[Study on construction of c-Met specific CAR-T cells and its killing effect on non-small cell lung carcinoma].

Objective: To produce chimeric antigen receptor T cells (CAR-T) targeting human hepatocyte growth factor/c-Met (HGF/c-Met) protein and detect its cytotoxicity against non-small cell lung cancer (NSCLC) cells H1975 in vitro. Methods: The whole gene sequence of c-Met CAR containing c-Met single-chain fragment variable was synthesized and linked to lentiviral vector plasmid, plasmid electrophoresis was used to detect the correctness of target gene. HEK293 cells were transfected with plasmid and the concentrated solution of the virus particles was collected. c-Met CAR lentivirus was transfected into T cells to obtain second-generation c-Met CAR-T and the expression of CAR sequences was verified by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot, and the positive rate and cell subtypes of c-Met CAR-T cells were detected by flow cytometry. The positive expression of c-Met protein in NSCLC cell line H1975 was verified by flow cytometry, and the negative expression of c-Met protein in ovarian cancer cell line A2780 was selected as the control. The cytotoxicity of c-Met CAR-T to H1975 was detected by lactate dehydrogenase (LDH) cytotoxicity assay at 1∶1, 5∶1, 10∶1 and 20∶1 of effector: target cell ratio (E∶T). Enzyme-linked immunosorbent assay (ELISA) was used to detect the release of cytokines such as TNF-α, IL-2 and IFN-γ from c-Met CAR-T co-cultured with H1975. Results: The size of band was consistent with that of designed c-Met CAR, suggesting that the c-Met CAR plasmid was successfully constructed. The results of gene sequencing were consistent with the original design sequence and lentivirus was successfully constructed. CAR molecules expression in T cells infected with lentivirus was detected by western blot and RT-qPCR, which showed c-Met CAR-T were successfully constructed. Flow cytometry results showed that the infection efficiency of c-Met CAR in T cells was over 38.4%, and the proportion of CD8(+) T cells was increased after lentivirus infection. The NSCLC cell line H1975 highly expressed c-Met while ovarian cancer cell line A2780 negatively expressed c-Met. LDH cytotoxicity assay indicated that the killing efficiency was positively correlated with the E∶T, and higher than that of control group, and the killing rate reached 51.12% when the E∶T was 20∶1. ELISA results showed that c-Met CAR-T cells released more IL-2, TNF-α and IFN-γ in target cell stimulation, but there was no statistical difference between c-Met CAR-T and T cells in the non-target group. Conclusions: Human NSCLC cell H1975 expresses high level of c-Met which can be used as a target for immunotherapy. CAR-T cells targeting c-Met have been successfully produced and have high killing effect on c-Met positive NSCLC cells in vitro.

Diabetes self-management education exposure and glycated haemoglobin levels among Marshallese participants in a randomized controlled study.

AIMS: A randomized control trial (RCT) of diabetes self-management education (DSME), undertaken by a community-based participatory research (CBPR) partnership between the University of Arkansas for Medical Sciences (UAMS) and the Marshallese community in Arkansas. The RCT examined the effect of hours of intervention exposure, with the hypothesis that increased exposure is one reason the Adapted-Family DSME was found to be more effective than the Standard DSME. METHODS: Some 221 Marshallese with type 2 diabetes were randomized to an Adapted-Family DSME group (in-home setting) (n = 110) or a Standard DMSE group (community setting) (n = 111). The Adapted-Family DSME included 10 h of education that covered the core self-care elements recommended by the American Diabetes Association (ADA) and American Association of Diabetes Educators' (AADE) recommendations. The Standard DSME included 10 h of intervention with all ADA and AADE core elements. RESULTS: The number of hours of intervention exposure in the Adapted-Family DSME arm (mean = 8.0; median = 10.0) was significantly higher than the number of hours of intervention received in the Standard DSME arm (mean = 1.5; median = 0.0). As hypothesized, higher exposure was associated with a significant reduction in HbA1c in a model including only study arm and exposure (P = 0.01), and in a model including study arm, exposure, and all demographic variables (P = 0.046). CONCLUSIONS: This finding is consistent with previous reviews that showed increased exposure to DSME produced improved glycaemic control and ≥ 10 h of DSME produces clinically meaningful reductions in HbA1c .

Production of transgenic calves expressing an shRNA targeting myostatin.

Myostatin (MSTN) is a well-known negative regulator of muscle growth. Animals that possess mutations within this gene display an enhanced muscling phenotype, a desirable agricultural trait. Increased neonatal morbidity is common, however, resulting from complications arising from the birth of offspring with increased fetal muscle mass. The objective of the current research was to generate an attenuated MSTN-null phenotype in a large-animal model using RNA interference to enhance muscle development without the detrimental consequences of an inactivating mutation. To this end, we identified a series of short interfering RNAs that demonstrated effective suppression of MSTN mRNA and protein levels. To produce transgenic offspring capable of stable MSTN suppression in vivo, a recombinant lentiviral vector expressing a short hairpin RNA (shRNA) targeting MSTN for silencing was introduced into bovine fetal fibroblasts. These cells were used as nucleus donors for somatic cell nuclear transfer (SCNT). Twenty blastocysts were transferred into seven recipient cows resulting in five pregnancies. One transgenic calf developed to term, but died following delivery by Caesarean-section. As an alternative strategy, microinjection of recombinant lentiviral particles into the perivitelline space of in vitro-produced bovine zygotes was utilized to produce 40 transgenic blastocysts that were transferred into 14 recipient cows, resulting in 7 pregnancies. Five transgenic calves were produced, of which three expressed the transgene. This is the first report of transgenic livestock produced by direct injection of a recombinant lentivirus, and expressing transgenes encoding shRNAs targeting an endogenous gene (myostatin) for silencing.

Evaluation of the effects of sire and dam calving group on age at first calving in Brahman heifers.

This study evaluated the possible effects of sire and dam calving groups on age at first calving in Brahman heifers. A total of 570 heifers born between the years 2004 and 2017 were exposed as yearlings to fertile bulls through time of pregnancy determination. A calving group was determined by calculating the mean (993 d) and standard deviation (187 d) of heifer age at first calving. Heifers considered to calve early (≤899 d; calving group = 1) or late (≥1087 d; calving group = 3) were at least half a standard deviation (94 d) away from the mean. All other heifers were considered to have an intermediate age at first calving (900-1086 d; calving group = 2). Of the 570 heifers, only heifers from a dam (n = 182) with a known age at first calving and from a sire (n = 35) with 5 or more daughters were kept to determine the effect of dam calving group and the effect of sire calving group on age at first calving and calving group in daughters, resulting in a total of 284 heifers available for analysis. Variables included were dam and sire calving groups of the heifer, heifer age at first calving, heifer calving group, heifer season of birth, and heifer year of birth. Data were analyzed using the GLM procedures of SAS and proportions were tested using Chi-square. Sire calving group did affect (P < 0.01) age at first calving and calving group in heifers, but dam calving group did not affect (P > 0.10) daughter age at first calving or calving group. Analysis of dam calving group and sire calving group effects identified a year of birth effect (P < 0.01) on daughter age at first calving and calving group, whereas there was no significant season of birth effect. The proportion of daughters calving early for sire calving groups differed significantly from the expected proportion (P < 0.01), whereas the proportion of daughters calving early for dam calving groups did not differ. An effort to produce a greater proportion of Brahman heifers capable of calving early will not be effective from the dam side but may be effective from the sire side.

Maternal nutrient restriction alters endocrine pancreas development in fetal heifers.

Maternal nutrient restriction during pregnancy alters fetal programming, which modifies the growth and health of the offspring in postnatal life. In cattle, nutrient restriction during pregnancy can be a result of environmental or economic factors, but little is known about how it alters the physiology of the fetus and affects future reproductive or growth efficiency. This study used female monozygotic twins, produced through in vitro fertilization and embryo splitting, to determine the effect of moderate maternal nutrient restriction on fetal development. Recipient Angus cross heifers pregnant with one twin were fed a diet meeting 100% National Research Council (NRC) total energy requirements (n = 4; control), whereas recipient heifers pregnant with the second twin were fed at 70% of NRC total energy requirements (n = 4; restricted) from gestational day (GD) 158 to GD 265 in Calan gate feeders. Recipient heifers were killed at GD 265. Change in maternal metabolic body weight was greater from zero in restricted heifers than controls (P < 0.05); restricted heifers lost weight during the nutrient restriction period. There was no difference in last rib back fat or rib eye area between groups (P > 0.10). There was no difference in fetal weight, uterine weight, or total placentome weight between groups (P > 0.10). The pancreas weight was reduced in restricted fetuses compared with control fetuses (P < 0.01), but there were no other differences in fetal organ weights (P > 0.10). Plasma insulin concentrations were reduced in restricted fetuses compared with controls (P < 0.01), but there was no effect of maternal diet on plasma glucose or glucagon concentrations in the fetus (P > 0.10). Histological analyses of the fetal pancreas revealed no differences in endocrine cell number or localization. Results indicate that a modest late gestation nutritional restriction impairs development of the fetal pancreas in the cow. Additional research will be needed to determine if these developmental changes lead to altered glucose and insulin homeostasis in the adult.

The Integrative Migraine Pain Alleviation through Chiropractic Therapy (IMPACT) trial: Study rationale, design and intervention validation.

INTRODUCTION: Approximately 15% of the US population experiences migraine, with women afflicted three times as often as men. While medications are often used as first-line treatments, up to 50% of people with migraine pursue complementary and integrative medicine. One promising non-pharmacological approach for migraine is chiropractic care, due to the co-occurrence of migraine disease and musculoskeletal tension and pain. To date, no large-scale trials have evaluated the impact of a comprehensive model of chiropractic care on migraine. METHODS: The Integrative Migraine Pain Alleviation through Chiropractic Therapy (IMPACT) study is a two-arm pilot pragmatic randomized clinical trial evaluating a multimodal chiropractic care intervention plus enhanced usual care (UC) vs. enhanced UC alone for adult women with episodic migraine. A total of 60 women aged 20-55 who meet criteria for episodic migraine will be randomly assigned to an evidence-informed, musculoskeletal focused multimodal chiropractic care (10 sessions over 14 weeks) plus enhanced UC vs. enhanced UC alone. Enhanced UC includes conventional care, migraine education materials, and biweekly check-in phone calls. Study specific aims include: 1) Determine safety and feasibility of the study design; 2) Provide preliminary data on the effectiveness of chiropractic care on migraine frequency, severity, duration and medication use; and 3) Provide preliminary estimates of the effects of chiropractic care on disability, health-related quality of life, and psychosocial well-being. DISCUSSION: Findings will be used to inform the design of a full-scale trial evaluating chiropractic care for women with episodic migraines.

Spinal manipulative therapy versus a low force mimic maneuver for women with primary dysmenorrhea: a randomized, observer-blinded, clinical trial.

Non-drug therapies for women with primary dysmenorrhea are primarily based on anecdotal evidence and small-scale clinical studies. This randomized, observer-blinded, clinical trial evaluated the efficacy of spinal manipulative therapy (SMT) in the treatment of women with primary dysmenorrhea. Women were recruited from the Chicago metropolitan area and evaluated for inclusion through four screening levels. One hundred thirty eight women, ages 18-45, with primary dysmenorrhea diagnosed by participating gynecologists, were randomly assigned to either SMT or a low-force mimic (LFM) maneuver. No treatment occurred at menstrual cycle 1. Treatment for both groups took place on day 1 of cycles 2, 3 and 4, and prophylactic treatment of three visits took place during the 7 days before cycles 3 and 4. Main outcome measures were the Visual Analog Scale (VAS) and plasma concentration of the prostaglandin F2alpha metabolite, 15-keto-13,14-dihydro-prostaglandin F2alpha (KDPGF2alpha), measured 15 min before treatment and 60 min after treatment on day 1 of four consecutive menstrual cycles. The Moos' Menstrual Distress Questionnaire (MDQ) was also administered after treatment on day 1 of each cycle. At cycle 2, the post-treatment VAS scores decreased for both groups, with no statistically significant difference in pre- to post-treatment scores between the two groups (P = 0.44). The changes in pre- to post-treatment KDPGF2alpha levels were not statistically different between the SMT and LFM groups (P = 0.15). No treatment effects were detected over the three cycles for VAS, KDPGF2alpha or MDQ (P = 0.65, P = 0.61 and P = 0.78, respectively). However, there were statistically significant linear time effects for VAS (P = 0.008), MDQ (P < 0.001), and borderline significance for KDPGF2alpha (P = 0.054); these decreases were not considered clinically meaningful. The LFM maneuver used in this study was designed to act as a 'placebo-like' control treatment in comparison with SMT. Although it is possible that the trial did not continue long enough for any placebo effect of the LFM to wash out, it seems more likely that this maneuver was indistinguishable from SMT. Therefore, the postulated superior benefit of high-velocity, short-lever, low-amplitude, high-force spinal manipulation to a low-force maneuver is not supported by the results of this study. 1999 International Association for the Study of Pain.

Epidemiology of alcohol use in a group of older American Indians.

PURPOSE: This study describes alcohol use in a group of older American Indians and the associations with demographic and health status characteristics, by gender. METHODS: Alcohol use was examined in a cross-sectional, population-based study of 161 American Indians, aged 45-76 years (a substudy of the Strong Heart Study). Alcohol use was measured by a questionnaire administered during a personal interview. Information about demographic characteristics and health status was ascertained from interviews and abstraction of medical records. RESULTS: A higher proportion of men than women had used alcohol heavily (71% vs. 28%). Men were more likely than women to drink currently (46% vs. 18%), to binge (26% vs. 5%), and to screen positive for alcoholism (77% vs. 43%). Among current drinkers, > 30% had diabetes, and the average score on the Short Michigan Alcoholism Screening Test (SMAST) was in the alcoholic range. Heavy drinking was associated with more symptoms of depression in women (P < 0.05) and fewer in men (P < 0.05). Alcoholism was positively associated with a history of heavy drinking in both men (P < 0.05) and women (P < 0.001). CONCLUSIONS: Alcohol use was common and varied by gender. Alcohol use also varied according to other sociodemographic and health status characteristics. Since many older American Indians with chronic illness are currently drinking, this age group may require enhanced alcohol control programs.

Blood pressure load--a better determinant of hypertension.

Noninvasive ambulatory blood pressure monitoring was used to evaluate the diagnosis of hypertension in 168 untreated patients with essential hypertension. On the basis of overall office blood pressure--the mean of 12 measurements, 2 in each of three positions (supine, sitting, and standing) on 2 consecutive days--133 patients were diagnosed as having hypertension (diastolic blood pressure of 90 mm Hg or higher) and 35 as having borderline hypertension (diastolic blood pressure of less than 90 mm Hg). The mean blood pressures for those with hypertension and borderline hypertension were 149/99 and 135/87 mm Hg, respectively. The mean ambulatory diastolic blood pressure was 90 mm Hg or higher in 123 patients during awake hours and in 91 patients during 24 hours. The diastolic blood pressure loads (percentage of ambulatory diastolic blood pressures more than 90 mm Hg) in patients with hypertension and borderline hypertension, respectively, were 69% and 43% during awake hours and 59% and 35% during 24 hours. The systolic blood pressure loads (percentage of systolic readings more than 140 mm Hg) during awake and 24 hours were 56% and 48%, respectively, in patients with established hypertension and 31% and 26%, respectively, in those with borderline hypertension. Thus, ambulatory blood pressure monitoring and blood pressure load provide useful information for diagnosing hypertension.

Chiropractic care for patients aged 55 years and older: report from a practice-based research program.

OBJECTIVE: To characterize patients aged 55 years and older and features of chiropractic care provided to them. DESIGN: Observational, practice-based research study. SETTING: Chiropractic offices in the United States and Canada, 1997-1998. PARTICIPANTS: Chiropractors in 96 practices in 32 states and two Canadian provinces collected data on 805 eligible patients aged 55 years and older during a 12-week study period. MEASUREMENTS: In addition to questionnaires on practice characteristics, patient demographics, chief complaints, and health habits, two standardized instruments were administered: for general health status, the Medical Outcomes Study 12-Item Short-Form Health Survey (SF-12); and for disability related to chronic pain, the Pain Disability Index (PDI). RESULTS: Of 805 study patients, 60.1% were women and' 94.7% were white. Overweight patients comprised 38.6% and obese 20.6% (n = 656) of the total; 9.7% of patients were hypertensive (n = 590). Smoking was reported by 12.7% and 50.2% reported regular exercise. The Physical Component Summary scores of the SF-12 seemed somewhat lower than population norms, whereas the Mental Component Summary scores differed very little from norms. Chief complaints were predominantly pain-related (72.3%), most commonly back pain (32.9%). The PDI mean baseline score for chronic patients was 16.3 (scale, 0-70), and 40.6% of study patients reported using at least one pain medication (prescription or nonprescription) more than three times per week. More than half of complaints (54.9%) had onsets more than 6 weeks before the baseline visit. For 66.6% of subjects, a chiropractor was the only provider for their current complaint. In addition to manipulation, most common features of care were recommendations on exercise (41.0%), heat or cold applications (40.8%), and food supplements (24.5%). At 4 weeks, 19.6% were discharged, 58.8% continued treatment, and 20.1% had discontinued care (self-discharged). For these three groups, those with higher PDI mean baseline scores showed more change at 4 weeks. For patients who were discharged by the doctor, the proportion of reported pain medication use decreased 7.3% from baseline to 4 weeks, increased for patients who discontinued care, and remained about the same for those continuing care. CONCLUSIONS: Further investigation of the PDI and a decrease in pain medication use as outcome measures seems warranted. The descriptive information in this study may assist providers of care to older adults to better understand their patients' use of chiropractic care.

Antihypertensive efficacy of lisinopril. Ambulatory blood pressure monitoring.

Noninvasive automatic ambulatory blood pressure monitoring during 24 hours in eight patients with moderate hypertension was used to determine the blood pressure response to lisinopril, an angiotensin-converting enzyme inhibitor. Office, 24-hour ambulatory, awake ambulatory, and sleep ambulatory diastolic blood pressures were decreased from 108 +/- 3, 98 +/- 8, 101 +/- 7, and 87 +/- 14 mm Hg, respectively, at baseline to 83 +/- 4 (P less than or equal to 0.0001), 82 +/- 7 (P less than 0.0001), 84 +/- 7 (P less than 0.0001), and 73 +/- 9 mm Hg (P less than 0.005), respectively, after 20 weeks of lisinopril treatment (dose range, 40 to 80 mg once daily). The diastolic blood pressure loads (percentages of ambulatory diastolic blood pressures more than 90 mm Hg) during 24 hours and during awake hours were 74% +/- 19% and 83% +/- 15%, respectively, at baseline and 24% +/- 19% (P less than 0.0001) and 29% +/- 21% (P less than 0.0001), respectively, during treatment. Heart rate was not altered by lisinopril. In conclusion, lisinopril is an effective antihypertensive agent for the treatment of moderate hypertension, and ambulatory blood pressures and diastolic blood pressure loads are useful for evaluating therapy for hypertension.

Systemic diseases associated with nonarteritic anterior ischemic optic neuropathy.

PURPOSE: We investigated, in nonarteritic anterior ischemic optic neuropathy, the prevalence of various systemic diseases before or at its onset and the incidence of subsequent morbidity and mortality. METHODS: We investigated prospectively the presence of systemic diseases before or at the onset of nonarteritic anterior ischemic optic neuropathy in 406 patients. The information was obtained by complete medical history and physical examination. The prevalence rates of systemic diseases in young, middle-aged, and elderly groups were compared with those in the general population. We also analyzed the influence of systemic diseases on subsequent morbidity and mortality. RESULTS: Compared with the prevalences reported in the general population, our patients in each of the three age groups showed a significantly higher prevalence of arterial hypertension (P < or = .02), diabetes mellitus (P < .01), and gastrointestinal ulcer (P < or = .02). Also, middle-aged and elderly patients showed a significantly higher prevalence of ischemic heart disease (P < .01) and thyroid disease (P < .01). Middle-aged patients had significantly higher rates of chronic obstructive pulmonary disease and cerebrovascular disease (P < or = .01). After onset of anterior ischemic optic neuropathy, patients with both arterial hypertension and diabetes mellitus had a significantly higher incidence of cerebrovascular disease (P < .01). CONCLUSIONS: Nonarteritic anterior ischemic optic neuropathy is a multifactorial disease in which some systemic diseases may act as predisposing factors and others as precipitating factors. Patients with anterior ischemic optic neuropathy show no significant increase in mortality, but those with both arterial hypertension and diabetes mellitus have significantly (P < .01) increased incidence of cerebrovascular disease.

The relationship between membrane status and fertility of boar spermatozoa after flow cytometric sorting in the presence or absence of seminal plasma.

The motility, viability (percent live), capacitation status and in vitro fertility of boar spermatozoa were examined, after staining with Hoechst 33,342 and flow cytometric sorting in the absence or presence of seminal plasma. Viability was higher in unstained controls and when seminal plasma was present in the medium used to collect spermatozoa from the cell sorter than when seminal plasma was absent or in the staining extender only, but motility was highest when seminal plasma was included in the extender only, compared with the controls and other treatments. The proportions of capacitated spermatozoa were increased by sorting, but were lower when seminal plasma was present, rather than absent, from the staining extender and the collection medium. Compared with unstained controls, extension and staining without sorting only increased the proportion of capacitated spermatozoa after washing in preparation for in vitro fertilization. The percentages of polyspermic, penetrated and cleaved oocytes were lower when inseminated with unsorted (stained) than control (unstained) spermatozoa, regardless of the presence or absence of seminal plasma. These parameters were higher for sorted than for control spermatozoa in the absence of seminal plasma, but in its presence penetration and cleavage were substantially lower. The proportions of capacitated spermatozoa were lower when seminal plasma was present in the collection medium only than in the staining extender or when it was absent altogether, but the former treatment substantially reduced the proportions of polyspermic, penetrated and cleaved oocytes, and the proportion of blastocysts. These findings indicate that sperm capacitation associated with flow cytometric sorting can be reduced by the inclusion of seminal plasma in the collection medium, but this treatment reduces the ability of spermatozoa to fertilize oocytes in vitro under these conditions.

Factors affecting use of chiropractic services in seven midwestern states of the United State.

Although chiropractic is used by approximately 10 percent of the U.S. population, predictors of its use have not been definitively described. Previous studies have suggested that chiropractic users differ from nonusers in a number of sociodemographic characteristics, but their findings are inconsistent, perhaps because of differences in populations sampled and dates of data collection, most of which are prior to 1990. Regional studies have been conducted in rural areas based on the premise that rural residents are more likely than non-rural residents to use chiropractice; however, this premise has not been definitively documented. The purpose of this study was to provide clarification of these sociodemographic predictors of chiropractic use in Illinois, Iowa, Minnesota, Missouri, Nebraska, South Dakota and Wisconsin by characterizing chiropractic users and nonusers in terms of sociodemographics, including rural or non-rural residence, and presence of low back pain. Data from 1,511 respondents to a 1994 population-based survey, conducted by the University of Iowa Social Science Institute, were analyzed. Unconditional logistic regression was used to derive odds ratios and 95 percent confidence intervals for univariate and multiple regression models. Overall, 15.1 percent of respondents had used chiropractic within the last year, most often for low back pain (57 percent). Chiropractic use was less likely in African Americans, Hispanics and Asians than whites, less likely by non-rural than rural residents, and less likely in Catholics than Protestants in states other than Iowa and South Dakota. Overall, 42.7 percent of workers with low back pain reported using chiropractic, and use increased with age but remained significantly related to race, rural or non-rural residence, state of residence and religious preference. Race, rural or non-rural residence, state of residence and religious preference, independently of low back pain, affect use of chiropractice in seven Midwestern states.

Use of complementary healthcare practices among chiropractors in the United States: a survey.

CONTEXT: Although chiropractic is the most commonly used complementary healthcare practice, chiropractors have not been surveyed in depth about their attitudes toward and practice of complementary and alternative therapies apart from spinal manipulation. OBJECTIVES: To examine attitudes among US chiropractors on the role of their profession in complementary healthcare and to gather data on the types of complementary healthcare practices chiropractors use. DESIGN: Descriptive, cross-sectional study using a mail-in survey. SETTING: United States. PARTICIPANTS: Random sample of US chiropractors stratified by zip code region. RESULTS: A total of 563 surveys were completed, for a response rate of 30%. Respondents were almost equally divided on their responses to the question "Do you feel that chiropractic should at the present time be termed 'complementary'?" Although many respondents (68%) believed that chiropractic was viewed as a therapeutic modality, more (82%) believed that it should be viewed as a complete system. The therapies most commonly used by respondents in their practice were acupressure (72%), massage (72%), mineral supplements (63%), and herbs (56%). CONCLUSION: The disparity between the established view of chiropractic as synonymous with spinal manipulation and the profession's view of chiropractic as a complete system indicates a need for better interprofessional communication.

In vitro production of pig embryos: comparisons of culture media and boars.

The utilization of in vitro produced pig embryos for commercial production or research is dependent upon the development of improved methodology. Our objective was to establish a consistent in vitro embryo production (IVP) system and subsequently utilize the procedures to evaluate culture system components and boar effects. To summarize the IVP system, 403 inseminated oocytes from a total of 2243 were analyzed across 17 replicates for maturation and fertilization efficiency, while 1838 zygotes were cultured in 26 replicates for developmental data. Penetration, cleavage and blastocyst development rates were determined at 18, 44 and either 144 or 168 h post insemination, respectively. Monospermic penetration averaged 31.8+/-7.3% while polyspermy was 30.8+/-17.2%. Cleavage rate was 44.9+/-16.1%, with 21.8+/-7.5% of fertilized oocytes and 51.9+/-15.9% of cleaved embryos developing to blastocysts. For culture medium comparison, fertilized oocytes were cultured in either BECM-6, BECM-7, NCSU-23 or NCSU-23aa and supplemented on Day 5 post insemination (pi) with 10% FCS. These treatments resulted in 4.0, 4.9, 19.8 and 13.6% (+/-3.2%) blastocysts by Day 7 pi, with an average cell number of 44.4+/-9.0, 65.1+/-8.2, 61.3+/-4.5 and 64.4+/-4.8, respectively. These IVP procedures consistently produced zygotes from semen of several different boars, capable of forming blastocysts in vitro. Comparison of developmental rates among the boars indicated that this system is variable among boars but not strictly boar-dependent. Culture media comparisons suggest that NCSU-23 yielded a higher percentage of blastocysts than the other media in this IVP system.

New commercial opportunities for advanced reproductive technologies in horses, wildlife, and companion animals.

As advanced reproductive technologies become more efficient and repeatable in livestock and laboratory species, new opportunities will evolve to apply these techniques to alternative and non-traditional species. This will result in new markets requiring unique business models that address issues of animal welfare and consumer acceptance on a much different level than the livestock sector. Advanced reproductive technologies and genetic engineering will be applied to each species in innovative ways to provide breeders more alternatives for the preservation and propagation of elite animals in each sector. The commercialization of advanced reproductive techniques in these niche markets should be considered a useful tool for conservation of genetic material from endangered or unique animals as well as production of biomedical models of human disease.

Verification of flow cytometorically-sorted X- and Y-bearing porcine spermatozoa and reanalysis of spermatozoa for DNA content using the fluorescence in situ hybridization (FISH) technique.

Flow cytometric sperm sorting based on X and Y sperm DNA difference has been established as the only effective method for sexing the spermatozoa of mammals. The standard method for verifying the purity of sorted X and Y spermatozoa has been to reanalyze sorted sperm aliquots. We verified the purity of flow-sorted porcine X and Y spermatozoa and accuracy of DNA reanalysis by fluorescence in situ hybridization (FISH) using chromosome Y and 1 DNA probe. Eight ejaculates from 4 boars were sorted according to the Beltsville Sperm Sexing method. Porcine chromosome Y- and chromosome 1-specific DNA probes were used on sorted sperm populations in combination with FISH. Aliquots of the sorted sperm samples were reanalyzed for DNA content by flow cytometry. The purity of the sorted X-bearing spermatozoa was 87.4% for FISH and 87.0% for flow cytometric reanalysis; purity for the sorted Y-bearing spermatozoa was 85.9% for FISH and 84.8% for flow cytometric reanalysis. A total of 4,424 X sperm cells and 4,256 Y sperm cells was examined by FISH across the 8 ejaculates. For flow cytometry, 5,000 sorted X spermatozoa and 5,000 Y spermatozoa were reanalyzed for DNA content for each ejaculate. These results confirm the high purity of flow sorted porcine X and Y sperm cells and the validity of reanalysis of DNA in determining the proportions of X- and Y-sorted spermatozoa from viewing thousands of individual sperm chromosomes directly using FISH.

Cloning to reproduce desired genotypes.

Cloned sheep, cattle, goats, pigs and mice have now been produced using somatic cells for nuclear transplantation. Animal cloning is still very inefficient with on average less than 10% of the cloned embryos transferred resulting in a live offspring. However successful cloning of a variety of different species and by a number of different laboratory groups has generated tremendous interest in reproducing desired genotypes. Some of these specific genotypes represent animal cell lines that have been genetically modified. In other cases there is a significant demand for cloning animals characterized by their inherent genetic value, for example prize livestock, household pets and rare or endangered species. A number of different variables may influence the ability to reproduce a specific genotype by cloning. These include species, source of recipient ova, cell type of nuclei donor, treatment of donor cells prior to nuclear transfer, and the techniques employed for nuclear transfer. At present, there is no solid evidence that suggests cloning will be limited to only a few specific animals, and in fact, most data collected to date suggests cloning will be applicable to a wide variety of different animals. The ability to reproduce any desired genotype by cloning will ultimately depend on the amount of time and resources invested in research.

Efficiency of weight gain of serial slaughtered bulls of a five-breed diallel.

Weight and feed consumption of 197 bulls were recorded monthly in a serial slaughter experiment continuing over a period of 24 mo. The bulls were produced in a modified five-breed diallel of the Angus, Brahman, Hereford, Holstein and Jersey breeds. Our objective was to estimate efficiency of feed conversion for weight gain for each breed and cross. The model included average weight gain per day (ADG) as a dependent variable; independent variables included breed-type, season, month within season, average feed intake per day (ADI), initial weight, weight to the .75 power, breed x ADI and season x ADI effects. The sum of the partial regression coefficients of ADG on ADI and on breed x ADI adjusted for season, month within season, initial weight, weight to the .75 power and season x ADI was interpreted to be an estimate of intrinsic or net efficiency of ADG. There were no significant differences among the various breed-types in intrinsic efficiency of ADG. Average heterosis for intrinsic efficiency of feed conversion was not significant. None of the contrasts among breed-types was significant for intrinsic efficiency of ADG (British vs dairy, 1.1 +/- 12.3 g/d; Brahman-dairy crosses vs British-dairy crosses, 32.7 +/- 12.9 g/d; British-Brahman crosses vs British-dairy crosses, 13.9 +/- 12.4 g/d; British-Brahman crosses vs straightbred British 3.2 +/- 14.8 g/d).