Racial and Ethnic Disparity in 4Ms among Older Adults Among Telehealth Users as Primary Care.

Telehealth has been widely accepted as an alternative to in-person primary care. This study examines whether the quality of primary care delivered via telehealth is equitable for older adults across racial and ethnic boundaries in provider-shortage urban settings. The study analyzed documentation of the 4Ms components (What Matters, Mobility, Medication, and Mentation) in relation to self-reported racial and ethnic backgrounds of 254 Medicare Advantage enrollees who used telehealth as their primary care modality in Southern Nevada from July 2021 through June 2022. Results revealed that Asian/Hawaiian/Pacific Islanders had significantly less documentation in What Matters (OR = 0.39, 95%, p = .04) and Blacks had significantly less documentation in Mobility (OR = 0.35, p < .001) compared to their White counterparts. The Hispanic ethnic group had less documentation in What Matters (OR = 0.18, p < .001) compared to non-Hispanic ethnic groups. Our study reveals equipping the geriatrics workforce merely with the 4Ms framework may not be sufficient in mitigating unconscious biases healthcare providers exhibit in the telehealth primary care setting in a provider shortage area, and, by extrapolation, in other care settings across the spectra, whether they be in-person or virtual.

Impact of Advance Care Planning on the Hospitalization-Associated Utilization and Cost of Patients with Alzheimer's Disease-Related Disorders Receiving Primary Care via Telehealth in a Provider Shortage Area: A Quantitative Pre-Study.

Telehealth has been adopted as an alternative to in-person primary care visits. With multiple participants able to join remotely, telehealth can facilitate the discussion and documentation of advance care planning (ACP) for those with Alzheimer's disease-related disorders (ADRDs). We measured hospitalization-associated utilization outcomes, instances of hospitalization and 90-day re-hospitalizations from payors' administrative databases and verified the data via electronic health records. We estimated the hospitalization-associated costs using the Nevada State Inpatient Dataset and compared the estimated costs between ADRD patients with and without ACP documentation in the year 2021. Compared to the ADRD patients without ACP documentation, those with ACP documentation were less likely to be hospitalized (mean: 0.74; standard deviation: 0.31; p < 0.01) and were less likely to be readmitted within 90 days of discharge (mean: 0.16; standard deviation: 0.06; p < 0.01). The hospitalization-associated cost estimate for ADRD patients with ACP documentation (mean: USD 149,722; standard deviation: USD 80,850) was less than that of the patients without ACP documentation (mean: USD 200,148; standard deviation: USD 82,061; p < 0.01). Further geriatrics workforce training is called for to enhance ACP competencies for ADRD patients, especially in areas with provider shortages where telehealth plays a comparatively more important role.

Post-Discharge Transitional Care Program and Patient Compliance With Follow-Up Activities.

We explore whether the frequency of post-hospital discharge transitional care calls affects patients' adherence to their discharge plans. We reviewed 1,000 call records of a post-discharge transitional care program run by a large U.S. tertiary care hospital in 2018-2019 and generated binary outcomes capturing patient self-reports of (1) scheduling or completing follow-up appointments, procedures, or prescription fills, (2) missing a scheduled event, and (3) reporting a new adverse clinical event. Our predictor variables captured callers' success in completing the first and each subsequent call with discharged patients. We ran linear probability models (LPM) for each binary outcome after controlling for sociodemographic and clinical characteristics. Results indicate successful completion of the first two calls was associated with the increased probability of scheduling or completing follow-up appointment (15.5% points, p < 0.01) and follow-up procedure (13.5% points, p < 0.01), and reporting new adverse event (5.9% points, p < 0.05). Our findings suggest that making the first phone call to patients, while crucial, may not be sufficient for effective care transition; making two to three phone calls seems to be more optimal, while further calls may have limited value.

Inpatient palliative care utilisation among patients with gallbladder cancer in the United States: A 10-year perspective.

OBJECTIVES: Gallbladder cancer (GBC) is a rare, poor-prognosis cancer with unique demographics, comorbidities and a paucity of research. This study investigated inpatient palliative care and its associations with demographics, comorbidities (e.g., obesity), length of stay and hospital charges in GBC in US hospitals (2007-2016). METHODS: Data were extracted from the National Inpatient Sample (NIS) database that contains deidentified clinical and nonclinical information for each hospitalisation. Inpatient palliative care utilisation was identified using the International Classification of Diseases (ICD-9 and ICD-10) codes (V66.7 and Z51.5). Generalised regression analysis was conducted with adjustment for variations in predictors. RESULTS: Of the 4921 reported GBC hospitalizations, only 10.3% encountered palliative care. Palliative care was associated with reduced hospital charges by $12,405 per hospitalisation (P < 0.0001) with no change in length of stay. Palliative care utilisation increased over time (P = 0.004). It was associated with age >80 years, with more severe disease, and in-hospital death (P < 0.0001). Obesity had a negative association with palliative care utilisation (P = 0.0029). DISCUSSION: Our novel study found that obese people were less likely to use palliative care services in GBC. Interventions are needed to increase palliative care consultation in GBC patients, particularly in obese patients.

The Outcome and Economic Viability of Embryo Production Using IVF and SOV Techniques in the Wagyu Breed of Cattle.

The Japanese black cattle breed (Wagyu) has an improved metabolism, which allows them to have a higher marbling score when compared with other cattle breeds. However, this may affect other aspects of the animal's physiology, including hormone secretion and their reproductive success, such as their response to synchronization protocols and embryo production. Therefore, the objectives of this study were to test a superovulation protocol (SOV) developed with low doses of FSH and to evaluate the outcome and economic viability of embryo production using the SOV and in vitro fertilization (IVF) approaches in the Wagyu cattle breed. For that, ten Wagyu cows were submitted to five SOVs over a period of 15 months using a standard protocol: CIDR + 3 mg estradiol benzoate (D0), 35 mg FSH (Folltropin®) a.m. and p.m. (D4), 35 mg Folltropin® a.m. and 20 mg p.m. (D5), 20 mg Folltropin® a.m. and 10 mg p.m. (D6), 10 mg Folltropin® and 0.5 mg cloprostenol, both a.m. and p.m., + CIDR removal (D7), 0.05 mg GnRH + insemination 12 and 24 h after (D8) and embryo collection + 0.5 mg of cloprostenol (D16). Thirty days after each SOV, a follicular aspiration was conducted to produce IVF embryos without any pre-synchronization using standard semen in the same group of animals. The average number of embryos produced was 7.63 ± 5.61 (SOV) and 4.52 ± 2.44 (IVF) (p = 0.303). There was no significant correlation between the number of embryos produced by the different techniques (SOV and IVF), indicating that cows that respond well to SOV did not respond well to IVF and vice versa (r = 0.379, p = 0.529). The total cost of each embryo produced by SOV was R$215.00 and R$410.00 for IVF. Therefore, cows that produce less than five embryos by SOV are not economically viable due their lack of response to FSH, and the use of IVF in those animals may be more effective.

Reducing the use of restrictive interventions by changing staff attitudes.

The use of restraint in health and social care services has had a high profile in recent years. The goal for these organisations is to reduce it, and use effective preventive interventions instead. Changing staff attitudes to restraint, as part of a comprehensive training programme, is important in reducing the use of restrictive interventions. This article describes an approach to implementing training programmes, and applies aspects of this to the challenge of creating changes in the attitude of staff.

Improved cost, health, and satisfaction with a health home benefit plan for self-insured employers and small physician practices.

We compared the impacts on total costs, health, and satisfaction among 615 adults enrolled 2 years in an employer's health home benefit plan to their baseline year in a standard preferred provider organization plan. The new plan combined strong continuity care incentives with nurse coaching support. After 24 months, total medical costs were 23% lower than the baseline year, biometric measures improved for more than 85% of members, and patient satisfaction exceeded 85%. Emergency department visits decreased by 16% and hospital days decreased by 48%. Health home benefit plans engaging small primary care physician practices and members in coordinated continuity care can deliver high value.

Quantitative genomics of 30 complex phenotypes in Wagyu x Angus F1 progeny.

In the present study, a total of 91 genes involved in various pathways were investigated for their associations with six carcass traits and twenty-four fatty acid composition phenotypes in a Wagyu×Angus reference population, including 43 Wagyu bulls and their potential 791 F(1) progeny. Of the 182 SNPs evaluated, 102 SNPs that were in Hardy-Weinberg equilibrium with minor allele frequencies (MAF>0.15) were selected for parentage assignment and association studies with these quantitative traits. The parentage assignment revealed that 40 of 43 Wagyu sires produced over 96.71% of the calves in the population. Linkage disequilibrium analysis identified 75 of 102 SNPs derived from 54 genes as tagged SNPs. After Bonferroni correction, single-marker analysis revealed a total of 113 significant associations between 44 genes and 29 phenotypes (adjusted P<0.05). Multiple-marker analysis confirmed single-gene associations for 10 traits, but revealed two-gene networks for 9 traits and three-gene networks for 8 traits. Particularly, we observed that TNF (tumor necrosis factor) gene is significantly associated with both beef marbling score (P=0.0016) and palmitic acid (C16:0) (P=0.0043), RCAN1 (regulator of calcineurin 1) with rib-eye area (P=0.0103), ASB3 (ankyrin repeat and SOCS box-containing 3) with backfat (P=0.0392), ABCA1 (ATP-binding cassette A1) with both palmitic acid (C16:0) (P=0.0025) and oleic acid (C18:1n9) (P=0.0114), SLC27A1(solute carrier family 27 A1) with oleic acid (C18:1n9) (P=0.0155), CRH (corticotropin releasing hormone) with both linolenic acid (OMEGA-3) (P=0.0200) and OMEGA 6:3 RATIO (P=0.0054), SLC27A2 (solute carrier family 27 A2) with both linoleic acid (OMEGA-6) (P=0.0121) and FAT (P=0.0333), GNG3 (guanine nucleotide binding protein gamma 3 with desaturase 9 (P=0.0115), and EFEMP1 (EGF containing fibulin-like extracellular matrix protein 1), PLTP (phospholipid transfer protein) and DSEL (dermatan sulfate epimerase-like) with conjugated linoleic acid (P=0.0042-0.0044), respectively, in the Wagyu x Angus F(1) population. In addition, we observed an interesting phenomenon that crossbreeding of different breeds might change gene actions to dominant and overdominant modes, thus explaining the origin of heterosis. The present study confirmed that these important families or pathway-based genes are useful targets for improving meat quality traits and healthful beef products in cattle.

Noninvasive monitoring of adrenocortical function in captive jaguars (Panthera onca).

Jaguars are threatened with extinction throughout their range. A sustainable captive population can serve as a hedge against extinction, but only if they are healthy and reproduce. Understanding how jaguars respond to stressors may help improve the captive environment and enhance their wellbeing. Thus, our objectives were to: (1) conduct an adrenocorticotrophic hormone (ACTH) challenge to validate a cortisol radioimmunoassay (RIA) for noninvasive monitoring of adrenocortical function in jaguars; (2) investigate the relationship between fecal corticoid (FCM) and androgen metabolite (FAM) concentrations in males during the ACTH challenge; and (3) establish a range of physiological concentrations of FCMs for the proposed protocol. Seven jaguars (3 M, 4 F) received 500 IU/animal of ACTH. Pre- and post-ACTH fecal samples were assayed for corticoid (M and F) and androgen metabolites (M) by RIA. Concentrations of FCMs increased (P80.01) after ACTH injection (pre-ACTH: 0.90 ± 0.12 µg/g dry feces; post-ACTH: 2.55 ± 0.25 µg/g). Considering pre- and post-ACTH samples, FCM concentrations were higher (P80.01) in males (2.15 ± 0.20 µg/g) than in females (1.30 ± 0.20 µg/g), but the magnitude of the response to ACTH was comparable (P>0.05) between genders. After ACTH injection, FAMs increased in two (of 3) males; in one male, FCMs and FAMs were positively correlated (0.60; P80.01). Excretion of FCMs was assessed in 16 jaguars (7 M, 9 F) and found to be highly variable (range, 80.11-1.56 µg/g). In conclusion, this study presents a cortisol RIA for monitoring adrenocortical function in jaguars noninvasively.

Pulsed GnRH secretion and the FSH secretory peaks that initiate ovarian antral follicular wave emergence in anestrous ewes.

In ewes, immunization against GnRH blocks LH pulses but mean serum FSH concentrations are only partly reduced; the fate of the FSH peaks that precede ovarian follicular waves has not been studied. In this study, we used immunization against GnRH to examine the need for pulsed GnRH secretion in the genesis of FSH peaks in the anestrous ewe. Six anestrous ewes were given a GnRH immunogen on Day 0 and a booster injection on Day 28. Control ewes (n=6) received adjuvant only. Transrectal ultrasonography was performed daily for 2 days prior to and 10 days following both the primary (Days -2 to 10) and booster (Days 26-38) injections and for a 13-day period beginning 26 days after booster injection (Days 54-66). Blood samples were collected daily. Intensive bleeding (every 12min for 7h) was performed on Days 9, 37, and 65 of the experimental period to characterize the pulsatile pattern of LH secretion. GnRH antibody titers were increased and LH pulses were abolished immediately after booster immunization (P<0.05). The number of FSH peaks, FSH peak concentration and amplitude and basal FSH concentrations were only decreased in immunized ewes in the period of observations starting 26 days after booster immunization (P<0.05); however, some peaks were still seen. The number of follicular waves was decreased in the period around booster immunization and no follicular waves were seen during the period starting 26 days after booster immunization in immunized ewes (P<0.05). In summary, in anestrous ewes, when pulsed LH secretion was abolished by immunization against GnRH, the peaks in serum concentrations of FSH that trigger ovarian follicular waves continued for a period of time. We concluded that although blocking the effects of GnRH gradually causes a diminution of FSH secretion, there is no acute requirement for GnRH in the regulation of FSH peaks. The existence of FSH peaks in the absence of follicular waves, and pulsed LH secretion, suggests that some endogenous rhythm may drive the occurrence of FSH peaks, independent of both changes in negative feedback by secretory products from ovarian antral follicles and GnRH.

CpG motif-based adjuvant as a replacement for Freund's complete adjuvant in a recombinant LHRH vaccine.

This study compared: (1) Freund's complete adjuvant and CpG oligodeoxynucleotide (ODN) 2006 in water-in-oil emulsion as adjuvants; and (2) increasing doses of a recombinant ovalbumin-LHRH (ova-LHRH) fusion protein as an antigen for a contraceptive vaccine. Treatment groups (n=8 heifers/group) were: one untreated control group; five groups receiving CpG ODN with different doses of ova-LHRH (1.5; 2.3; 3.4; 5.1; and 7.6 mg); and one group receiving 3.4 mg ova-LHRH in Freund's. Heifers were immunized at weeks 0 and 14. All vaccine treatments caused gonadal regression and estrus suppression. CpG ODN is a suitable replacement for Freund's for LHRH immunization.

The effectiveness of a CpG motif-based adjuvant (CpG ODN 2006) for LHRH immunization.

A recombinant ovalbumin-luteinizing hormone-releasing hormone (ova-LHRH) antigen has been developed for immunocontraception. In this study, a novel immunostimulant for ova-LHRH immunization, CpG oligodeoxynucleotide (ODN) 2006, was compared against Mycobacterium butyricum. Also, the immunogenicity of ova-LHRH after lyophilization and exposure to organic solvents was assessed. Rats received either ova-LHRH solubilized in urea; lyophilized ova-LHRH; lyophilized ova-LHRH exposed to methylene chloride; or lyophilized ova-LHRH exposed to ethyl acetate. Immunogenicity of lyophilized ova-LHRH was reduced compared with solubilized ova-LHRH. Exposure to ethyl acetate further decreased immunogenicity of ova-LHRH. CpG ODN 2006 was a more effective immunostimulant than M. butyricum for LHRH immunization.

A simplified QTL mapping approach for screening and mapping of novel AFLP markers associated with beef marbling.

Genome screening of quantitative trait loci (QTL) for a complex trait is usually costly and highly laborious, as it requires a large number of markers spanning the whole genome. Here we present a simplified approach for screening and mapping of QTL-linked markers for beef marbling using a WagyuxLimousin F(2) reference population. This simplified approach involves integration of the amplified fragment length polymorphism (AFLP) with DNA pooling and selective genotyping and comparative bioinformatics tools. AFLP analysis on two high and two low marbling DNA pools yielded ten visually different markers. Among them, four were confirmed based on individual AFLP validation. Sequencing and in silico characterization assigned two of these AFLP markers to bovine chromosomes 1 (BTA1) and 13 (BTA13), which are orthologous to human chromosomes HSA21q22.2 and HSA10p11.23 with both regions harboring QTL for obesity-related phenotypes. Both AFLP markers showed significantly large additive genetic effects (0.28+/-0.11 on BTA1 and 0.54+/-0.21 on BTA13) on beef-marbling score (BMS) (P<0.05). Overall, this approach is less time consuming, inexpensive and in particular, suitable for screening and mapping QTL-linked markers when targeting one or a few complex traits.

A novel nuclear-encoded mitochondrial poly(A) polymerase PAPD1 is a potential candidate gene for the extreme obesity related phenotypes in mammals.

People with obesity, especially extreme obesity, are at risk for many health problems. However, the responsible genes remain unknown in >95% of severe obesity cases. Our previous genome-wide scan of Wagyu x Limousin F2 cattle crosses with extreme phenotypes revealed a molecular marker significantly associated with intramuscular fat deposition. Characterization of this marker showed that it is orthologous to the human gene KIAA1462 located on HSA10p11.23, where a major quantitative trait locus for morbid obesity has been reported. The newly identified mitochondrial poly(A) polymerase associated domain containing 1 (PAPD1) gene, which is located near this marker, is particularly interesting because the polymerase is required for the polyadenylation and stabilization of mammalian mitochondrial mRNAs. In the present study, both cDNA and genomic DNA sequences were annotated for the bovine PAPD1 gene and ten genetic markers were detected in the promoter and exon 1 region. Among seven markers assayed on approximately 250 Wagyu x Limousin F2 animals, two single nucleotide polymorphisms (SNPs) in the promoter region were significantly associated with intramuscular fat (P<0.05). However, there was a significant interaction (P<0.05) between a third SNP, which causes an amino acid change in coding exon 1, and each of these two promoter SNPs on intramuscular fat deposition. In particular, the differences between double heterozygous animals at two polymorphic sites and the slim genotype animals exceeded 2.3 standard deviations for the trait in both cases. Our study provides evidence for a new mechanism--the involvement of compound heterosis in extreme obesity, which warrants further examination.

Evaluation of candidate gene effects for beef backfat via Bayesian model selection.

Candidate gene approaches provide tools for exploring and localizing causative genes affecting quantitative traits and the underlying variation may be better understood by determining the relative magnitudes of effects of their polymorphisms. Diacyglycerol O-acyltransferase 1 (DGAT1), fatty acid binding protein (heart) 3 (FABP3), growth hormone 1 (GH1), leptin (LEP) and thyroglobulin (TG) have been previously identified as genes contributing to genetic control of subcutaneous fat thickness (SFT) in beef cattle. In the present research, Bayesian model selection was used to evaluate effects of these five candidate genes by comparing competing non-nested models and treating candidate gene effects as either random or fixed. The analyses were implemented in SAS to simplify the programming and computation. Phenotypic data were gathered from a F(2) population of Wagyu x Limousin cattle. The five candidate genes had significant but varied effects on SFT in this population. Bayesian model selection identified the DGAT1 model as the one with the greatest model probability, whether candidate gene effects were considered random or fixed, and DGAT1 had the greatest additive effect on SFT. The SAS codes developed in the study are freely available and can be downloaded at: http://www.ansci.wsu.edu/programs/.

Significant associations of the mitochondrial transcription factor A promoter polymorphisms with marbling and subcutaneous fat depth in Wagyu x Limousin F2 crosses.

Mitochondrial transcription factor A (TFAM), a nucleus-encoded protein, regulates the initiation of transcription and replication of mitochondrial DNA (mtDNA). Decreased expression of nuclear-encoded mitochondrial genes has been associated with onset of obesity in mice. Therefore, we hypothesized genetic variants in TFAM gene influence mitochondrial biogenesis consequently affecting body fat deposition and energy metabolism. In the present study, both cDNA (2259 bp) and genomic DNA (16,666 bp) sequences were generated for the bovine TFAM gene using a combination of in silico cloning with targeted region PCR amplification. Alignment of both cDNA and genomic sequences led to the determination of genomic organization and characterization of the promoter region of the bovine TFAM gene. Two closely linked A/C and C/T single nucleotide polymorphisms (SNPs) were found in the bovine TFAM promoter and then genotyped on 237 Wagyu x Limousin F(2) animals with recorded phenotypes for marbling and subcutaneous fat depth (SFD). Statistical analysis demonstrated that both SNPs and their haplotypes were associated with marbling (P=0.0153 for A/C, P=0.0026 for C/T, and P=0.0004 for haplotype) and SFD (P=0.0200 for A/C, P=0.0039 for C/T, and P=0.0029 for haplotype), respectively. A search for transcriptional regulatory elements using MatInspector indicated that both SNPs lead to a gain/loss of six putative-binding sites for transcription factors relevant to fat deposition and energy metabolism. Our results suggest for the first time that TFAM gene plays an important role in lipid metabolism and may be a strong candidate gene for obesity in mammals.

Testicular development, ultrasonographic and histological appearance of the testis in ram lambs immunized against recombinant LHRH fusion proteins.

Sixteen native ram lambs weaned at 10 wk of age were divided into two groups. Eight animals were immunized against LHRH with a mixture of two fusion proteins: ovalbumin-LHRH-7 and thioredoxin-LHRH-7. The immunized lambs received a primary immunization plus two booster immunizations at 4 and 12 wks. Animals in the control group (n=8) were not treated. Scrotal measurements and blood samples were taken at 2-week intervals. Beginning at 25 wk of age, semen was collected and sexual behaviour was evaluated on a weekly basis. At 35 and 37 wk of age testes and accessory glands of all animals were subjected to ultrasound scanning. At 37 wk of age animals were slaughtered and testes were evaluated histologically. Serum LHRH antibodies (P<0.01) were detected in animals of the immunized group which had reduced serum testosterone concentrations (P<0.01). Testicular development was suppressed in the immunized animals (P<0.01). Immunized animals exhibited mounting activity 5 wks later than control animals. No mature spermatozoa containing ejaculates were collected from immunized animals. Control animals had moderately echogenic ultrasonographic appearance at 37 wk age, whereas immunized animals had hypoechogenic images. Mean seminiferous tubule diameter in immunized lambs was significantly smaller than that in control lambs. Basal membrane was thickened and hyalinized; there was an increase in peritubular connective tissue. No proliferating spermatogonia or mature spermatozoa were present in the tubules in these animals. There were no differences in the ultrasonographic appearance of prostate and vesicular gland between control and immunized animals. The LHRH recombinant fusion proteins were effective in immunological castration in ram lambs when started at 10 wk of age as noted by differences in serum testosterone, testicular histology and ultrasonographic appearance of testis and weight of accessory sex glands. Determining the effects of immunization on ultrasonographic appearance of the testis related to time after immunization requires further investigations.

Establishment of spermatogenesis in neonatal bovine testicular tissue following ectopic xenografting varies with donor age.

Ectopic testicular xenografting can be used to investigate spermatogenesis and as an alternative means for generating transgenic spermatozoa in many species. Improving the efficiency of spermatogenesis in xenografted testicular tissue will aid in the application of using this approach. The present study was conducted to evaluate age-related differences in the establishment of spermatogenesis in grafted testicular tissue from bulls between 2 and 16 wk of life. Testicular tissue was ectopically xenografted under the skin on the backs of castrated nude mice and subsequently evaluated for growth, testosterone production, and establishment of spermatogenesis 24 wk after grafting. The greatest weight increases occurred in donor tissue from calves of the ages 2, 4, and 8 wk compared with the ages of 12 and 16 wk. Recipient mouse serum testosterone concentration was at normal physiological levels 24 wk after grafting and no significant differences were detected between recipients grafted with testicular tissue from bull calves of different ages. The development of germ cells to elongated spermatids were observed in seminiferous tubules of grafts from donor calves of the ages 4, 8, 12, and 16 wk but not observed in grafts from 2-wk donors, which contained round spermatids as the most advanced germ cell stage. Grafts from 8-wk donors contained a significantly higher (10-fold) average percentage of seminiferous tubules with elongated spermatids than all other donor ages. These data demonstrate differences in the ability of testicular tissue from donor animals of different ages to establish spermatogenesis following ectopic testicular xenografting.

Biological activity of cryopreserved bovine spermatogonial stem cells during in vitro culture.

Functional roles of spermatogonial stem cells in spermatogenesis are self-renewing proliferation and production of differentiated daughter progeny. The ability to recapitulate these actions in vitro is important for investigating their biology and inducing genetic modification that could potentially lead to an alternative means of generating transgenic animals. The objective of this study was to evaluate the survival and proliferation of frozen-thawed bovine spermatogonial stem cells in vitro and investigate the effects of exogenous glial cell line-derived neurotrophic factor (GDNF). In order to accomplish this objective we developed a bovine embryonic fibroblast feeder cell line, termed BEF, to serve as feeder cells in a coculture system with bovine germ cells. Bovine spermatogonial stem cell survival and proliferation in vitro were evaluated by xenogeneic transplantation into the seminiferous tubules of immunodeficient mice. Bovine germ cells cocultured for 1 wk resulted in significantly more round cell donor colonies in recipient mouse testes compared to donor cells transplanted just after thawing. Bovine germ cells cocultured for 2 wk had fewer colony-forming cells than the freshly thawed cell suspensions or cells cultured for 1 wk. Characterization of the feeder cell line revealed endogenous expression of Gdnf mRNA and protein. Addition of exogenous GDNF to the culture medium decreased the number of stem cells present at 1 wk of coculture, but enhanced stem cell maintenance at 2 wk compared to cultures without added GDNF. These data indicate that frozen-thawed bovine spermatogonial stem cells survive cryopreservation and can be maintained during coculture with a feeder cell line in which the maintenance is influenced by GDNF.

Spermatogenesis and germ cell transgene expression in xenografted bovine testicular tissue.

The present study was conducted to evaluate the development of spermatogenesis and utility of using electroporation to stably transfect germ cells with the beta-galactosidase gene in neonatal bovine testicular tissue ectopically xenografted onto the backs of recipient nude mice. Bull testicular tissue from 4-wk donor calves, which contains a germ cell population consisting solely of gonocytes or undifferentiated spermatogonia, was grafted onto the backs of castrated adult recipient nude mice. Testicular grafts significantly increased in weight throughout the grafting period and the timing of germ cell differentiation in grafted tissue was consistent with postnatal testis development in vivo relative to the bull. Seminiferous tubule diameter also significantly increased with advancing time after grafting. At 1 wk after grafting, gonocytes in the seminiferous cords completed migration to the basement membrane and differentiated germ cell types could be observed 24 wk after grafting. The presence of elongating spermatids at 24 wk confirmed that germ cell differentiation occurred in the bovine tissue. Leydig cells in the grafted bovine tissue were also capable of producing testosterone in the castrated recipient mice from 4 wk to 24 wk after grafting at concentrations that were similar to levels in intact, nongrafted control mice. The testicular tissue that had been electroporated with a beta-galactosidase expression vector showed tubule-specific transgene expression 24 wk after grafting. Histological analysis showed that transgene expression was present in both Sertoli and differentiated germ cells but not in interstitial cells. The system reported here has the potential to be used for generation of transgenic bovine spermatozoa.