Jerry J. Reeves

Spermatogenesis and Germ Cell Transgene Expression in Xenografted Bovine Testicular Tissue

Abstract The present study was conducted to evaluate the development of spermatogenesis and utility of using electroporation to stably transfect germ cells with the β-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 β-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.

Establishment of Spermatogenesis in Neonatal Bovine Testicular Tissue Following Ectopic Xenografting Varies with Donor Age

Abstract 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

Abstract 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.

Effects of breed and sire on carcass characteristics and fatty acid profiles of crossbred wagyu and angus steers.

In a two-year experiment, 54 steers sired by seven Wagyu bulls [American Wagyu Association (AWA) sire numbers 331, 384, 388, 411, 429, 433 and 488] and 15 steers sired by two Angus bulls, all out of Angus-Hereford cows, were used to evaluate the effects of sire and breed on carcass characteristics and fatty acid composition. Steers were given ad-libitum access to a high-concentrate diet (15 % alfalfa cubes and 85 % barley supplement) for at least 170 days. Breed and individual sire effects were analysed. Wagyu-sired steers had higher marbling, maturity and quality scores, more estimated kidney, pelvic and heart fat, larger longissimus dorsi muscle areas, lower fat thicknesses and yield grades than Angus-sired steers (p < 0.05). Steers sired by 388, 411 and 433 had lower fat thicknesses than steers sired by Angus, 429 and 488 (p < 0.05). Steers sired by 384 and 388 had higher marbling scores per cm subcutaneous fat than steers sired by Angus, 429 and 488, and lower fat thickness per 100 kg of carcass weight than Angus-sired steers (p < 0.05). For both subcutaneous fat and longissimus dorsi muscle, Wagyu-sired steers had higher (p < 0.05) percentages of 14:0, 14:1, 16:0, 16:1, and lower percentages of 18:0 than Angus-sired steers. The genetic differences in carcass characteristics among Wagyu sires may enable us to select for improved marbling with less fat in the Wagyu breed. Some statistically significant (p < 0.05) but small differences existed in fatty acid profiles between breeds and among sires.

Effects of biological source on cooking and palatability attributes of beef produced for the Japanese market

Boneless beef loin samples from five biological sources (Japanese Wagyu, American Wagyu (3 4 - 7 8 Wagyu), Angus, Longhorn and US Choice) were evaluated for cooking and palatability attributes as shabu-shabu, steaks and roasts. Japanese Wagyu beef was superior in palatability compared to Angus, Longhorn and US Choice beef when prepared as shabu-shabu or as steaks. Very palatable beef was produced for the Japanese market when the Wagyu breed and a controlled, extended feeding period were utilized. The results were more equivocal when the beef was prepared as roasts, but it is unlikely that a substantial demand for roasts will develop in Japan due to high retail costs and traditions in cookery.

Development of recombinant ovalbumin-luteinizing hormone releasing hormone as a potential sterilization vaccine

The objective was to develop an immunogenic chimeric ovalbumin-LHRH (ova-LHRH) molecule using genetic engineering. Hybrid ova-LHRH genes with either four or seven LHRH inserts were constructed by cassette mutagenesis and oligonucleotide mismatch mutagenesis. Recombinant ova-LHRH proteins were over-expressed in E. coli strain BL21 (DE3) using a pET expression system, which expresses a target protein with a C-terminal His-Tag. The C-terminal His-Tag allows purification by metal chelation chromatography. The antigenicity and biological effects of these recombinant proteins were tested in mice. In experiment 1, 17 female 7 wk old BALB/c mice were randomly divided into three groups. Six mice were injected with 50 microg of the recombinant ovalbumin (ova) protein. Five mice were injected with 50 microg of the recombinant protein with four LHRH inserts (ova-LHRH-7). Six mice were injected with 50 microg of the recombinant protein with seven LHRH inserts (ova-LHRH-7). One primary immunization using Freunds complete adjuvant was followed by one booster using incomplete adjuvant. Mice were killed 2 wk after the booster, blood collected, and the reproductive tract removed and weighed. Only ova-LHRH-7 decreased (P < 0.01) uterine-ovarian weight (89+/-11 mg) vs control (138+/-6 mg) and ova-LHRH-4 (126+/-16 mg). The genetically engineered molecule with seven LHRH inserts induced LHRH antibody titers which were significantly correlated (r = -0.79) with biological response. In experiment 2, the recombinant ova-LHRH-7 was evaluated at two doses with the adjuvants Zmax and Immumax. Seventy female 6-8 wk old BALB/c mice were randomly divided into seven groups of 10 mice each. Anti-LHRH titers were detected in all of the ova-LHRH-7 immunized mice. Significant decreases were shown in uterine-ovarian weight of the mice by the immunization with 30 microg of ova-LHRH-7 and Zmax (P < 0.005) or 10 microg of ova-LHRH-7 with Immumax (P < 0.025). These data show that the recombinant ova-LHRH-7 protein could have potential as an effective sterilization vaccine.

Growth and carcass characteristics of pasture fed LHRH immunocastrated, castrated and intact Bos indicus bulls

The effectiveness of a luteinizing hormone-releasing hormone (LHRH) fusion protein vaccine or surgical castration, at two years of age, on growth and carcass characteristics of Bos indicus bulls was evaluated. Seventy Nelore-cross bulls were divided into three groups: (1) immunized, (2) castrated and (3) intact control. At slaughter (three years of age), intact bulls had higher body weights, ADG, carcass weights, and muscle percentage compared to immunized and surgically castrated animals. Both castrated and immunized animals had greater marbling and percent carcass fat than the intact bulls. Average tenderness scores were inferior for intact bulls compared to immunized and castrated animals, but these differences were not significant (P>0.05). Juiciness, flavor, thawing, nor cooking losses differed significantly among the three groups. Immunocastration was effective in producing carcass traits similar to that of surgical castration. Therefore, immunization with LHRH fusion proteins appears to have practical utility in the management and castration of grazing bulls.

Testis Tissue Explant Culture Supports Survival and Proliferation of Bovine Spermatogonial Stem Cells

Abstract The present study was designed to evaluate the survival and proliferation of bovine spermatogonial stem cells in an explant culture system over a 2-wk period. Explants of calf testicular parenchyma were placed on 0.45-μm pore membranes in culture and maintained for 1–2 wk. Histological examinations of fresh (t0) and cultured tissues revealed morphologically normal seminiferous tubules. Germ cell numbers/tubule increased (P ≤ 0.05) during culture when compared with t0, yet germ cell differentiation was not observed. Testosterone was present in medium throughout the culture period, indicating functional Leydig cells. Sertoli, spermatogonial, and spermatogonial stem cell viability was evaluated by reverse transcription-polymerase chain reaction for cell-specific gene expression of stem cell factor, protein gene product 9.5, and glial cell line-derived neurotrophic factor family receptor-α1, respectively. Results demonstrated the expression of all genes at t0, 1 wk, and 2 wk of culture. Single-cell suspensions were prepared from the testicular tissues at t0 and during culture and transplanted into nude mouse testes to investigate spermatogonial stem cell viability. One month after transplantation, colonies of round bovine cells were identified in all mouse testes analyzed, indicating survival of spermatogonial stem cells. The average number of resulting colonies in recipient testes was significantly (P ≤ 0.05) higher following 1 wk of culture compared with t0 and was numerically higher at 2 wk of culture compared with t0. This increase in colony numbers over time in culture indicates spermatogonial stem cell proliferation in vitro. This explant culture system appears to provide an environment that supports survival and proliferation of bovine spermatogonial stem cells.

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 F2 population of Wagyu × 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/.

Effects of time on feed and post-mortem aging on palatability and lipid composition of crossbred Wagyu beef

Twenty-seven Wagyu-sired steers were fed for 90 (14 steers) or 170 (13 steers) days to study the effects of time on feed on palatability and fatty acid composition, and the effects of post-mortem aging time (2, 4 or 10 days) on palatability. Hot carcass weight, fat thickness, longissimus dorsi muscle area, yield grade, estimated kidney, pelvic and heart fat and maturity score were increased (p < 0.05) by an additional 80 days on the high concentrate feed, but marbling was not changed (p > 0.05). Feeding the high concentrate diet for 170 days increased Warner-Bratzler shear force values (p < 0.05) and tended to decrease tenderness (p > 0.05), flavor intensity and connective tissue scores. For the 90 day feeding group, 4 days of aging improved connective tissue score (p < 0.05) and tended to increase (p > 0.05) tenderness scores and decrease shear force, compared with 2 days of aging. For the 170 day feeding group, 10 days of aging improved (p < 0.05) shear force and all sensory attributes except flavor intensity, compared to 2 days of aging. An additional 80 days on feed decreased (p < 0.05) stearic acid and total saturated fatty acids (SFA) and generally increased (p < 0.05) monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), MUFA:SFA, and PUFA:SFA in subcutaneous fat and longissimus dorsi muscle. The cholesterol content of fat and muscle increased (p < 0.05) as time on feed increased. Ninety days on a high concentrate diet was adequate for yearling crossbred Wagyu steers to produce highly acceptable carcasses. The additional 80 days on feed produced little or no overall benefit and the steers became overfinished and less tender. Ten days post-mortem aging improved (p < 0.05) all palatability attributes except flavor intensity.