The effect of exogenous follicle stimulating hormone (FSH) and endogenous plasma leptin concentrations on the pregnancy rate of beef heifers subjected to fixed-timed artificial insemination (FTAI).

In this study, 53 crossbred beef heifers were used to test the hypotheses that administration of exogenous FSH 2 days following CIDR insertion and administration of estradiol would increase the pregnancy rate in heifers synchronized for FTAI and that plasma leptin concentrations in beef heifers would be higher for heifers that became pregnant to FTAI. The heifers used in this study had a median age of 440 days, an average weight of 324 kg, an average body condition score of 5.1 and a mean reproductive tract score of 3.1. Heifers were stratified by weight and BCS into two groups and then treatments were randomly allotted to each group: (1) control (n=28) or (2) FSH (n=27). Both groups were administered 200mg estradiol benzoate (EB) and received an intravaginal controlled internal drug-releasing device (CIDR) on day 0. On day 2, females in the FSH treatment group were administered 20 mg of FSH, while the control group received 1 ml of saline. On day 7 all females were administered 25 µg PGF2α and the CIDR was removed. Then 24h following CIDR removal all females were administered 1mg EB and 24h later were subjected to FTAI. Pregnancy diagnosis was performed via transrectal ultrasonography 43 days following insemination. Blood samples were collected via jugular venipuncture on days 2, 6-10, 13 and 52 and plasma leptin concentrations were determined by radioimmunoassay. Pregnancy rates were higher (P=0.01) for FSH-treated females (60%) compared with females not receiving FSH (25%). Circulating plasma leptin concentrations were higher (P=0.0051) for pregnant females compared with females that did not become pregnant following FTAI during the experiment. Mean plasma leptin concentration was also higher (P=0.04) from day 2 to day 9 during the synchronization protocol in heifers that became pregnant compared with heifers that did not become pregnant from FTAI. There was no difference (P=0.38) in reproductive tract scores for heifers that became pregnant compared with heifers that did not become pregnant from FTAI. Circulating leptin concentrations were not different (P=0.11) for females receiving FSH compared with females in the non FSH-treated group. Circulating leptin concentrations were affected by sampling day (P<0.0001). However, there was no interaction between sampling day and pregnancy status (P=0.80), treatment and pregnancy status (P=0.14) or treatment and sampling day (P=0.12). These results indicate that the administration of FSH on day 2 of the synchronization protocol may increase pregnancy rates in beef heifers and that increased circulating concentrations of plasma leptin during the synchronization protocol may be indicative of subsequent pregnancy outcome.

Daily or thrice weekly handling of eland antelope (Taurotragus oryx): effects on serum cortisol level.

We evaluated the cortisol response of adult female eland (n=8) that were handled in hydraulic chute daily or 3×/week. Females were divided into two groups and each group (n=4) successively received two estrous cycle synchronization treatments: (1) two injections of prostaglandin (PG-PG) F2α at 11 day intervals and (2) oral administration of altrenogest for 7 days and an injection of PGF2α on day 7 (Alt-PG). Blood samples were collected 3×/week during the synchronization (Synch) and expected luteal phase (Nonintensive) periods, and daily during the expected time of induced (Intensive 1) or natural (Intensive 2) estrus. Overall, mean cortisol levels were highest during Intensive 1, followed by Intensive 2, Synch and Nonintensive periods. Individual eland were the most significant source of variation for cortisol level. The frequency of handling and the synchronization treatment significantly affected cortisol levels in 3/8 and 4/8 females, respectively. In conclusion, in response to increased frequency of handling, eland cortisol levels rose transiently and returned to baseline within few days after more intensive handling. Thus, the eland females were tolerant to and recovered from the effects of repeated daily handling.

The effect of equine chorionic gonadotropin (eCG) on pregnancy rates of white-tailed deer following fixed-timed artificial insemination.

Control of the white-tailed doe's reproductive cycle is not well documented. The objective was to determine the effects of giving equine chorionic gonadotropin (eCG) at progesterone device removal on fixed time artificial insemination (FTAI) pregnancy rates in white-tailed does. All does (n = 74) were synchronized with a vaginal progesterone implant (CIDR; 0.3 g progesterone), inserted on Day 0 (without regard to stage of estrous cycle), removed 14 days later, and subjected to FTAI, on average, 60 h post-CIDR removal. Of these, 34 were given 200 IU (im) of eCG at CIDR removal. Overall, FTAI pregnancy rate was 50% across 2 yrs (effect of year, P = 0.35). Administration of eCG at CIDR removal did not affect (P = 0.16) pregnancy rate (eCG = 59%; no eCG = 43%). Pregnancy rates were not affected by vulva score or doe disposition. Does that were ≤ 4 yrs old were more likely (P = 0.01) to become pregnant than does > 4 yrs of age. Does inseminated ≥ 60.5 h after CIDR removal were 22 times more likely (P = 0.002) to become pregnant to FTAI than does inseminated < 60.5 h. When frozen-thawed semen was deposited in the cervix or uterus, does were 17 times more likely (P = 0.005) to become pregnant compared with those receiving intravaginal insemination. Fecundity was not different (P = 0.73) across treatment groups (1.6 ± 0.11; no eCG vs. 1.7 ± 0.10; eCG). Furthermore, fecundity of does pregnant to FTAI was not different (P = 0.72) compared with does pregnant to clean-up bucks (1.7 ± 0.08; AI does vs. 1.7 ± 0.09; clean-up bucks). In summary, white-tailed does were successfully inseminated using a 14 days FTAI protocol, eCG may not be essential for acceptable pregnancy rates, and increased pregnancy rates may result when FTAI is done ≥ 60.5 h after progesterone device removal.

Persistent breeding-induced endometritis after hysteroscopic insemination in the mare.

Low-dose insemination has been proposed to reduce persistent breeding-induced endometritis (PBIE) in mares with delayed uterine clearance (DUC). Others proposed that hysteroscopic insemination induces an exaggerated inflammatory response and should be avoided in DUC mares. The objectives here were to evaluate presence and severity of PBIE in normal and DUC mares after hysteroscopic insemination with fresh semen, and to determine if hysteroscopy could be used in DUC mares without inducing excessive inflammation. Reproductively normal (n = 4) and DUC (n = 5) mares received four treatments in random order: uterine body insemination (UB, 1 × 10(9) spermatozoa, 20 ml), hysteroscopic insemination (HYST, 5 × 10(6) spermatozoa, 0.5 ml), sham hysteroscopic insemination (SHAM, semen extender, 0.5 ml) and hysteroscopic infusion of seminal plasma (SP, 0.5 ml). Significantly more DUC (50%) mares than normal (14%) mares accumulated intrauterine fluid 24 h post-treatment. The difference in fluid accumulation between DUC (40%) mares and normal (7%) mares was also significant 48 h post-treatment. Fluid scores were not significantly different between treatments in normal mares. However, treatments HYST and SHAM resulted in significantly higher fluid scores 24 h but not 48 h post-treatment in DUC mares. There was no effect of treatment or mare group on the percentage and total number of neutrophils in uterine fluid 48 h post-treatment. Percentage of neutrophils was correlated with duration of hysteroscopy in normal mares, with procedures lasting ≥ 9 min associated with PBIE. There was no effect of mare group, treatment or duration of hysteroscopy on pregnancy rate. Hysteroscopy induces a transient inflammation that is not more severe than that after conventional artificial insemination, suggesting no contraindication to its use in DUC mares.

Progesterone enhances in vitro development of bovine embryos.

Increased pregnancy rates in cattle given progesterone (P4) prior to 5 d after breeding have recently been reported. The objective was to determine if this increase in pregnancy rate could be attributed to a direct positive effect of P4 on the developing embryo. In Experiment 1, 280 bovine oocytes were inseminated in vitro and at Day 3 (insemination=Day 0), good quality 8 cell embryos (n=206) were randomly allocated to be cultured in either CR1aa+serum with 0 or ∼15 ng/mL (n=102 and n=104, respectively). In Experiment 2, 881 bovine oocytes were used; on Day 3, good quality 8 cell embryos (n=511) were randomly allocated to either the control (CR1aa+FCS, n=168), vehicle (CR1aa+FCS+ethanol, n=170), or P4 treatment (CR1aa+FCS+∼15 ng/mL P4 in ethanol, n=173). On Day 7, in both experiments, there were increased numbers of blastocysts developing in the P4 group (Experiment 1, 59% and Experiment 2, 71%) compared to the vehicle (Experiment 2, 53%) or control (40 and 62% in Experiments 1 and 2, respectively). The addition of P4 (8%) stimulated the rate of embryo development (early blastocysts or more advanced stages on Day 6) compared to vehicle (3%) and control (0%) and the P4 group had more hatched or hatching blastocysts (33%) on Day 9 compared to the control or vehicle group (21 or 22%). Additionally, the P4 group had greater embryo diameter and significantly more Grade 1 blastocysts on Day 7. In conclusion, P4 had a direct, positive effect on developing bovine embryos cultured in vitro.

Distribution of sexes within the left and right uterine horns of cattle.

In cattle, limited data are available regarding the sex ratio of the offspring in relation to the horn of gestation. Therefore, the objective of this study was to evaluate the sex ratio of fetuses gestated in the left and right uterine horns of cattle (Bos taurus, Bos indicus and crosses). The distribution of male and female fetuses in the left and right uterine horn was analyzed on gravid, abattoir-derived reproductive tracts and artificially inseminated crossbred cows. The total number of fetuses/calves and the sex of the fetuses/calves gestated in each uterine horn were used as the end point for side comparisons using the Glimmix Procedure. Of 64 gravid reproductive tracts evaluated, 29 (45.3%) pregnancies occurred in the left uterine horn, whereas 35 (54.7%) occurred in the right. The sex ratio (% males) of fetuses in the left uterine horn (37.9%) was significantly lower than the sex ratio detected in the right uterine horn (65.7%). Of 113 pregnancies evaluated in artificially inseminated heifers, 53 (46.9%) occurred in the left uterine horn, whereas 60 (53.1%) occurred in the right uterine horn. The sex ratio of calves gestated in the left uterine horn (35.8%) was significantly lower than the sex ratio of calves gestated in the right uterine horn (63.3%). In conclusion, in these experiments, a significantly greater proportion of males were gestated in the right uterine horn of cattle and a greater proportion of females in the left uterine horn. Further investigation is needed to determine the mechanisms underlying the observed disparity of the expected sex ratio within the uterine horns of cattle.

Comparison of the permeability properties and post-thaw motility of ejaculated and epididymal bovine spermatozoa.

There are very few experimental reports on the comparative water transport (membrane permeability) characteristics of ejaculated and epididymal mammalian spermatozoa during freezing. In the present study, we report the effects of cooling ejaculated and epididymal bovine sperm from the same males with and without the presence of a cryoprotective agent, glycerol. Water transport data during freezing of ejaculated and epididymal bovine sperm suspensions were obtained at a cooling rate of 20 degrees C/min under two different conditions: (1) in the absence of any cryoprotective agents, CPAs and, (2) in the presence of 0.7 M glycerol. Using values published in the literature, we modeled the spermatozoa as a cylinder of length 39.8 microm and a radius of 0.4 microm with an osmotically inactive cell volume, V(b), of 0.61 V(o), where V(o) is the isotonic cell volume. The subzero water transport response is analyzed to determine the variables governing the rate of water loss during cooling of bovine spermatozoa, i.e. the membrane permeability parameters (reference membrane permeability, L(pg) and activation energy, E(Lp)). The predicted best-fit permeability parameters ranged from, L(pg)=0.021-0.038 microm/min-atm and E(Lp)=27.8-41.1 kcal/mol. The subzero water transport response and consequently the subzero water transport parameters are not significantly different between the ejaculated and epididymal bovine spermatozoa under corresponding cooling conditions. If this observation is found to be more generally valid for other mammalian species as well, then in the future the sperm extracted from the testes of a postmortem male could be optimally cryopreserved using procedures similar to those derived for ejaculated sperm.

Effects of culture medium and protein supplementation on mRNA expression of in vitro produced bovine embryos.

Numerous studies have reported aberrant gene expression levels attributed to suboptimal in vitro culture conditions. This study investigated the effects of different culture systems and protein sources on the developmental competence of in vitro production (IVP) embryos measured by cleavage and blastocyst rates, cell number, and relative abundance of POU5F1 (OCT4), nanog, GJA1 (connexin 43), and SLC2A1 (GLUT1) transcripts when compared to in vivo embryos. Experiment 1 compared IVP embryos cultured in either synthetic oviductal fluid (SOFaa) or potassium simplex optimized medium supplemented with amino acids (KSOMaa). Experiment 2 compared the same two culture systems with and without the addition of calf serum (CS). Results from both experiments indicated that despite similar developmental rates, significant differences were observed at the mRNA level. In Experiment 1, OCT4 was the only transcript to have a mean abundance level significantly higher in KSOMaa blastocysts when compared with both SOFaa and in vivo embryos. The same pattern of upregulation of OCT4 mRNA was noted in Experiment 2. There were no significant alterations of the ICM specific transcript nanog in either experiment. In contrast to reports by others, connexin 43 mRNA was not expressed at detectable levels in in vivo embryos analyzed in our studies. Blastocysts cultured in SOFaa with CS or KSOMaa had a significant upregulation of GLUT1 mRNA when compared with other treatments and in vivo embryos. Until differences between IVP and in vivo embryos are minimized, aberrations in IVP will continue to arise.

In vitro production of cattlexbuffalo hybrid embryos using cattle oocytes and African buffalo (Syncerus caffer caffer) epididymal sperm.

Interspecies hybridization of bovids occurs between domestic cattle and at least three other species; American bison (Bison bison), yak (Bos grunniens) and banteng (Bos banteng). Birth of a cattlexbuffalo (Bubalus bubalis) hybrid has reportedly occurred in Russia and in China, but these reports were not authenticated. Such hybrids could be important in improving livestock production and management of diseases that impede production in tropical Africa. This study investigated hybridization between cattle and its closest African wild bovid relative, the African buffalo (Syncerus caffer caffer). In an attempt to produce cattlexbuffalo hybrid embryos in vitro, matured cattle oocytes were subjected to a standard in vitro fertilization (IVF) procedure with either homologous cattle (n=1166 oocytes) or heterologous African buffalo (n=1202 oocytes) frozen-thawed epididymal sperm. After IVF, 67.2% of the oocytes inseminated with the homologous cattle sperm cleaved. In contrast, fertilization with buffalo sperm resulted in only a 4.6% cleavage rate. The cleavage intervals were also slower in hybrid embryos than in the IVF-derived cattle embryos. Of the cleaved homologous cattle embryos 52.2% progressed to the morula stage compared with 12.7% for the buffalo hybrid embryos. No hybrid embryos developed beyond the early morula stage, while 40.1% of the cleaved cattlexcattle embryos developed to the blastocyst stage. Transfer of buffalo hybrid IVF embryos to domestic cattle surrogates resulted in no pregnancies at 60 days post-transfer. This study indicates that interspecies fertilization of cattle oocytes with African buffalo epididymal sperm can occur in vitro, and that a barrier to hybridization occurs in the early stages of embryonic development. Chromosomal disparity is likely the cause of the fertilization abnormalities, abnormal development and subsequent arrest impairing the formation of hybrid embryos beyond the early morula stage. Transfer of the buffalo hybrid embryos did not rescue the embryos from development arrest.

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.

In vitro production of llama (Lama glama) embryos by intracytoplasmic sperm injection: effect of chemical activation treatments and culture conditions.

Assisted reproductive technologies in the llama (Lama glama) are needed to provide alternative methods for the propagation, selection and genetic improvement; however, recovery of adequate quantity and quality of spermatozoa for conventional IVF is problematic. Therefore, an effort was made to adapt the intracytoplasmic sperm injection (ICSI) procedure for the in vitro production of llama embryos. The specific objectives of this study were: (1) to determine in vitro maturation rates of oocytes recovered by transvaginal ultrasound-guided oocyte aspiration (TUGA) or flank laparotomy; (2) to evaluate the effects of activation treatments following ICSI; (3) to evaluate the development of llama ICSI embryos in CR1aa medium or in an oviduct cell co-culture system. Llamas were superstimulated by double dominant follicle reduction followed by oFSH administered in daily descending doses over a 3-day interval. Oocytes were harvested by flank laparotomy or TUGA and matured in vitro for 30 h. Mature oocytes were subjected to ICSI followed by no chemical activation (Treatment A), ionomycin only (Treatment B) or ionomycin/DMAP activation (Treatment C). More oocytes were recovered by flank laparotomy procedure compared with TUGA (94% versus 61%, P<0.05) and a greater number of oocytes harvested by flank laparotomy reached the metaphase-II stage (77% versus 44%, P<0.05). After ICSI, the proportion of cleaved and 4-8-cell stages embryos was significantly greater when injected oocytes were activated with ionomycin/DMAP combination (63% and 38%, respectively, P<0.05). The co-culture of ICSI embryos with llama oviduct epithelial cells resulted in progression to morula (25%) and blastocyst (12%) stages; whereas, all embryos cultured in CR1aa medium arrested at the 8-16-cell developmental stage.

Effect of glycerol and cholesterol-loaded cyclodextrin on freezing-induced water loss in bovine spermatozoa.

Recent experimental data show that incubating bovine sperm with cholesterol-loaded cyclodextrin (CLC) before cryopreservation increases the percentages of motile and viable cells recovered after freezing and thawing, compared with control sperm. In the present study, we report the effect of incubating bovine sperm with CLC on the subzero water transport response and the membrane permeability parameters (reference membrane permeability (L(pg)) and activation energy (E(Lp))). Water transport data during freezing of bovine sperm cell suspensions were obtained at a cooling rate of 20 degrees C/min under three different conditions: 1. in the absence of cryoprotective agents (CPAs); 2. in the presence of 0.7 M glycerol; and 3. in the presence of 1.5 mg/ml CLC and 0.7 M glycerol. With previously published values, the bovine sperm cell was modeled as a cylinder of length 39.8 microm and radius 0.4 microm, with osmotically inactive cell volume (V(b)) of 0.61 V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained data, the best-fit water transport parameters (L(pg) and E(Lp)) were determined. The predicted best-fit permeability parameters ranged from L(pg) = 0.02 to 0.036 microm/min-atm and E(Lp) = 26.4 to 42.1 kcal/mol. These subzero water transport parameters are significantly different from the suprazero membrane permeability values (obtained in the absence of extracellular ice) reported in the literature. Calculations made of the theoretical response of bovine spermatozoa at subzero temperatures suggest that the optimal cooling rate to cryopreserve bovine spermatozoa is 45-60 degrees C/min, agreeing quite closely with experimentally determined rates of freezing bovine spermatozoa.

Transfer of inner cell mass cells derived from bovine nuclear transfer embryos into the trophoblast of bovine in vitro-produced embryos.

Presence of placental tissues from more normal noncloned embryos could reduce the pregnancy failure of somatic cloning in cattle. In this study, inner cell mass (ICM) cells of in vitro-produced (IVP) embryos was replaced with those of nuclear transfer (NT) embryos to reconstruct bovine blastocysts with ICM and trophoblast cells from NT and IVP embryos, respectively. A total of 65 of these reconstructed embryos were nonsurgically transferred to 20 recipient beef females. Of those, two females were diagnosed pregnant by ultrasonography on day 30 of gestation. One pregnancy was lost at 60-90 days of gestation, and the other recipient cow remained pregnant at day 240 of gestation; however, this female died on day 252 of gestation. Gross pathology of the internal organs of the recipient female, a large fetus, and a large placental tissue mass suggested the massive size of the fetus and placental tissue were likely involved in terminating the life of the recipient female. Biopsy samples were harvested from the skin of the dead recipient cow, the fetus and from cotyledonary tissue. Microsatellite DNA analysis of these samples revealed that the genotype of the fetus was the same as that of the NT donor cells and different from that of the recipient cow. Correspondingly, neither the fetus nor recipient cow had the same genotype with that of the fetal cotyledonary tissue. These results present the first known documented case of a bovine somatic NT pregnancy with nonclone placental tissues after transfer of a blastocyst reconstructed by a microsurgical method to exchange of ICM cells and trophoblast tissue between NT and IVP blastocysts.

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 10min after the first ejaculation. Semen was processed using the standard Salamon cryopreservation procedure in a Tris-yolk-glycerol extender, frozen in 0.5ml plastic straws using liquid nitrogen (LN(2)) vapor and stored in LN(2). Each ejaculate was evaluated for volume, sperm concentration/ml (x10(9)/ml), number of spermatozoa/ejaculate (x10(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 10min later. The mean semen volume, sperm concentration and number of spermatozoa per ejaculate obtained from the first ejaculate were significantly greater (P< or =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 degrees Celsius), cooled (5 degrees Celsius) and frozen (-196 degrees Celsius) post-thawed spermatozoa was less (P< or =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 10min 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.

Health and reproductive profiles of malaria antigen-producing transgenic goats derived by somatic cell nuclear transfer.

Nuclear transfer (NT) using transfected primary cells is an efficient approach for the generation of transgenic goats. However, reprogramming abnormalities associated with this process might result in compromised animals. We examined the health, reproductive performance, and milk production of four transgenic does derived from somatic cell NT. Goats were derived from two fetal cell lines, each transfected with a transgene expressing a different version of the MSP-1(42) malaria antigen, either glycosylated or non-glycosylated. Two female kids were produced per cell line. Health and growth of these NT animals were monitored and compared with four age-matched control does. There were no differences in birth and weaning weights between NT and control animals. The NT does were bred and produced a total of nine kids. The control does delivered five kids. The NT does expressing the glycosylated antigen lactated only briefly, probably as a result of over-expression of the MSP-1(42) protein. However, NT does expressing the non-glycosylated antigen had normal milk yields and produced the recombinant protein. These data demonstrated that the production of healthy transgenic founder goats by somatic cell NT is readily achievable and that these animals can be used successfully for the production of a candidate Malaria vaccine.

Banteng (Bos javanicus) embryos and pregnancies produced by interspecies nuclear transfer.

The banteng (Bos javanicus), a member of the bovidae family, is currently listed as threatened by the IUCN Red List and it is estimated the total world population is <10,000 animals. In exotic or endangered species, the lack of oocytes and recipients precludes the use of traditional somatic cell nuclear transfer (NT), and an approach such as interspecies NT may be the only alternative to produce embryos and offspring. A total of 348 enucleated domestic bovine oocytes were reconstructed with either male (Treatment A) or female (Treatment B) adult banteng fibroblasts and a total of 103 bovine oocytes were parthenogenically activated as a control (Treatment C). There was no significant difference in fusion rate (68 versus 77%) between Treatments A and B. Of fused couplets, those in Treatment A had greater (P < 0.05) cleavage (67 versus 51%) and blastocyst (28 versus 15%) rate than Treatment B. Of a total of 24 blastocysts transferred into 12 domestic cattle recipients from Treatment A, two pregnancies (17%) were established with heart beats detectable at 30 day by rectal ultrasonography. No pregnancies resulted from the transfer of 14 blastocysts from Treatment B. Both pregnancies were subsequently lost, one between 30 and 60 days and the second between 60 and 90 days of gestation. The bovine cytoplast supported mitotic cleavage of banteng karyoplasts, and was capable of reprogramming the nucleus to achieve blastocyst stage embryos and pregnancies in exotic bovids.

Subzero water transport characteristics of boar spermatozoa confirm observed optimal cooling rates.

Incomplete understanding of the water transport parameters (reference membrane permeability, L(pg), and activation energy, E(Lp)) during freezing in the presence of extracellular ice and cryoprotective agents (CPAs) is one of the main limiting factors in reconciling the difference between the numerically predicted value and the experimentally determined optimal rates of freezing in boar (and in general mammalian) gametes. In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the water transport during freezing of boar spermatozoa. Water transport data during freezing of boar sperm cell suspensions were obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and 6% (v/v) glycerol. Using previously published values, the boar sperm cell was modeled as a cylinder of length 80.1 microm and a radius of 0.31 microm with an osmotically inactive cell volume, V(b), of 0.6 V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained data, the best-fit water transport parameters (L(pg) and E(Lp)) were determined. The "combined-best-fit" parameters at 5 and 20 degrees C/min for boar spermatozoa in the presence of extracellular ice are: L(pg) = 3.6 x 10(-15) m(3)/N. s (0.02 microm/min-atm) and E(Lp) = 122.5 kJ/mole (29.3 kcal/mole) (R(2) = 0.99); and the corresponding parameters in the presence of extracellular ice and glycerol are: L(pg)[cpa] = 0.90 x 10(-15) m(3)/N. s (0.005 microm/min-atm) and E(Lp)[cpa] = 75.7 kJ/mole (18.1 kcal/mole) (R(2) = 0.99). The water transport parameters obtained in the present study are significantly different from previously published parameters for boar and other mammalian spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The theoretically predicted optimal rates of freezing using the new parameters ( approximately 30 degrees C/min) are in close agreement with previously published but experimentally determined optimal cooling rates. This analysis reconciles a long-standing difference between theoretically predicted and experimentally determined optimal cooling rates for boar spermatozoa.

Concentrations of nitric oxide in equine preovulatory follicles before and after administration of human chorionic gonadotropin.

In the present study, follicular fluids of estrous mares treated with saline solution (Control) or nitric oxide synthase (NOS) inhibitors were analyzed for nitric oxide (NO), estradiol-17beta (E2) and progesterone (P4) concentrations before and 36h after administration of human chorionic gonadotropin (hCG). Follicular fluids obtained before (0h) hCG administration from control mares had lower concentrations of NO than those obtained 36h after administration of hCG (58.3+/-17.8 micromol versus 340.4+/-57.7 micromol; P<0.05). A similar pattern was also noted for intrafollicular P4 in control mares, which had lower concentrations of intrafollicular P4 before hCG than 36h post-hCG administration (P<0.05). As expected, E2 concentrations of control follicles sampled before hCG administration were higher than those sampled 36h post-hCG administration (P<0.05). However, the E2 concentrations in follicles of mares treated with the NOS inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine (AG) did not decrease after hCG administration, unlike those in control mares (P>0.10). In addition, mares treated with NOS inhibitors had lower intrafollicular concentrations of NO and P4 than control mares, both before and after hCG administration (P<0.05). Increased intrafollicular concentrations of NO in control, hCG-stimulated mares provide evidence for the presence of an NO-generating system in the equine preovulatory follicle that is likely upregulated following administration of hCG.

Comparison of hybrid and purebred in vitro-derived cattle embryos during in vitro culture.

Frozen-thawed spermatozoa collected from a beef bull (Japanese Black) were used for in vitro fertilization (IVF) of matured oocytes obtained from dairy (Holstein) and beef (Japanese Black) females. Embryos were examined for fertilization, cleavage rate, interval between insemination and blastocyst production (experiment I), total cell number per embryo and sex ratio during blastocyst formation (experiment II), and blastocyst production rate of zygotes that developed to 2-, 4-, and 8-cell stages at 48h post-fertilization (experiment III). Fertilized oocytes were cultured in vitro on a cumulus cell co-culture system. The fertilization and cleavage rate of oocytes groups were similar, however, the blastocyst production rate was greater (P<0.05) in hybrid than from purebred embryos (27% versus 20%). Development of blastocysts produced from hybrid embryos developed at a faster rate than blastocysts produced from the straightbred embryos. In hybrid embryos, blastocyst production was significantly greater on day 7 (56%) and gradually decreased from 20% on day 8 to 17% on day 9. In contrast, blastocyst production rate from the purebred embryos was lower on day 7 (17%), increasing on day 8 to 59% and then decreased on day 9 to 24%. The total number of cells per embryo and sex ratio of in vitro-produced blastocysts were not different between hybrid and purebred embryos. The number of blastocysts obtained from embryos at the 8-cell stage of development by 48h post-fertilization (94%) was greater (P<0.01) than the number of zygotes producing blastocysts that had developed to the 4-cell stage (4%) and the 2-cell stage (2%) during the same interval. These results show that the blastocyst production rate and developmental rate to the blastocyst stage were different between hybrid and purebred embryos, and that almost all of the in vitro-produced blastocysts were obtained from zygotes that had developed to the 8-cell stage 48h post-fertilization.