Pregnancy recognition and abnormal offspring syndrome in cattle.

Development of the post-hatching conceptus in ruminants involves a period of morphological expansion that is driven by complex interactions between the conceptus and its intrauterine environment. As a result of these interactions, endometrial physiology is altered, leading to establishment of the pregnancy and continued development of the placenta. Disruption of normal fetal and placental development can occur when embryos are exposed to manipulations in vitro or when inappropriate endocrine sequencing occurs in vivo during the pre- and peri-implantation periods. The present review addresses the development of the post-hatching bovine conceptus, its interactions with the maternal system and changes in development that can occur as a result of in vivo and in vitro manipulations of the bovine embryo.

Assessment of ovulation synchronization protocols in goats and use of pregnancy specific protein B (PSPB) enzyme linked immunsorbent assay (ELISA) to determine kid number at birth.

The efficacy of several protocols for ovulation synchronization and timed artificial insemination (TAI) in goats was examined. In addition, the relationship between levels of pregnancy specific protein B (PSPB) during gestation assessed with a commercially available ELISA and the number of offspring at birth was determined. In Experiment 1, 70 does were randomized into four treatments: (1) breed by estrus [BBE], (2) 6-d treatment with a new [C6N], (3) once-used [C61], or (4) twice-used Controled Internal Drug Release (CIDR) device [C62)]. BBE does received two 15 mg doses of prostaglandin-F2α (PGF) at a 10-d interval and were bred 12 h after estrus onset. CIDR groups received a CIDR for 6 d with 15 mg PGF given at CIDR removal. TAI was performed 48 h after CIDR removal and does were given 50 µg GnRH. All does were inseminated with a single dose of frozen semen using a non-surgical, transcervical technique. Pregnancy rates for the BBE, C6N, C61 and C62 treatment groups were 39% ± 12%, 64% ± 12%, 77% ± 12% and 57% ± 12%, respectively, and did not differ. Reuse of CIDRs, even with reuse extending for a total of 21 d, was as effective as new CIDRs for synchronization of ovulation. In Experiment 2, 68 does were randomized into four treatments: (1) BBE, (2) C6N, (3) NC.Synch [NCS], (4) modified NCS [NCSM]. The BBE and C6N groups were as described for Experiment 1. The NCS and NCSM groups received 15 mg PGF on Day 1, 50 µg GnRH on Day 8 and 15 mg PGF on Day 15 (NCS) or Day 15.5 (NCSM). Does were bred by TAI at 72 h (NCS) or 60 h (NCSM) after the second PGF injection. All does in the NCS and NCSM groups received 50 µg GnRH at TAI. Pregnancy rates were 53% ± 12%, 30% ± 11%, 50% ± 11% and 41% ± 12% for does in the BBE, C6N, NCS and NCSM group, respectively, and did not differ. In Experiment 3, 62 does pregnant to TAI were bled at Days 48 and 85 post-insemination for PSPB. Data on kid numbers and birth weights were subsequently recorded. At Day 48 of gestation, PSPB levels for does birthing singletons were lower than for does birthing twins or triplets (25.0 ± 0.1a, 28.8 ± 0.1b and 30.7 ± 0b ng/mL, respectively, abP<0.05). At Day 85 of gestation, PSPB levels were progressively greater for does birthing singletons versus twins versus triplets (27.0 ± 0.1a, 28.5 ± 0.1b and 31.6 ± 0c ng/mL, abcP<0.05). In conclusion, PSPB concentrations detected using a commercially available ELISA at Day 48 or 85 of gestation could distinguish does carrying single versus multiple fetuses.

The role of transcription in EGF- and FSH-mediated oocyte maturation in vitro.

Understanding mechanisms responsible for meiotic resumption in mammalian oocytes is critical for the identification of strategies to enhance developmental competence of in vitro-matured oocytes. Improvement of in vitro oocyte maturation systems is dependent on a better understanding of mechanisms that regulate oocyte maturation both in vivo and in vitro as well as on the identification of methods to manipulate the meiotic progression of oocytes matured in vitro in a physiological manner. The purpose of this review is two-fold: first, to examine the mechanisms that underlie the acquisition of oocyte developmental competence and regulation of oocyte maturation in vivo and in vitro; second, to present data examining the role of transcription in mediating the ability of EGF and FSH to induce oocyte maturation in vitro. Results presented support the conclusions that (1) EGF-induced oocyte maturation does not require nascent gene transcription in both mice and domestic cats; (2) FSH requires gene transcription to induce oocyte maturation in both species; (3) EGF must be present in the maturation medium to optimize the effectiveness of FSH to promote oocyte maturation; (4) the mechanism used by FSH to induce oocyte maturation in vitro appears to predominate over that used by EGF when both EGF and FSH are present in maturation medium used for either murine or feline cumulus oocyte complexes.

Identification of candidate mRNAs associated with gonadotropin-induced maturation of murine cumulus oocyte complexes using serial analysis of gene expression.

In cultured cumulus oocyte complexes (COC), FSH induces gene transcription required for germinal vesicle breakdown (GVBD). Experiments were performed to determine the critical period when gene transcription is required for GVBD and to identify candidate mRNAs involved. Experiment I: murine COC were cultured 4 h in the presence of FSH with 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) added at different intervals after the start of culture. COC cultured with FSH underwent GVBD (82 +/- 7%). When DRB was added at 0, 5, or 10 min after culture initiation, oocyte maturation was blocked (17 +/- 7, 14 +/- 6, and 21 +/- 6% GVBD, respectively). When DRB was added after 15, 20, or 30 min, progressively more COC underwent GVBD (37 +/- 6, 39 +/- 6, and 66 +/- 6%, respectively). The critical period of transcription required for GVBD occurred between 15 and 30 min after culture initiation. Experiment II: COC were cultured for 25 min in the presence (plusDRB) or absence (minusDRB) of DRB. SAGE libraries were generated from COC RNA of each treatment group. A total of 48,431 and 45,367 tags were sequenced for the plusDRB and minusDRB libraries, respectively. Criteria used to identify transcripts of interest included a total tag count of at least 10 across both libraries and a threefold or greater difference in expression between libraries. Using these criteria, 39 and 27 transcripts were identified as differentially expressed at the P < or = 0.01 and P

In situ localization of mRNA for the interferon, ovine trophoblast protein-1, during early embryonic development of the sheep.

The embryonic interferon, ovine trophoblast protein-1 (oTP-1), is considered to be the major protein signal by which the developing ovine conceptus communicates its presence to the mother in order to provide extension of luteal progesterone secretion critical for the establishment of pregnancy. The objective of the present study was to examine the distribution of mRNA for oTP-1 in developing ovine embryos by using in situ hybridization. A total of 11 ovine embryos were collected on days 11, 13, 15, 17, and 23 of gestation (n = 1, 2, 3, 3, and 2, respectively) and were subjected to either immediate paraformaldehyde fixation or culture for 24 h followed by fixation. Fixed embryos were embedded in paraffin and oTP-1 mRNA levels determined by in situ hybridization with a 35S-labeled cDNA probe specific for the 3'-untranslated region of the oTP-1 mRNA. Controls included parallel hybridizations with a 35S-labeled gamma-actin cDNA to detect actin mRNA (positive control) and with 35S-labeled plasmid cDNA (negative control). Hybridization signals were detected by autoradiography and quantified by computer-assisted video image analysis. Ovine TP-1 mRNA levels in tissue were low but detectable on day 11, rose 6.5-fold to peak concentrations on day 13, and declined in a linear fashion through day 23. A low, detectable signal was present in portions of chorionic tissue on day 23. Messenger RNA was localized solely to the trophectoderm and did not appear in the extraembryonic endoderm, yolk sac, and embryonic disc. The relative hybridization signal for actin mRNA was approximately 12-fold lower than that for oTP-1 mRNA on day 13. However, by day 17 oTP-1 and actin mRNA hybridization signals were similar. In conclusion, oTP-1 mRNA is localized in the trophectoderm of the developing embryo, being produced between days 11 and 23 of gestation with peak amounts produced per cell at approximately day 13 of gestation.

Maternal serum progesterone concentration and early conceptus development of bovine embryos produced in vivo or in vitro.

The hormone progesterone is essential for proper embryonic development. The objective of this study was to examine the relationship between recipient serum concentrations of progesterone, at the time of embryo transfer and at conceptus recovery, on conceptus development from in vivo- or in vitro-produced embryos. Embryos were produced in vivo by superovulation of Holstein cows (IVO; n = 17) or in vitro with either serum-containing (IVPS; n = 27) or serum-restricted medium (IVPSR; n = 34). Single grade I blastocysts from each embryo production system were transferred into heifers on day 7 of development. Conceptuses were recovered on day 17 of gestation and classified as complete, degenerated, or no conceptus. Compared with the IVO group, in vitro-produced embryos had more (P = 0.055) degenerated conceptuses (IVO, 0%; IVPS, 18.5%; and IVPSR, 20.6%). There were no differences in progesterone concentrations at the time of transfer when recipients received either male or female embryos (P > 0.05). Progesterone concentrations in recipients receiving in vivo-produced embryos were higher (P < 0.05; 3.74 ± 0.4 ng/mL; least-squares mean ± standard error of the mean) on day 7 compared with those receiving in vitro-produced embryos (IVPS, 2.4 ± 0.2; IVPSR, 2.58 ± 0.3 ng/mL). However, there was no difference in progesterone concentration on day 7 between treatment groups for heifers from which short conceptuses (≤194 mm) were recovered on day 17. In contrast, when longer (>194 mm) conceptuses were recovered on day 17, heifers receiving in vitro-produced embryos had lower (P = 0.05) serum concentrations of progesterone on day 7 compared with those receiving in vivo-produced embryos (IVPS, 2.2 ± 0.5; IVPSR, 2.3 ± 0.5; IVO, 3.9 ± 0.5 ng/mL). In conclusion, differences in autonomy may exist between in vitro- and in vivo-produced embryos during the period of conceptus elongation with in vitro-produced embryos relying more on intrinsic factors to influence elongation.

Identification of cDNAs encoding bovine cyclin B and Cdk1/Cdc2.

cDNA sequences encoding homologs of cyclin B and Cdk1/Cdc2 were isolated from bovine blastocyst-stage embryos produced in vitro. The bovine CycB sequence is 1548 nucleotides (nt) in length and contains the conserved motif 'FLRRXSK', characteristic for known cyclin B proteins. The deduced protein contains 427 amino acids (aa) and has an estimated mass of 47,653 Da. The bovine cdk1/cdc2 sequence is 1275 nt in length and contains the highly conserved motif 'EGVPSTAIREISLLKE'. The deduced protein contains 297 aa (33,931 Da).

Developmental capacity of bovine cumulus oocyte complexes after transcriptional inhibition of germinal vesicle breakdown.

UNLABELLED: Oocytes cultured in the presence of FSH and the transcriptional inhibitor, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), remain in meiotic arrest at the germinal vesicle (GV) stage. The objectives of this study were to assess the kinetics of maturation and the developmental capacity of bovine cumulus oocyte complexes (COC) following release from prolonged meiotic arrest by DRB. In Experiment I, COC were cultured for 20 h in Tissue culture medium (TCM)-199 supplemented with 10% estrus cow serum (ECS), 5 microg/ml FSH and 1 microg/ml estradiol in the presence of 120 microM DRB. COC were then released from meiotic arrest and cultured for 20 h in DRB-free medium. CONTROL COC were cultured for 20 h in DRB-free medium, with culture initiated concomitant to the release of DRB-treated COC from meiotic arrest. Nuclear maturation was assessed after 0, 5, 10, 15, and 20 h of culture in DRB-free medium. The proportion of DRB-arrested oocytes reaching metaphase II (MII) following 20 h culture in DRB-free medium was not significantly different from controls ( 96+/-4% versus 99+/-4%). In Experiment II, COC were cultured for 20 h in TCM-199 supplemented with 10% ECS, 10 microg/ml LH, 5 microg/ml FSH, and 1 microg/ml estradiol in the presence or absence of 120 microM DRB. COC in the DRB-treated group were then washed and matured coincident with a second group of control COC for 20 h in DRB-free medium. COC in both groups were fertilized and then randomly assigned to one of two culture systems: TCM-199 + 10%ECS or mSOF + 0.6% fatty acid-free BSA. Development was assessed at 72 h post insemination (hpi), 168 hpi (Day 7) and 216 hpi (Day 9). In this experiment, culture with DRB-arrested oocyte maturation at the GV stage (DRB, 85+/-3% GV; CONTROL, 2+/-3% GV; P<0.001 ). Following release from arrest, maturation and fertilization, the proportion of COC that cleaved by 72 hpi was decreased by treatment with DRB (DRB: 78+/-3% versus CONTROL: 90+/-3%; P<0.05). However, no effect of DRB was found on the proportion of cleaved zygotes that reached the blastocyst stage on either Day 7 or Day 9 of culture (Day 7: DRB 16+/-2% versus CONTROL, 21+/-2%; Day 9: DRB 23+/-3% versus CONTROL, 31+/-3%). More embryos reached the blastocyst stage in the TCM-199/serum culture system compared to the mSOF/BSA system on both Days 7 and 9 (Day 7: TCM-199, 23+/-2% versus mSOF, 13+/-2%, P<0.05; Day 9: TCM-199, 32+/-3% versus mSOF, 22+/-3%, P<0.05 ). In summary, bovine COC maintained in meiotic arrest for 20 h by culture in the presence of the transcriptional inhibitor DRB retained their capacity to develop to the blastocyst stage after fertilization in vitro.

Effect of gonadotropins on the ability of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) to inhibit germinal vesicle breakdown in bovine oocytes.

The transcriptional inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), prevents germinal vesicle breakdown (GVBD) in bovine oocytes only in the presence of gonadotropins. The objectives of this study were to examine the ability of gonadotropins to facilitate transcriptional inhibition of GVBD in bovine oocytes and to examine the effect of gonadotropins on transcriptional inhibition of cumulus expansion. Cumulus-oocyte complexes (COC) from 2 to 7 mm follicles were cultured in TCM-199 with 1 microg/ml estradiol, 50 microg/ml gentamicin and hormonal treatments for 20 to 24 h at 39 degrees C in 5% CO2 in air. After culture, COC were assessed for degree of cumulus expansion and oocytes were then denuded, fixed and stained to determine stage of meiosis. In the presence of LH and FSH the proportion of oocytes arrested at germinal vesicle (GV) stage was significantly increased with DRB treatment (58 vs 3% GV for LH/FSH + DRB vs LH/FSH-DRB; P < 0.001). However, maximal inhibition of GVBD by treatment with DRB could also be achieved in the presence of FSH alone (60% GV for FSH + DRB). The ability of DRB to block GVBD was significantly reduced in the presence of LH alone (20 to 28% GV for LH + DRB; P < 0.05 vs FSH + DRB), and treatment with DRB did not block GVBD in the presence of hCG (6.8 to 13.3% GV for hCG + DRB; P < 0.001 vs FSH + DRB). Inhibition of cumulus expansion by treatment with DRB occurred in the presence of either FSH or LH. Based on these results, it is suggested that DRB prevents GVBD in cultured bovine COC by interfering with a transcriptional event mediated primarily by FSH.

Influence of in vitro systems on embryo survival and fetal development in cattle.

In vitro systems are commonly used for the production of bovine embryos. Comparisons between in vivo and in vitro produced embryos illustrate that the morphology of preimplantation-stage embryos differ significantly, the survival of embryos and fetuses is decreased, the size distributions of the populations of conceptuses and fetuses are altered throughout gestation, and placental development is significantly changed. Taken together these findings indicate that exposure to some in vitro environments during the first 7 days of life can profoundly influence fetal and placental development in cattle. An understanding of how in vitro oocyte maturation, in vitro fertilization, and embryo culture systems influence both fetal and placental development should result in systems that consistently produce normal embryos, fetuses, and calves.

A comparison of Menezo's B2 and tissue culture Medium-199 for in vitro production of bovine blastocysts.

The objectives of this study were, first, to evaluate the effectiveness of 2 culture media, Menezo's B2 (B2) and Tissue Culture Medium-199 (M-199), for the production of bovine blastocysts in a commercial embryo transfer program; and, second, to characterize the stage of development, quality grade and cell number of blastocysts produced in each medium. One-cell bovine embryos were produced using in vitro maturation and fertilization procedures. After fertilization, the embryos were co-cultured on Buffalo rat liver (BRL) cell monolayers in either B2 or M-199+1% BSA (M-199) medium. Both media were supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin. Embryo cultures were continued undisturbed to either Day 7 or Day 8 post-insemination. In the Day 7 cultures, all blastocysts were removed for evaluation on Day 7, and the remaining embryos were cultured for a further 24 h. Any additional blastocysts that formed were removed for evaluation and designated as Day 8 disturbed embryos. All blastocysts were classified for stage and quality grade. Embryos were fixed and stained for determination of cell number. Overall, the proportion of blastocysts was greater (P = 0.0003) with B2 medium (46%) than with M-199 (33%). This was due to a larger (P = 0.0001) proportion of blastocysts produced in B2 medium when cultures were left undisturbed for 8 d (50 vs 28% for B2 vs M-199). The proportion of blastocysts on Day 7 of culture tended to differ (P = 0.073) between media (33 vs 24% for B2 vs M-199). In addition, there were more (P = 0.007) blastocysts at advanced stages of development in B2 medium on Day 7. There was no effect of type of medium on the distribution of embryo quality grades on any day examined. The number of cells per blastocyst did not differ between media but did vary significantly (P < .05) with both stage and grade. In conclusion, B2 medium was superior to M-199 medium when used in a co-culture system with BRL cells for the production of bovine blastocysts.

Effect of estrus synchronization with norgestomet on the integrity of oocytes from persistent follicles in beef cattle.

Our objective was to determine whether oocyte integrity is compromised when oocytes are recovered from progestogen-induced persistent follicles. Beef cows were presynchronized using PGF2alpha (PGF). Cows detected in estrus after PGF were assigned to either NOR (one 6-mg norgestomet implant for 10 d starting on d 16 of cycle; day 0 = estrus; n = 112) or CON (control, no implant [n = 128] and presynchronized 8 d later than NOR). All cows received 25 mg of PGF at the end of treatment (NOR, d 26; CON, d 18). Treatments produced persistent preovulatory follicles (NOR) or normal preovulatory-size follicles (CON), which were measured via ultrasonography 1 d before slaughter. Ovaries were collected from all animals (NOR, d 27; CON, d 19) along with random (RAN) ovaries from cattle slaughtered on the same days. Cumulus oocyte complexes (COC) were aspirated from the preovulatory follicles with recovery rates of 63% across treatments. Small follicles (2 to 7 mm diameter) from NOR, CON, and RAN cows were also aspirated to recover COC. Preovulatory follicles were larger (19.5+/-.9 vs. 13.6+/-.4 mm, P<.05), serum P4 was lower (.4+/-.1 vs. 3.9+/-.2 ng/mL, P<.05), and serum E2 was higher (28.7+/-1.6 vs. 7.6+/-.8 pg/mL, P<.05) in NOR than in CON cows. Cumulus oocyte complexes recovered from preovulatory follicles (62 NOR, 64 CON) were matured, fertilized, and cultured in vitro for comparison of embryonic development. A subset (24 NOR, 34 CON) of COC were assigned morphological quality grades. A separate set of recovered COC (10 NOR, 15 CON) was fixed within 1 h after recovery for assessment of the stage of meiosis. Treatments did not differ for oocyte quality grade or stage of meiosis. However, COC from NOR cows had more layers of cumulus cells (P<.05), and more of those COC had undergone cumulus expansion (29.2 vs. 5.9%, P<.05 for NOR vs. CON, respectively). Development of cleaved embryos to the morula and blastocyst stages from preovulatory follicles (22.6% NOR, 18.9% CON) or small follicles (42% NOR, 40% CON, 42% RAN) did not differ with treatment. Oocyte quality and in vitro developmental competence were not compromised for oocytes from induced persistent follicles compared with oocytes from normal preovulatory follicles. Increased expansion of cumulus cells associated with oocytes from progestogen-induced persistent follicles may be relevant to the reduction of in vivo fertility associated with such follicles.

Role of luteinizing hormone in regulating luteal function in ruminants.

The technique of hypothalamic-pituitary stalk-disconnection was used to reinvestigate the roles of luteinizing hormone (LH) and prolactin in the regulation of luteal function in ewes. Stalk-disconnection was performed on d 5 of the estrous cycle and ewes were administered either saline (control), LH at 40 micrograms at 4-h intervals, 2 mg of alpha-ergocryptine at 12-h intervals or both LH and ergocryptine. The treatment regimen for LH was designed to mimic luteal phase concentrations of this hormone. Blood samples were collected from all stalk-disconnected and 6 sham-disconnected ewes at 4-h intervals beginning at 0600 h on the day of surgery for determination of serum concentrations of prolactin, cortisol and progesterone. Corpora lutea were collected from control ewes on d 5 of the estrous cycle and from the stalk-disconnected and sham-disconnected ewes on d 12 of the cycle. The luteal tissue was weighed, a slice taken for morphometric analysis of cell numbers, sizes and types and luteal progesterone content was determined. The weight and progesterone content of corpora lutea collected from stalk-disconnected ewes were similar to those observed in control ewes on d 5 of the cycle but less (P less than .05) than those in control ewes on d 12 of the cycle. However, serum concentrations of progesterone were unaffected by stalk-disconnection. Luteinizing hormone replacement therapy increased both the weight and progesterone content of corpora lutea in stalk-disconnected ewes to values similar to those observed in control ewes on d 12. Treatment of stalk-disconnected ewes with alpha-ergocryptine reduced serum concentrations of prolactin by greater than 95% but was without effect on the parameters of luteal function measured. The number of small steroidogenic luteal cells in any of the stalk-disconnected ewes was not different from that observed in control ewes. However, treatment of stalk-disconnected ewes with LH was followed by an increase (P less than .05) in the diameter of small luteal cells. The number of large luteal cells was greater (P less than .05) in LH-treated, stalk-disconnected ewes than in intact control ewes on d 12 of the estrous cycle. The mean diameter of large luteal cells was not affected by treatment with LH.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of vitamin A injection before mating on oocyte development, follicular hormones, and ovulation in gilts fed high-energy diets.

Previous research revealed that treatment with vitamin A approximately 5 d before ovulation may increase litter size in weaned sows and improve embryonal survival in gilts fed high-energy diets that reduced embryonal survival. For the current study, the hypothesis was that administration of vitamin A before ovulation would alter development of follicles and oocytes in a way favorable to enhanced embryonal survival. (Landrace x Large White) x (Duroc x Hampshire) gilts (n = 44) were fed 11.0 Mcal ME x gilt(-1) x d(-1) beginning 7 d after second estrus and given (i.m.) corn oil or 1 x 10(6) IU of vitamin A (retinyl palmitate) on d 15 after second estrus. Gilts were checked for estrus every 4 h, mated naturally at third estrus, and assigned randomly to undergo midventral laparotomy beginning at 24 to 28, 28 to 32, 32 to 36, or 36 to 40 h after onset of third estrus. At laparotomy, ovulated oocytes and early-stage embryos were recovered from oviducts, and ovaries were removed for aspiration of oocytes and granulosa cells from unovulated follicles. Oocytes and embryos were stained for assessment of stage of development. Granulosa cells were cultured to assess their ability to secrete progesterone. Follicular fluid was assayed for progesterone, estradiol-17beta, IGF-I, and PGF2alpha. Treatment with vitamin A altered development of oocytes and embryos by decreasing the percentage at the germinal vesicle stage and increasing the percentage at advanced stages. Mean stage of development was increased by vitamin A, but variation in stage was decreased. Among follicles matched by meiotic stage of oocyte, follicular fluid concentrations of progesterone, IGF-I, and PGF2alpha were greater in vitamin A-treated gilts than in controls, but treatment with vitamin A in vivo did not affect LH-stimulated or unstimulated secretion of progesterone by granulosa cells in vitro. These data provide evidence that vitamin A may influence embryonic development by advancing resumption of meiosis and altering follicular hormonal environment during follicle maturation.

Determination of concentration of albendazole sulfoxide in plasma and uterine fluid of heifers.

OBJECTIVES: To determine time and concentration of peak plasma and uterine fluid concentrations of albendazole (ABZ) sulfoxide (ABZSO) in heifers after oral administration of ABZ. SAMPLE POPULATION: 25 young Angus and Simmental heifers maintained on pasture with ad libitum access to hay and water. PROCEDURE: Heifers were assigned at random to ABZ or control (water) groups, and were drenched with ABZ suspension at a dosage of 15, 30, 60, or 120 mg/kg of body weight, or with water. Plasma was collected hourly, from 14 to 25 hours after administration of ABZ or water. After a drug-withdrawal period, heifers were synchronized for estrus and drenched with 60 mg of ABZ/kg, or water (50 ml). Each uterine horn was flushed. All samples were extracted and subjected to high-performance liquid chromatography analysis. RESULTS: For all groups, highest mean +/- SEM plasma concentration of ABZSO was observed between 15 and 16 hours after ABZ administration, at 2.0 +/- 0.4 micrograms/ml (15 mg/kg), 5.3 +/- 1.0 micrograms/ml (30 mg/kg), 7.4 +/- 1.5 micrograms/ml (60 mg/kg), and 11.1 +/- 2.7 micrograms/ml (120 mg/kg). Mean concentration for all uterine horn fluid samples was 265 +/- 25 ng/ml/horn; range was 79 to 546 ng/ml/horn. The only significant (P = 0.0006) source of variation was the technician performing the flush. Mean concentration for each technician was 184 +/- 24 ng/ml/horn and 345 +/- 35 ng/ml/horn. CLINICAL RELEVANCE: Teratogenic and embryotoxic effects of ABZSO differ for ewes and heifers. Albendazole sulfoxide is detectable in the uterus of heifers; however, ABZSO peaks in heifer plasma earlier and at a lower concentration than that reported for ewes, perhaps contribution to differences in susceptibility at similar dosages.

Expression of antisense of insulin-like growth factor-2 receptor RNA non-coding (AIRN) during early gestation in cattle.

The insulin-like growth factor type 2 receptor (IGF2R) regulates fetal growth by removing IGF2 from circulation. In mice, expression of the Igf2r gene is only imprinted after implantation and is associated with expression of the antisense non-coding (nc)RNA, Airn. The objectives of this study were, first, to determine if bovine AIRN was expressed during developmentally important stages of gestation, and second, to determine if expression of bAIRN was affected by method of embryo production. Control reactions confirmed that sequence verified bAIRN PCR amplicons resulted from RNA within the sample and not from genomic DNA contamination. IGF2R mRNA was expressed in all fetal liver samples at Days 35-55 and 70 of gestation as well as in 8 of 9 Day 15 conceptuses, 10 of 10 Day 18 conceptuses, and in all day 7 blastocyst pools. bAIRN was expressed in all samples of fetal liver at Days 35-55 and 70 of gestation. The proportion of conceptuses that expressed bAIRN increased from 1 of 9 at Day 15 of gestation to 8 of 10 at Day 18 of gestation. No bAIRN was expressed in any blastocyst pools. The relative level of bAIRN was greater (P<0.05) in fetal liver from embryos produced in vivo compared to that from embryos produced in vitro. In summary bAIRN was not expressed in blastocyst-stage embryos, was expressed in an increasing proportion of embryos around the time of maternal recognition of pregnancy and was expressed following implantation. Furthermore, relative levels of bAIRN in bovine fetal liver can be altered by method of embryo production.

Undergraduate performance in a domestic animal laboratory taught via distance education.

The objective of this study was to determine if laboratory modules of an undergraduate animal anatomy course offered in distance education (DistEd) format were as effective as face-to-face (F2F) format in helping students learn. Students (n = 159) completed an anatomy pretest as well as a presurvey to assess prior DistEd experience. Alternating each week, laboratory topics were presented either as F2F or as virtual DistEd laboratories. Two laboratory examinations were administered and included material from both laboratory formats (DistEd and F2F). Questions from the pretest were also included and used to generate the posttest scores. At the end of the semester, students completed a postsurvey to determine if DistEd was a viable alternative to F2F. Student grades on each examination were compared using an ANOVA model that included main effects of presentation method (DistEd, F2F), semester (fall, spring), and their interaction. Learning was evaluated based on the performances of students on pre- and posttests using unpaired t-tests. There was an increase (P < 0.0001) in anatomy post- vs. pretest scores for both semesters, indicative of student learning, although there was no effect of presentation method (F2F or DistEd). On exam 1, students achieved greater scores in fall 2008 (P < 0.0001) on material presented via DistEd compared with that presented as F2F. However, in spring 2009 students scored better on material presented as F2F. There was no effect of presentation method on exam 2 scores for either semester. Based on the postsurvey, 79.3% of students in fall 2008 and 52% of students from spring 2009 agreed that DistEd laboratories were a viable alternative to F2F laboratories. The results of this study support the conclusion that anatomy material can be taught effectively by distance education methods.

Expression of messenger RNAs for insulin-like growth factors and their receptors in bovine fetuses at early gestation from embryos produced in vivo or in vitro.

The objective of this study was to determine the effects of in vitro embryo production on physical development and levels of expression of mRNAs for insulin-like growth factor (IGF) ligands (IGF1, IGF2), their receptors (IGF1R, IGF2R), and IGF binding protein-2 (IGFBP2) in bovine fetuses during early gestation. In vivo embryos were recovered from superovulated Holstein cows. For production of embryos in vitro, Holstein oocytes were matured, fertilized, and subsequently cultured in M199 with 10% serum to 168 hpi. On Day 70 of gestation, fetuses (in vivo, n = 14; in vitro, n = 13) were recovered, serum samples collected, and physical measurements recorded. Semi-quantitative RT-PCR assays were used to determine the levels of expression of mRNAs for IGF1, IGF2, IGF1R, and IGF2R in fetal liver and skeletal muscle. Western blots were used to assess levels of IGFBP2 in fetal serum. Fetal body weight did not differ with treatment; however, production of embryos in vitro was associated with decreased crown-nose length and a tendency for increased paired kidney weight, which became significant when expressed on a per bodyweight basis. There was no effect of treatment on levels of IGFBP2 in fetal serum. Levels of IGF1 mRNA in fetal liver were decreased (P < 0.001) in the in vitro group. Levels of IGF2R mRNA in both liver and skeletal muscle were also decreased (P < 0.01) in fetuses from the in vitro group. In summary, fetuses at Day 70 of gestation from embryos produced in vitro had shortened crown-nose length and increased kidney weight on a per bodyweight basis, as well as decreased expression of mRNAs for IGF1 in liver and IGF2R in both liver and skeletal muscle, compared with fetuses from embryos produced in vivo. In conclusion, in vitro embryo culture was associated with subtle changes in fetal development as well as altered expression of both imprinted and non-imprinted genes.

Effect of season on fresh and cryopreserved stallion semen.

The objective of this study was to determine the effect of season on sperm quality variables, expression of the fertility-related protein SP22 and selected mRNA transcripts in fresh and cryopreserved stallion sperm. Four stallions were collected in each of the four seasons: summer, fall, winter and spring. Ejaculates were divided and then evaluated for motility, morphology, SP22 staining and expression of selected mRNAs as either fresh semen samples or cryopreserved samples. A significant interaction between season and cryopreservation status was found for total and progressive sperm motility. RNA yield from sperm was not affected by any variable examined. There was no effect of season or cryopreservation on the relative amounts of mRNA for PGK2, TPX1, TIMP3 or ACTB. There was a tendency (P=0.1) for an effect of stallion on the relative amount of ACTB mRNA. The proportion of sperm immunostained for SP22 over the equatorial segment was affected (P<0.05) by stallion. In addition, there was an interaction (P<0.05) between season and cryopreservation status on the percentage of sperm staining for SP22 on the equatorial segment. The correlation among total motility, progressive motility and SP22 immunostaining was much greater (P<0.05) during the breeding season (March and June) than during the non-breeding season (September and December). Based on data analyzed, semen collected in the Northern Hemisphere between March and June may be best suited for cryopreservation.

Transfer of bovine embryos produced in vivo or in vitro: survival and fetal development.

The objectives of the present experiment were to compare survival after transfer of bovine embryos produced in vivo with those produced in vitro and to examine the physical characteristics of fetuses produced from these transfers. Embryos produced in vivo (Holstein x Angus) were recovered from uterine flushings of superovulated heifers 7 days after first artificial insemination, and embryos produced in vitro (Holstein x beef breeds) were collected 7 days after insemination. Embryos were paired by source (in vivo, in vitro), stage (compact morula, blastocyst), and quality grade (excellent = 1, good = 2), and transferred nonsurgically to recipient heifers on Day 7 (+/- 1 day) of the estrous cycle. Pregnancy status was monitored by determination of serum progesterone concentrations, ultrasonography, and palpation through 7 mo of gestation, at which time fetuses were recovered. In comparison with grade 1 embryos produced in vivo, the risk of embryonic death after transfer was similar for grade 2 embryos produced in vivo (p = 0.56) and for grade 1 embryos produced in vitro (p = 0.88). By contrast, grade 2 embryos produced in vitro were at greater (p = 0.04) risk of embryonic death. Embryo loss was associated (p = 0.01) with increased serum concentrations of progesterone in recipients at the time of transfer. At 7 mo of gestation, fetuses from embryos produced in vitro were heavier (p = 0.02) than fetuses from embryos produced in vivo and had skeletal measurements that were disproportionate (p < or = 0.04) to body weight.