Biomedical subjects

Carol L Keefer

Publications and source records attributed to Carol L Keefer.

5 recordsLinked to original sources

PubMed2006

Transgenesis using nuclear transfer in goats.

Nuclear transfer (NT) using transgenic donor cells is an efficient means for generation of transgenic founder goats, especially in regard to the number of animals required to produce a transgenic founder expressing the protein of interest. Vectors can be designed for organ-specific expression and secretion of recombinant proteins within the target tissue. Furthermore, donor cells can be selected for gender, genetically modified to introduce the transgene of interest and screened for incorporation of the transgene into the genome before use in NT. This chapter describes methods for production of transgenic donor cells, subsequent NT embryo production, and transfer into recipients.

Animals↗

PubMed2005

Telomere length analysis in goat clones and their offspring.

Incomplete epigenetic reprogramming of the donor genome is believed to be the cause behind the high rate of developmental mortality and post-natal anomalies observed in animal clones. It appears that overt phenotypic abnormalities are not transmitted to their progeny suggesting that epigenetic errors are corrected in the germline of clones. Here, we show variation in telomere lengths among Nigerian dwarf goat clones derived from different somatic cell types and that the offspring of two male clones have significantly shorter telomere lengths than age-matched noncloned animals. Telomere lengths were significantly shorter in skin biopsies of goat clones derived from adult granulosa cells compared to those measured for controls. Telomere lengths were highly variable in male goat clones reconstructed from fetal fibroblasts but their mean terminal repeat fragment (TRF) length was within normal range of normal goats. However, in the progeny of two male clones, mean TRF lengths were considerably shorter than age-matched controls for both skin and leukocyte samples. Evidence for possible inheritance of shortened telomeres was obtained by measuring telomere lengths in testicular biopsies obtained from the clones, which when compared with those from noncloned animals of a similar age were significantly shorter. The offspring exhibited telomere lengths intermediate to the TRF values obtained for their cloned fathers' and age-matched control testes. These results demonstrate that telomere length reprogramming in clones is dependent on the type of donor cell used and that the progeny of clones may inherit telomere length alterations acquired through the cloning procedure.

Aging↗

PubMed2003

Developmental competence of prepubertal and adult goat oocytes cultured in semi-defined media following laparoscopic recovery.

With an increased interest in transgenic animal production, the caprine species offers many advantages, and the prepubertal goat is a potential source of large numbers of oocytes for in vitro embryo production. The aim of the present study was to evaluate the follicular response and recovery of oocytes from prepubertal and adult goats following ovarian stimulation and laparoscopic recovery, and their developmental competence following culture in semi-defined media. Oocytes were collected over a 15-week period from prepubertal goats (3-7 months old) and adult controls (2-4 years old) that had been subjected to estrus synchronization and ovarian stimulation. Following insemination, zygotes were cultured for 96h in G1.2 followed by an additional 120h in G2.2. Morulae and blastocysts were scored using light microscopy on Days 7 and 9 followed by fluorescent staining for cell counts on Day 9 (216h postinsemination). The mean numbers of follicles aspirated and oocytes recovered were significantly greater for prepubertal than for adult goats (P<0.01). The number of oocytes recovered from prepubertal goats was observed to decline significantly with increasing age of the animals (P<0.05). The proportion of oocytes that matured and cleaved did not differ significantly between prepubertal and adult goats. Furthermore, no significant differences in morulae development (percentage of those cleaved), 5% versus 4%, or blastocyst development, 6% versus 7%, were observed for prepubertal and adult derived oocytes (P>0.1), respectively. Mean cell number per blastocyst also did not differ significantly. In conclusion, higher yields of oocytes were obtained from gonadotrophin-primed, prepubertal does than from adults, while in vitro development was similar.

Animals↗

PubMed2003

Cryopreservation of goat oocytes and in vivo derived 2- to 4-cell embryos using the cryoloop (CLV) and solid-surface vitrification (SSV) methods.

This study evaluated the efficiency and toxicity of two cryopreservation methods, solid-surface vitrification (SSV) and cryoloop vitrification (CLV), on in vitro matured oocytes and in vivo derived early stage goat embryos. In the SSV method, oocytes were vitrified in a solution of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4% trehalose. Microdrops containing the oocytes were cryopreserved by dropping them on a cold metal surface that was partially immersed in liquid nitrogen. In the cryoloop method, oocytes were transferred onto a film of the CLV solution (20% DMSO, 20% EG, 10mg/ml Ficoll and 0.65 M sucrose) suspended in the cryoloop. The cryoloop was then plunged into the liquid nitrogen. In vivo derived embryos were vitrified using the same procedures. The SSV microdrops were warmed in a solution of 0.3M trehalose and those vitrified with CLV were warmed with incubation in 0.25 and 0.125 M sucrose. Oocytes and embryos vitrified by the SSV method had a significantly lower survival rate than the control (60 and 39% versus 100%, respectively; P<0.05), while the survival rate of CLV oocytes and embryos (89 and 88%, respectively) did not differ from controls. Cleavage and blastocyst rates of the surviving vitrified oocytes (parthenogenetically activated) and embryos (cultured for 9 days) were not significantly different (P>0.05) from the control nor did they differ between vitrification methods. Embryos vitrified with the CLV method gave rise to blastocysts (2/15). Our data demonstrated that the two vitrification methods employed resulted in acceptable levels of survival and cleavage of goat oocytes and embryos.

Animals↗

PubMed2003

Influence of GnRH administration on timing of the LH surge and ovulation in dwarf goats.

This study was conducted to determine whether or not exogenous gonadotropin releasing hormone (GnRH) alters the timing or improves the synchrony of estrus, the LH surge, and ovulation following estrous synchronization in dwarf goats, and to assess the effects of season on these parameters. In January and June, estrus was synchronized in 12 Pygmy and Nigerian Dwarf goats with a 10-day progestagen sponge, 125 microg cloprostenol i.m. 48 h before sponge removal, and 300 IU equine chorionic gonadotrophin (eCG) i.m. at sponge removal. Six of the 12 goats were given 50 microg GnRH i.m. 24h after sponge removal. Onset of estrus was monitored using two males. Samples for plasma LH were collected at 2 h intervals beginning 22 h after sponge removal and ending at 48 h in January and at 58 h in June. Time of ovulation time was confirmed by laparoscopy at 36, 50, 60, and 74 h in January and at 50, 60, and 74 h in June. Administration of GnRH had no significant effect on the onset of estrus; however, it reduced the interval from sponge removal to the LH surge and improved the synchrony of the LH surge (P<0.05). Treatment with GnRH also reduced the interval from sponge removal to ovulation and improved the synchrony of ovulation (P<0.05). Season had a significant effect on the timing and the synchrony of estrus with and without GnRH treatment (P<0.05). A seasonal shift was also observed in the timing of the LH surge in the absence of GnRH treatment (P<0.05). Further research is required to determine the optimum time for GnRH administration and the minimum effective dose in dwarf goats.

Administration, Intravaginal↗