Gulf Coast Native Research Articles

PSVI-20 Effects of castration age on growth, carcass, and sensory characteristics of Gulf Coast Native sheep.

PSVI-20 Effects of castration age on growth, carcass, and sensory characteristics of Gulf Coast Native sheep.

Isolation, culture and characterisation of somatic cells derived from semen and milk of endangered sheep and eland antelope

Semen and milk are potential sources of somatic cells for genome banks. In the present study, we cultured and characterised cells from: (1) cooled sheep milk; (2) fresh, cooled and frozen-thawed semen from Gulf Coast native (GCN) sheep (Ovis aries); and (3) fresh eland (Taurotragus oryx) semen. Cells attached to the culture surface from fresh (29%), cooled (43%) and slow-frozen (1 degrees C/min; 14%) ram semen, whereas no attachment occurred in the fast-frozen (10 degrees C/min) group. Proliferation occurred in fresh (50%) and cooled (100%) groups, but no cells proliferated after passage 1 (P1). Eland semen yielded cell lines (100%) that were cryopreserved at P1. In samples from GCN and cross-bred milk, cell attachment (83% and 95%, respectively) and proliferation (60% and 37%, respectively) were observed. Immunocytochemical detection of cytokeratin indicated an epithelial origin of semen-derived cells, whereas milk yielded either fibroblasts, epithelial or a mixture of cell types. Deoxyribonucleic acid microsatellite analysis using cattle-derived markers confirmed that eland cells were from the semen donor. Eland epithelial cells were transferred into eland oocytes and 12 (71%), six (35%) and two (12%) embryos cleaved and developed to morulae or blastocyst stages, respectively. In conclusion, we have developed a technique for obtaining somatic cells from semen. We have also demonstrated that semen-derived cells can serve as karyoplast donors for nuclear transfer.

Segregation of natural and experimental gastrointestinal nematode infection in F 2 progeny of susceptible Suffolk and resistant Gulf Coast Native sheep and its usefulness in assessment of genetic variation

Gastrointestinal nematode parasitism is a concern to small ruminants worldwide. Productivity has been compromised because such nematodes, particularly Haemonchus contortus, have developed resistance to available anthelmintics. Some sheep breeds and lines within breeds are relatively resistant to infection, a trait that may be useful for developing control strategies. Suffolk sheep, which are susceptible to infection, were crossed with Gulf Coast Native sheep, which are more resistant to infection, to produce F 1 progeny. F 1 rams were bred to F 1 ewes which produced 227 F 2 offspring. These F 2 offspring were evaluated for variability in infection levels, based on fecal egg count (FEC) and blood packed cell volume (PCV), under two natural infection conditions (one at weaning and another after a summer grazing period) and one experimental infection. The range of both FEC and PCV was large for all three infection periods with annual variation. Overall, the range for the three infection periods, respectively, were 167–149,933, 0–31,400 and 17–114,667 eggs per gram (EPG) of feces and 8.7–37.0%, 7.3–33.0% and 8.3–36.0%. This segregation of infection is what would be expected of F 2 progeny from susceptible and resistant parent breeds. Heritabilities of FEC and PCV for the three infection periods, respectively, were 0.15, 0.29 and 0.12, and 0.11, 0.22 and 0.12. Based on segregation of infection, larger heritabilities and maternal environment effects that declined after weaning, the summer natural infection was probably the best model for assessing genetic variation.

Effect of CD4 + T lymphocyte depletion on resistance of Gulf Coast Native lambs to Haemonchus contortus infection

It has been reported that CD4 + T lymphocytes are important in acquired immunity to gastrointestinal nematode infection. Whether these lymphocytes are also involved in the immune response of naturally resistant Gulf Coast Native (GCN) sheep to Haemonchus contortus infection remains to be defined. The objective of this study was to determine the role of CD4 + T lymphocytes in this resistance. Ten GCN lambs were randomly assigned to a control ( n = 5) or a treatment ( n = 5) group. The treatment consisted of a series of IV injections with mouse anti-ovine CD4 + T lymphocyte monoclonal antibodies for a period of 3 weeks. After the second treatment, all lambs were experimentally infected with 10,000 H. contortus infective larvae by oral inoculation. All lambs were monitored for fecal egg counts, blood packed cell volumes, white blood cell differential counts and serum antibody responses on a weekly basis. Fluorescence-activated cell sorter (FACS) analysis was done biweekly to enumerate CD4 + T lymphocytes in peripheral blood. Necropsies were performed at the end of the study and 10% of the contents of the gastrointestinal tract were preserved for nematode enumeration and identification. Also at necropsy, mesenteric lymph nodes were extracted and FACS analysis was run on lymphoid cells. Mean fecal egg counts on day 21 and 28 post-infection and nematode counts at necropsy of the treated group were significantly ( p < 0.05) higher than that of the control group. Percent CD4 + T lymphocytes in peripheral blood was significantly ( p < 0.05) lower in the treatment group than in the control group from day 9 to the end of the study. No differences were found in blood packed cell volumes, white blood cell differential counts, antibody titer or lymph node CD4 + lymphocytes between groups. Lambs depleted of their CD4 + T lymphocytes were more susceptible to H. contortus infection than undepleted lambs. The results of this study suggest that CD4 + T lymphocytes are associated with the natural resistance of GCN sheep to H. contortus infection.

Quality and freezing qualities of first and second ejaculates collected from endangered Gulf Coast Native rams

The Gulf Coast Native sheep, or Louisiana Native sheep, is an endangered previously feral domestic sheep population of European origin that has been under natural selection pressure for reproductive survival in their transplanted range while roaming in the southern Gulf Coast Region of the United States. This sheep population has an increased natural resistance to internal parasites, breeds year-around and has a greater percentage of live lambs as compared with other breeds of sheep raised in similar environments. To preserve the genetic diversity of this important feral sheep population, semen was collected by electro-ejaculation and subjected to cryopreservation for subsequent storage in a genome resource bank. Unrelated rams ( n = 5) were collected 3 days-a-week, allowing at least 2 days of rest between collections. Two ejaculates were obtained from each ram per collection day, with the second collection conducted 10 min after the first ejaculation. Semen was processed using the standard Salamon cryopreservation procedure in a Tris–yolk–glycerol extender, frozen in 0.5 ml plastic straws using liquid nitrogen (LN 2) vapor and stored in LN 2. Each ejaculate was evaluated for volume, sperm concentration/ml (×10 9/ml), number of spermatozoa/ejaculate (×10 9), sperm progressive motility (%) for pre-cooled semen, cooled semen and semen after thawing. For the five rams, each semen variable for the first ejaculate was compared with that of the second ejaculate collected 10 min later. The mean semen volume, sperm concentration and number of spermatozoa per ejaculate obtained from the first ejaculate were significantly greater ( P ≤ 0.01) than those of the second ejaculate (comparisons being 1.62 and 1.06; 3.2 and 1.5; 5.4 and 1.8, respectively). Overall, the mean motility of pre-cooled (22 °C), cooled (5 °C) and frozen (−196 °C) post-thawed spermatozoa was less ( P ≤ 0.01) in the first ejaculate (71.5, 64.8 and 34.1%, respectively) compared with that of the second ejaculate (75, 72.4 and 44.1%, respectively). Conversely, no differences were detected in loss in the percent progressive motility of sperm from cooled sperm to post-thaw sperm from the first and second ejaculates. In summary, our findings suggest sperm collected during the second ejaculate 10 min after the first ejaculate of rams survives thawing with a greater rate of progressive motility than that of the first ejaculate. The ability to collect two consecutive ejaculates in a short period by electro-ejaculation could be valuable for gamete resource banking and preserving genetic diversity of the Gulf Coast Native sheep.

Glucose metabolism and insulin sensitivity in Gulf Coast Native and Suffolk ewes during late gestation and early lactation

Two experiments were conducted to evaluate possible differences in glucose metabolism and insulin responsiveness in Gulf Coast Native (GCN) and Suffolk (SFK) ewes. In Experiment 1, 30 GCN and 41 SFK ewes in late gestation were used to identify hyperglycemia and hyperinsulinemia. Blood samples were obtained via jugular venipuncture and analyzed for plasma insulin, glucose, thyroxine, albumin, and plasma urea nitrogen (PUN) concentrations. In Experiment 2, at 2-week postpartum, 19 GCN and 19 SFK ewes were administered frequently sampled intravenous glucose tolerance tests (FSIGT) consisting of a pulse dose of glucose (500 mg kg −1 BW) followed by an infusion of insulin (0.03 IU kg −1 BW). Blood samples were collected via jugular catheter at timed intervals over a 3 h period after glucose infusion. Samples from the FSIGT were analyzed for insulin and glucose concentrations, and the minimal model computer program was then used to assess glucose effectiveness ( S G), an estimate of insulin-independent glucose disappearance; insulin sensitivity ( S I), an estimate of insulin-dependent glucose disappearance; and acute insulin response (AIR glucose), insulin secretion relative to glucose administration. Concentrations of albumin, thyroxine, and PUN were measured in samples collected prior to glucose infusion. In Experiment 1, insulin concentrations were greater ( P<0.01) in SFK ewes while plasma glucose concentrations were not different between breeds. Concentrations of albumin ( P<0.01), thyroxine ( P<0.05), and PUN ( P<0.05) were greater in SFK ewes. In Experiment 2, S G and S I did not differ between breeds, while AIR glucose tended to be greater in GCN ewes. There were no differences in plasma albumin, thyroxine, or PUN concentrations. These data indicate slight differences in insulin sensitivity between GCN and SFK ewes during late gestation. However, there were no apparent differences in glucose metabolism between these breeds during the early lactation period.

61ISOLATION AND CULTURE OF SOMATIC CELLS OBTAINED FROM SEMEN AND MILK OF GULF COAST NATIVE SHEEP

In addition to the sperm found in semen, somatic cells (SC) are frequently present. Furthermore, SC are also present in fresh milk. These SC could also be a potential source of biomaterial for the preservation of endangered species. In the present study, the possibility of isolating SC from fresh, cooled and frozen semen, as well as from fresh milk, was evaluated in the endangered Gulf Coast Native sheep. Semen from 7 mature rams was collected by electro-ejaculation and a 0.5-mL sample from each ejaculate was then diluted in PBS (22°C). The remainder of the ejaculate was cooled (4°C), extended in a TRIS-yolk-glycerol extender, loaded into 0.5-mL plastic straws and frozen. Semen from the same collection (2–5 straws) was also diluted in PBS and allotted to the cooled semen treatment group (4°C). Sheep milk samples (n=26) from 5 Gulf Coast Native (GCN) ewes and 11 crossbred ewes (GCN × Suffolk) were collected and stored at 4°C. Samples of fresh (0.5mL), cooled (2–5 straws) and frozen–thawed (2–5 straws) semen and refrigerated milk (15–50mL) were washed with PBS, centrifuged at 500g for 10min and the re-suspended pellet cultured in tissue culture dishes containing 2mL of DMEM with 15% fetal bovine serum (FBS) plus 100μM nonessential amino acids, supplemented with antibiotics and incubated at 38.5°C in 5% CO2 in air. After 24h, the semen sample culture dishes were rinsed twice with fresh culture medium to remove remaining sperm cells and then returned to the the incubator. After 3 to 7d of incubation, groups of epithelial-like cell colonies were detected in 1 sample from the fresh semen group (1 of 7 rams, 14.3%) and 4 semen samples from the cooled group (4 of the 7 of rams, 57%). These cells subsequently proliferated until a monolayer was observed; the cells were then harvested and plated (in a single culture dish) for further passage. One cell line survived from the cooled semen group, which was frozen in DMEM medium with 15% FBS and 10% DMSO and then stored in LN2. No cells were derived from the frozen-thawed semen group. Colonies of fibroblast-like or epithelial-like cells were detected after culture of the milk samples. Of the 26 colonies, 23 (88%) initially plated cells, 11 (41%) proliferated to the 1st passage, 5 (19%) resulted in cell lines that were frozen and 2 (8%) of these subsequently proliferated during post-thaw incubation. The genetic status of the cell lines that proliferated until cryopreservation was assessed by PCR amplification of microsatellite markers. Results confirmed that the cell populations derived from both the semen and milk samples originated from the appropriate donor animals. Also, immuno-histochemical analyses for vimentin and cytokeratin were performed on these cell populations to determine the morphological cell type. Cells derived from semen samples were identified as epithelial cells by the presence of cytokeratin, whereas the milk samples produced epithelial and fibroblast cells. In the present study, we demonstrated that it is possible to establish a somatic cell line from cooled semen, as well as from fresh milk, in an endangered sheep. This approach could provide cell lines from valuable animals when other means of obtaining cells are not possible. This project was partially funded by the J. Bennett Johnston Science Foundation.

108CRYOPRESERVATION OF GERMPLASM FROM HERITAGE BREEDS OF DOMESTIC LIVESTOCK: SEASONAL EFFECTS ON SUPEROVULATION RESPONSE AND EMBRYO PRODUCTION

There is an urgent need to preserve the genetic diversity of rare breeds of domestic livestock. However, it is unknown if conventional superovulation methods are effective in unimproved breeds. The establishment of germplasm cryostorage banks requires the ability to produce viable preimplantation stage embryos. To our knowledge, we describe here the first successful production and cryopreservation of embryos from Gulf Coast Native (GCN) sheep and Tennessee Myotonic (TM) goats. Mature GCN ewes and TM does were synchronized (prostaglandin F2α, 7.5–10mg i.m.) and all TM does, and some GCN ewes, were treated with progesterone (20mg sq, 5×on alternate days, prior to FSH treatment, then 10mg i.m. on second day of FSH treatment). Animals were superovulated (FSH, 50–40–30mg bid i.m., decreasing over 3 days) both in April (cycling) and June (anestrous). Females were bred by natural service with a proven male over 24hr, and embryos were collected surgically from the uterus on Day 7. FSH-stimulated cycling TM does produced a total of 15.3±3.0CL and 10.7±7.2 morulae/animal, whereas FSH-treated anestrous TM does yielded 9.0±2.8CL and 7.0±8.4 embryos per animal. Recovered goat embryos were morphologically of good-excellent quality, and were cryopreserved using a conventional multi-step freezing protocol. Cycling GCN ewes responded to FSH simulation and produced 9.7±2.5CL per animal, whereas the ovaries of non-cycling ewes did not respond to exogenous FSH treatment (0CL). This report documents that TM does will effectively respond to FSH superovulation treatment and produce viable embryos during the breeding season and during the anestrous period. In contrast, GCN ewes appear to respond only to exogenous FSH during the breeding season. Collectively, these results demonstrate that the TM and GCN landrace breeds of goats and sheep display differential responses to hormone manipulation; yet, with breed-optimized protocols, preimplantation stage embryos can be obtained for long-term cryostorage of germplasm of heritage breeds of livestock. Supported by and in partnership with SVF Foundation.

Differential responses to an oral urea load test in small ruminants: species and breed effects

An experiment was conducted to determine the differential responses to an oral urea load test (OULT) in 1–2-year-old sheep and goats. Six non-bred ewes from each breed [Suffolk (SFK), Gulf Coast Native (GCN), Katahdin (KAT), and St. Croix (SCX)] and six non-bred does [Boer Goat Cross (BGC)] were individually penned and fed a concentrate-base diet at 120% of NRC (1985) requirement for maintenance along with bermudagrass hay ad libitum for 2 weeks. At the end of the 2-week-period, half of the animals from each group were fitted with jugular vein catheters, and blood was collected at 15 min intervals for 360 min. At the 45 min mark, the OULT (0.4 g urea/kg BW) was administered via a stomach tube. Ruminal samples were collected before and after the OULT was administered. The experiment was repeated with the remaining animals on the following day. Blood was analyzed for plasma ammonia-N, urea-N, glucose, insulin, and l-carnitine. Ruminal fluid was collected via stomach tube and analyzed for pH and ammonia-N. Comparisons were made for each group of animals to test for breed effect, and all sheep data were pooled to compare with goats and to test for species effect. After OULT administration, ruminal pH increased from 6.51 to 7.05 ( P<0.0001), and there was a seven-fold increase in ruminal ammonia-N 30 min post-OULT ( P<0.0001; 16.2 versus 92.5 mmol/l). Plasma urea-N and ammonia-N levels increased over time in response to the OULT ( P<0.0001), but there were no breed or species effects, breed×time, or species×time interactions ( P>0.10). A breed×time ( P<0.0001) and species×time interactions ( P<0.0001) as well as a species effect ( P=0.05) were observed for glucose in response to the OULT with GCN plasma glucose increasing the most. Likewise, there was a breed effect ( P<0.01) and a trend ( P=0.14) toward a breed×time interaction for insulin, although there was no species effect ( P>0.10) or species×time interaction ( P>0.10). There were no breed-related differences ( P>0.10) in plasma l-carnitine. The data suggest that breeds and species can respond differently to an OULT and when conducting or interpreting experiments using this type of test, breed and species should be considered.

Growth and metabolic characteristics of Suffolk and Gulf Coast Native yearling ewes supplemented with chromium tripicolinate

An experiment was conducted to investigate differences in production and physiological criteria in yearling ewe lambs from two distinct breeds fed a concentrate–base diet (13.5% CP) supplemented with 0 (BAS) or 370 ppb (CRP) chromium tripicolinate. The breeds used were Suffolk (SFK, n=8, BW 58.9±1 kg), representing a breed selected for meat production, and Gulf Coast Native sheep (GCN, n=8, BW 44.9±1 kg), representing a breed selected for adaptability and wool production. Lambs were fed the BAS diet for 10 days. On day 0 of the experimental period, the lambs were stratified by BW within breed and randomly assigned to the experimental diets. On days 0, 11 and 22, after 18 h of feed deprivation, lambs were bled via jugular venipuncture and weighed. Moreover, on day 22, lambs were catheterized and an i.v. glucose tolerance test (IVGTT; 500 mg glucose kg −1 BW) was conducted. Statistical analyses of plasma metabolite and hormone measurements were conducted on their concentrations, as well as the relative changes (percent change) of these measurements for days 11 and 22 compared with day 0. Growth rate, feed intake and glucose and insulin kinetics in response to an IVGTT were not affected ( P>0.10) by CRP. Relative to BAS, CRP decreased plasma NEFA concentration ( P<0.04) and percent change ( P<0.015), and insulin ( P<0.09) and thyroxine (T 4; P<0.05) percent change. When comparing breed effects, SFK exhibited higher ( P<0.0001) daily feed intake (2.3 vs. 1.7 kg DM d −1), ADG (331 vs. 95 g d −1) and gain-to-feed ratio (0.15 vs. 0.06). Compared with GCN, SFK had higher plasma NEFA ( P<0.01), triiodothyronine ( P<0.07) and T 4 ( P<0.05), and lower IGF-I ( P<0.04) and urea N ( P<0.07). In response to the IVGTT, SFK exhibited faster plasma glucose and insulin clearance rates ( P<0.06) and had reduced area under the response curves ( P<0.07) compared with GCN. Diet or breed did not affect ( P>0.10) plasma glucose, albumin, triacylglycerol or cortisol concentrations. CRP reduced plasma NEFA, but did not affect glucose kinetics. There were major differences in physiological measurements between the two distinct breeds of sheep that could explain the differences observed in performance.

Epidemiology of gastrointestinal nematode parasitism in Suffolk and Gulf Coast Native sheep with special emphasis on relative susceptibility to Haemonchus contortus infection

An eight-year study was conducted to define the epidemiology of gastrointestinal nematode infection in Suffolk and Gulf Coast Native (Native) breeds of sheep, and to determine if the Native sheep is more resistant to infection. For the initial three years, each breed grazed separate pastures where anthelmintic treatments were administered to individual animals on a salvage basis. For the last five years, both breeds grazed concurrently; anthelmintic treatments were administered to individual animals on a salvage basis for the first three years, and to all animals, when treatment criteria were met, for the last two years. The fecal egg count (FEC) and blood packed cell volume (PCV) were monitored, and tracer lamb nematode burdens were determined. Overall, FEC for both breeds increased in the spring (periparturient rise) for most years and in the summer for all years. Under separate grazing conditions, Native ewes and lambs had consistently lower infection levels than Suffolk ewes and lambs. During the haemonchosis season (June–September) each year, Suffolk ewe and lamb PCV decreased, and Native ewe and lamb PCV remained relatively stable. The salvage treatment protocol resulted in 27 treatments for Suffolk and one for Native ewes; similarly for lambs, 13 for Suffolk and zero for Native. Tracer lambs grazed with their respective breed, and the FEC and mean total nematode burden corresponded with the pattern of infection for their respective breed. The predominant nematodes found in Suffolk and Native tracer lambs were Haemonchus contortus and Trichostrongylus spp., respectively. Under concurrent grazing conditions, the same seasonal repeatable pattern of infection was present and was exhibited by both breeds, with the Native ewes and lambs being consistently and significantly (p ≤ 0.05) lower for FEC and higher for PCV. The salvage treatment protocol resulted in 57 and zero treatments for Suffolk and Native ewes, respectively; for lambs, 46 and 11. Tracer lamb nematode burdens again corresponded to their respective breed pattern of infection, with H. contortus and Trichostrongylus spp. being predominant in Suffolk and Native lambs, respectively. Data from all tracer lambs showed a relatively low level of hypobiosis (H. contortus only), and, although there was no consistent hypobiosis season, the tendency was for a higher level to occur in the fall. These results showed that the classic repeatable seasonal pattern of gastrointestinal nematode infection occurred in both breeds of sheep, and that Native sheep were more resistant to infection (specifically H. contortus) than Suffolk sheep.