairpin RNA (shRNA) that targeted PRR15 mRNA for degradation, an shRNA containing a three-nucleotide mismatch to PRR15 mRNA, or a lentivirus expressing no shRNA. After infection, blastocysts were transferred into recipient ewes and collected back on Day 15 of gestation. Although the majority of the control and mismatched shRNA-treated conceptuses elongated and survived to Day 15, none of the embryos treated with the lentivirus expressing shRNA against PRR15 mRNA elongated, and most died. In conclusion, expression of PRR15 mRNA occurred during a narrow window of conceptus development, and degradation of PRR15 mRNA led to conceptus demise or abnormal development.

Experimental design, power and sample size for animal reproduction experiments.

The present paper concerns statistical issues in the design of animal reproduction experiments, with emphasis on the problems of sample size determination and power calculations. We include examples and non-technical discussions aimed at helping researchers avoid serious errors that may invalidate or seriously impair the validity of conclusions from experiments. Screen shots from interactive power calculation programs and basic SAS power calculation programs are presented to aid in understanding statistical power and computing power in some common experimental situations. Practical issues that are common to most statistical design problems are briefly discussed. These include one-sided hypothesis tests, power level criteria, equality of within-group variances, transformations of response variables to achieve variance equality, optimal specification of treatment group sizes, 'post hoc' power analysis and arguments for the increased use of confidence intervals in place of hypothesis tests.

Dietary factors affecting iron status of children residing in rural West Virginia.

This cross-sectional study was designed to determine the prevalence of iron deficiency among a group of infants (6 to 11.9 months) and toddlers (12 to 24 months) and to examine the relationship between dietary intake and iron status. Participants were recruited from WIC clinics in counties where the prevalence of anemia was high (>10%). Twenty-four hour recalls were used to determine dietary intake. Blood was analyzed for iron studies. Dietary factors were examined for their association with iron status using logistic regression analysis. No infants were iron deficient, but 12/39 (31%) toddlers were found to be iron deficient. Ferritin was significantly higher in infants compared to toddlers (44.2 microg/L v. 19.2 microg/L, p<0.001). Milk and calcium intakes were inversely associated with iron status. Each additional serving of meat increased the odds of normal iron status by about 30%. Meat intake may help to prevent iron deficiency during the transition to table foods.

Prostaglandin F2α 7 d prior to initiation of the 7-d CO-synch + CIDR protocol failed to enhance estrus response and pregnancy rates in beef heifers.

To determine the effects of administration of 25 mg of PGF2α 7 d prior to the initiation of the 7-d CO-Synch + controlled internal drug release (CIDR) fixed-time AI (TAI) protocol, 985 Bos taurus beef heifers were enrolled in a completely randomized design at 9 locations from April to July of 2016. Within location, all heifers were randomly assigned to 1 of 2 treatments: 1) CONTROL (n = 496); 100 µg injection of GnRH and a CIDR insert for 7 d [day 7], administration of 25 mg of PGF2α at CIDR removal [day 0], followed by a second injection of GnRH and TAI 54 ± 2 h later; or 2) PRESYNCH (n = 489); same as CONTROL but heifers received an additional injection of 25 mg of PGF2α 7 d prior [day 14] to CIDR insertion. Estrous detection patches were applied to all heifers on day 14 and were evaluated for estrual activity on day 7. Similarly, estrus alert patches were placed on all heifers on day 0 and evaluated for estrual activity at the time of TAI. Pregnancy was diagnosed via transrectal ultrasonography between 35 and 55 d after TAI. The percentage of heifers exhibiting estrus between days 14 and 7 was greater (P < 0.001) for the PRESYNCH (70.1 ± 2.4%) than the CONTROL (41.1 ± 2.3%) treatment, whereas the percentage of heifers exhibiting estrus between day 0 and TAI was greater (P < 0.001) for the CONTROL (55.6 ± 2.4%) than the PRESYNCH (39.7 ± 2.5%) treatment. Estrus response rates differed (P < 0.001) among locations. Pregnancy rates to TAI differed (P = 0.023) among locations; however, they did not differ (P = 0.739) between CONTROL and PRESYNCH treatments (45.4 ± 2.5 vs. 43.2 ± 2.5%, respectively). Final breeding season pregnancy rates did not differ (P = 0.811) between treatments. Therefore, an injection of PGF2α 7 d prior to initiation of the 7-d CO-Synch + CIDR protocol failed to improve pregnancy rates to TAI in replacement beef heifers.

Aspiration of oocytes from transitional, cycling, and pregnant mares.

The aim of this study was to compare the efficacy of three approaches for recovering equine oocytes via transvaginal ultrasound-guided follicular aspiration. Fourteen mares were used as oocyte donors during the spring transition period and physiologic breeding season, and 11 mares were bred for use as oocyte donors during early gestation. In all mares, large (>20 mm) and small (10-20 mm) follicles were aspirated in eight rounds every 10-11 days. In each of the four rounds during the transition period, half the mares received 12.5 mg eFSH once daily for 4 days prior to aspiration. For each of the four rounds during the cycling season, half the mares received 12.5 mg eFSH twice daily for 3 days prior to aspiration. Pregnant mares were aspirated on days 25, 40 and 55 of gestation and received no eFSH. There were more large (>20 mm) follicles in cycling controls (2.25+/-0.27) and cycling FSH-treated (2.64+/-0.27) mares than in transitional FSH-treated mares (1.18+/-0.27). The number of oocytes recovered from small (10-20 mm) follicles varied by mare (P<0.05) and averaged 1.08+/-0.22 per aspiration for transitional mares and 1.23+/-0.22 per aspiration for cycling mares (P>0.1). The number of oocytes per aspiration from large follicles was greater in cycling FSH-treated mares (0.46+/-0.09) than in transitional control mares (0.11+/-0.09). In pregnant mares, more large follicles were present at day 25 than at any other time, and the number of oocytes per aspiration from large follicles was greater at day 25 (0.73+/-0.16) than at day 55 (0.04+/-0.18). When compared across all seasons and treatments, the day 25 pregnant mares yielded the greatest number of oocytes per aspiration (2.91+/-0.66 per mare).

Metabolic regulation of in-vitro-produced bovine embryos. I. Effects of metabolic regulators at different glucose concentrations with embryos produced by semen from different bulls.

The toxic and/or beneficial effects of four metabolic regulators on embryo development were evaluated. In-vitro-produced compact morulae were cultured for 3 days in a chemically defined medium + bovine serum albumin (BSA; CDM-2) plus regulators (4991 total embryos). Phenazine ethosulfate (PES), phloretin (PL), pyrroline-5-carboxylate (P5C), and sodium azide (NaN3) were evaluated at four doses each in factorial combinations with four concentrations of glucose: 0, 0.5, 2, and 8 mm. Phenazine ethosulfate at 0.9 microm resulted in poorer development than lower or no PES. Phloretin was, in general, detrimental for embryo development, but most markedly at the highest dose (270 microm). Pyrroline-5-carboxylate had little effect on post-compaction embryos at the doses studied, 9 to 81 microm. Sodium azide at the concentrations used (3, 9, and 27 microm) had little effect on embryo development compared with controls. Concentrations of glucose had little effect on development of embryos. A fifth metabolic regulator, 2,4-dinitrophenol (DNP), was studied at various doses at pre-morula or morula-blastocyst stages cultured in 2 mm glucose. Embryos (2189 total) cultured in 90 microm DNP developed more slowly and were darker than embryos cultured at lower doses. Embryos cultured in 30 microm DNP had a higher blastocyst rate (48.3%) than controls (34.9%). In the last experiment using G1.2/G2.2 media, DNP (30 microm) resulted in a marked decrease in embryonic development when embryos were exposed at the zygote to 8- to 16-cell stages but had little effect when morulae were exposed for 2 days. The dose-response information for these metabolic regulators is crucial for designing future experiments.

Metabolic regulation of in vitro-produced bovine embryos. II. Effects of phenazine ethosulfate, sodium azide and 2,4-dinitrophenol during post-compaction development on glucose metabolism and lipid accumulation.

The objective was to compare effects of three metabolic regulators on development of post-compaction bovine embryos. In-vitro-produced 8- to 16-cell embryos were allocated to treatments for 72 h in G2.2 medium as follows: 0.3 microm phenazine ethosulfate (PES); 27 microm sodium azide (NaN3); 30 microm 2,4-dinitrophenol (DNP); and control, no regulator. Treatments responded similarly for blastocyst rates and embryo quality responses (P > 0.1). The PES treatment resulted in higher glucose metabolism than the NaN3 treatment (18.5 v. 14.5 pmol per embryo per h, P < 0.05), and both did not differ from DNP or the control. The PES treatment tended to result in more flux of glucose through the pentose phosphate pathway (PPP) than the control (50.5 v. 21.5%, P < 0.11). The NaN3 treatment caused more glucose uptake than the PES treatment (38.9 v. 13.1 pmol per embryo per h, P < 0.01), but neither differed from the control or DNP treatment (P > 0.1). Glycolysis for the PES treatment was 187%, which was higher than any of the other groups (88-94%; P < 0.01). There were fewer medium + large lipid granules in the cytoplasm of PES-treated embryos than any other group, including the in vitro control (P < 0.01). However, in vivo control embryos had still fewer large and medium-sized lipid granules (P < 0.01) than the PES treatment. Developmental competence to Day 14 after embryo transfer was similar among treatments. The PES treatment increased glucose metabolism, tended to increase the PPP flux of glucose and clearly reduced accumulation of lipids in embryos produced in the chemically defined media used. Use of PES in culture media may be a promising approach to improving in vitro production of embryos.

Effects of ergot alkaloids on food preference and satiety in rabbits, as assessed with gene-knockout endophytes in perennial ryegrass (Lolium perenne).

Neotyphodium species are fungal endophytes best known for their protection of grass hosts and production of bioactive metabolites including ergot alkaloids. Perennial ryegrass-Neotyphodium sp. Lp1 symbiota that have altered ergot alkaloid profiles (resulting from knockouts in two different endophyte genes) were fed, along with controls, to rabbits to test the effects of ergot alkaloids on food preference and satiety. Interestingly, rabbits dramatically preferred plants that were endophyte-infected but free of ergot alkaloids over endophyte-free plants (P = 0.01). Accumulation of ergot alkaloids of the clavine class counteracted the added appeal of endophyte-infected plants. In satiety tests, consumption of ergovaline (the ultimate ergot pathway product in wild-type endophyte), but not of several other ergot alkaloids, during an initial meal had a negative effect on subsequent rabbit chow consumption (P < 0.05). The data indicate that clavines were sufficient to reduce the appeal of endophyte-infected grasses, whereas only ergovaline reduced appetite.

Modifying oocytes and embryos to improve their cryopreservation.

Numerous studies indicate that in vitro-produced bovine embryos do not survive cryopreservation as well as those produced in vivo. Furthermore, embryos cultured in vitro in the absence of blood serum are more cryotolerant than embryos cultured in media containing serum. Although in vivo-produced embryos are more cryotolerant, there appear to be breed differences. Most if not all of these observations are correlated with cytoplasmic lipid content of embryos; more and larger lipid droplets are associated with reduced cryotolerance. This review concerns strategies for modifying oocytes and embryos to increase cryosurvival. Reduction of cytoplasmic lipid content of embryos with phenazine ethosulfate (PES), a compound that oxidizes NADPH, even improved cryotolerance of bovine embryos cultured in the absence of serum. Whether cytoplasmic lipid content per se or associated changes in lipid composition of cell membranes are responsible for differences in cryotolerance is unknown. Increasing cholesterol content of membranes of sperm and oocytes via cholesterol-loaded cyclodextrin also appears to improve cryotolerance. While lipids have been emphasized and appear to be important, non-lipid aspects of cell composition also likely affect cryotolerance, and might be modified to improve cryotolerance. Additional research on mechanisms of variation in cryotolerance will be applicable to circumvent cryo-intolerance attributable to variation associated with the individual animal, breed, species, cell type, and factors such as nutrition and season of the year.