In vitro embryo production after microinjection and ovarian dynamics following transvaginal follicular oocyte aspiration

Ultrasound-guided transvaginal follicular aspiration of oocytes from live cows combined with IVM, IVF and in vitro culture (IVC) is a procedure for producing preimplantation-stage bovine embryos and a source of oocytes for pronuclear microinjection of DNA for producing transgenic cattle. This experiment was designed to compare in vitro embryo development rates between oocytes derived from transvaginal follicular aspiration and those obtained from cows at slaughter. Nine cows were subject to a twice-weekly aspiration. Oocytes were aspirated with a 5 MHz ultrasound transducer packaged in a vaginal probe equipped with a dorsal-mounted needle guide (16-ga). All visible follicles (>2 mm) were punctured with a 17-ga, 55-cm needle at each aspiration session and the contents removed under vacuum suction. Oocytes underwent IVM/IVF/IVC. Microinjection of DNA was performed during the pronuclear stage of development, and the zygotes were co-cultured on Buffalo Rat Liver (BRL) cells in modified M199 at 39 degrees C in 5% CO2 and air. After 7 d in culture, embryos were removed and scored for development. A Chi-square analysis was used to compare transvaginal follicular-derived oocytes (microinjected and not) and slaughterhouse-derived, matured in transit oocytes (SHDMT; microinjected and not). Nonmicroinjected embryos resulting from IVF of transvaginal aspiration-derived oocytes developed to blastocysts at a higher rate than SHDMT oocytes (40.0 vs 30.8%; P < 0.05). There was no difference in development rates between the microinjected groups (aspiration = 15.9% vs SHDMT = 12.8%). Higher proportions of the embryos generated from the aspirated oocytes were of excellent or good quality following culture (P < 0.05). In the present experiments the effects of microinjection may overshadow some effects of ova source, but transvaginal follicular aspiration may provide a more consistent, synchronous population of oocytes than those derived from commercial slaughter house sources for use with in vitro systems.     

Effects of ultrasound-guided transvaginal follicular aspiration on oocyte recovery and hormonal profiles before and after GnRH treatment

Endocrine changes and recovered oocytes were evaluated during 16 wk of ultrasound-guided transvaginal follicular aspiration (TVFA) and prior to and following administration of GnRH at the cessation of aspiration. Nonlactating previously aspirated (PAC, n = 4) and non-aspirated, (AC, n = 4) Holstein cows were subjected to 16 wk of twice-weekly aspiration. Four control cows (OAC) were aspirated 1 time only at the final TVFA session (wk 16). Jugular blood samples were collected from all cows during aspiration, before and after the final TVFA session, and during an 18-d period following cessation of aspiration. Ovarian activity was monitored in all cows after cessation of aspiration for 18 d. The PAC and AC cows averaged 3.4 +/- 1.2 (+/- SE) and 6.8 +/- 1.2 oocytes per session, respectively. Progesterone concentrations during TVFA did not differ between the PAC and AC (0.8 +/- 0.1 and 0.9 +/- 0.1 ng/mL, respectively). Progesterone concentration in OAC was 4.5 +/- 0.2 ng/mL before TVFA, while the PAC and AC averaged 0.5 +/- 0.2 and 0.3 +/- 0.2 ng/mL, respectively, at 16 wk. At Week 16 LH was 1.0 +/- 0.2 ng/mL and it increased to 7.5 +/- 0.1 ng/mL after GnRH treatment. The LH concentration before the final aspiration session was higher at peak amplitude in PAC than in AC groups and peak length was longer in OAC than in AC cows (P < 0.07). Between 18 and 24 h after the last aspiration there were more LH peaks and greater peak frequencies in PAC than in OAC cows (P < 0.07), and the interval between peaks was longer in PAC and AC cows (P < 0.10) than in OAC cows. Mean FSH concentrations were lower (P < 0.01) for OAC than for PAC and AC groups at 20 and 24 h after the last aspiration. Follicle numbers after GnRH varied most among treatment groups for follicles < 9 mm, with the PAC, AC and OAC averaging 5.1 +/- 1.0, 5.1 +/- 1.0, and 3.8 +/- 1.0 follicles/d, respectively. Progesterone concentrations increased to 1.1 +/- 0.3 ng/mL in PAC cows and 2.5 +/- 0.3 and 3.4 +/- 0.3 ng/mL in AC and OAC groups, respectively, during the 18-d period. These results suggest that long-term TVFA affects progesterone, LH and FSH profiles and ovarian dynamics in cows.     

Secretion of recombinant human fibrinogen by the murine mammary gland

Transgenic animals secreting individual chains and assembled fibrinogen were produced to evaluate the capacity of the mammary gland for maximizing assembly, glycosylation and secretion of recombinant human fibrinogen (rhfib). Transgenes were constructed from the 4.1kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the A, B and fibrinogen chains. Transgenic mice secreted fully assembled fibrinogen into milk at concentrations between 10 and 200 g/ml, with total secretion of subunits approaching 700 g/ml in milk. Partially purified fibrinogen was shown to form a visible and stable clot after treatment with human thrombin and factor XIII. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the B and chains were rate limiting. Both the B and chains were glycosylated when co-expressed and the degree of saccharide maturation was dependent on expression level, with processing preferred for chains over B chains. Also, the subunit complexes ₂, A₂ and the individual subunits A, B and were found as secretion products. When the B was secreted individually, the glycosylation profile of the molecule was of a mature complex saccharide indicating recognition of the molecule by the glycosylation pathway without association with other fibrinogen chains. To date secretion of B chain has been not observed in any cell type, suggesting that the secretion pathway in mammary epithelia is less restrictive than that occurring in hepatocytes and other cells previously used to study fibrinogen assembly.     

Milk yield, energy balance, hormone, follicular and oocyte measures in early and mid-lactation Holstein cows

The purpose of the study was to determine the influence of energy status on metabolic and endocrine measures, follicular development, and the quality of oocytes obtained from cows during early and mid-lactation (ML). We selected Holstein cows at calving to be assigned to the early lactation (EL) group (n = 8), while we assigned cows at about day 90 postpartum to the ML group (n = 7). We obtained blood samples twice weekly from 4 weeks before aspiration to the aspiration periods for metabolite and hormone determinations. We performed ultrasound-guided transvaginal follicular aspiration (TVFA) twice weekly on all cows for a 10-week period. We obtained follicular fluid from the largest follicle > 10 mm in diameter for hormone determinations. We analyzed data by ANOVA, using the general linear model (GLM) procedures. Energy balance was positive (2.43 +/- 0.32 Mcal/kg) for ML cows and negative (-1.55 +/- 0.33 Mcal/kg) for EL cows. Serum progesterone (P4) for ML cows decreased rapidly from the first aspiration session (2.7 +/- 0.1 ng/ml) and reached a nadir at Week 8 (0.33 +/- 0.1 ng/ml), while follicular fluid P4 increased from 0.9 +/- 0.5 to 5.6 +/- 0.05 ng/ml. Serum and follicular fluid P4 remained relatively constant over the entire aspiration period for EL cows. Follicular fluid insulin-like growth factor I (TGF-I) concentrations increased linearly for EL and ML cows, but the increase was more rapid (159 +/- 36 to 200 +/- 36 ng/ml) for ML cows than for EL cows (145 +/- 36 to 164 +/- 36 ng/ml). Serum IGF-I followed the same pattern for ML cows but declined for EL cows. Early lactation cows experienced a rapid decrease in serum nonesterified fatty acids (NEFA; 0.32 +/- 0.2 to 0.22 +/- 0.2 meq/l), while serum NEFA concentrations were relatively stable (0.19 +/- 0.2 to 0.22 +/- 0.2 meq/l) for ML cows over the aspiration period. The number of follicles obtained from the twice weekly aspiration sessions increased linearly for both EL and ML cows (P < 0.05) over the 10-week period. However, the number of follicles increased from 14.2 +/- 0.5 (Day 119) to 18.1 +/- 0.5 (Day 190) in the ML cows, compared to the changes from 14.9 +/- 0.3 (Day 32) to 15.7 +/- 0.5 (Day 90) for the EL cows. These results indicate that cows are physiologically under more production stress during EL, but increasing follicular fluid and serum IGF-I throughout ML may reflect potential differences in follicle and oocyte measures, compared to cows in EL.     

Bovine embryo development after IVF with spermatozoa having abnormal morphology

The study was conducted to evaluate the effects of scrotal insulation on semen samples collected from bulls on embryonic development after IVF. Semen samples were obtained and cryopreserved from four Holstein bulls before and after a scrotal insulation period of 48 h (Day 0). Three types of samples were used for IVF: (1) semen from the test bulls collected 5 d prior to scrotal insulation (pre-insult); (2) semen from Day 13 (2-week post-insult; 2-week PI); and (3) semen from Day 20 (3-week PI). After 18 h of sperm-oocyte co-incubation, the zygotes were cultured for 8 d when a developmental score (0=degenerate, 1=2-cell embryo through 5=blastocyst) was assigned to each embryo. The post-thaw morphological evaluation of sperm samples revealed a decrease (P<0.01) in the percentages of normal spermatozoa in the 3-week PI samples in comparison with the pre-insult samples for Bulls I and III (74-22.3% and 67.7-0.5 %, respectively). The percentage of vacuolated spermatozoa increased significantly for Bull II. The cleavage and blastocyst formation rates and embryo development scores were affected (P<0.01) by the interaction of bull by sample collection time. For Bulls I and III (severe responders) the scrotal insulation effects persisted from the time of cleavage through blastocyst formation. In contrast, the cleavage and blastocyst formation rates for Bulls II and IV were unaffected, despite high percentages of vacuolated spermatozoa present in the post-insult samples for Bull II. In conclusion, the use of scrotal insulation to elevate scrotal temperature was an effective method to obtain semen samples with high percentages of abnormal spermatozoa. The decrease in embryonic development after IVF when using spermatozoa with morphological abnormalities seemed to be multifaceted and related to changes in head morphology.     

The incidence of apoptosis after IVF with morphologically abnormal bovine spermatozoa

Normal embryonic development depends on the maintenance of a population of normal healthy cells within each embryo. The aim of this study was to use a combination of apoptotic measures to assess differences in embryo quality after IVF with semen samples with high percentages of abnormal spermatozoa. Semen samples were obtained and cryopreserved from four Holstein bulls before (5 day prior) and after (2 week-post-insult; 2 week-PI and day 20; 3 week-PI) a scrotal insulation period of 48 h (day 0). The swim-up sperm separation method was used. The post-thaw morphology revealed a decrease (P < 0.01) in the percentages of normal spermatozoa in the 3 week-PI samples in comparison with the pre-insult samples for Bulls I and III (74-22.3 and 67.7-0.5%, respectively). The percentage of vacuolated spermatozoa increased significantly for Bull II. After 18 h of sperm-oocyte co-incubation, zygotes were cultured and subpopulations were removed from culture at day 8 and subjected to either the TUNEL or caspase assay. On day 8, caspase intensity increased significantly for both Bull I (217+/-147) and Bull III (229+/-98) for the 3 week-PI embryo groups compared to the equivalent embryo groups for Bull II (98+/-115) and Bull IV (90+/-111). In conclusion, the inability to consistently measure apoptosis with TUNEL alone complicated the assessment of differences in embryo quality. Thus, it is uncertain exactly when during early pre-implantation development the differences in embryo quality are first manifest. Despite discrepancies, our results clearly indicated a difference in the embryo quality between embryos obtained after IVF with semen samples from bulls that had an intense response to scrotal insulation.     

Evaluation of systems for collection of porcine zygotes for DNA microinjection and transfer

Crossbred gilts and sows (n=116) were used for the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Four synchronization and superovulation procedures were used: 1) sows were observed for natural estrous behavior; 1000 IU human chorionic gonadotrophin (hCG) was administered at the onset of estrus (NAT); 2) cyclic gilts were synchronized with 17.6 mg altrenogest (ALT)/day for 15 to 19 days followed by superovulation with 1500 IU pregnant mares serum gonadotropin (PMSG) and 500 IU hCG (LALT); 3) gilts between 11 and 16 days of the estrous cycle received 17.6 mg ALT for 5 to 9 days and PMSG and hCG were used to induce superovulation (SALT); and 4) precocious ovulation was induced in prepubertal gilts with PMSG and hCG (PRE). A total of 505 DNA microinjected embryos transferred into 17 recipients produced 7 litters and 50 piglets, of which 8 were transgenic. The NAT sows had less (P < 0.05) ovarian activity than gilts synchronized and superovulated by all the other procedures. Synchronization treatments with PMSG did not differ (P > 0.05) in the number of corpora hemorrhagica or unovulated follicles, but SALT and PRE treaments had higher ovulation rates than LALT (24.7 +/- 2.9, 24.3 +/- 1.8 vs 11.6 +/- 2.7 ovulations; X +/- SEM). The SALT and PRE treatments yielded 12.3 +/- 2.6 and 17.7 +/- 1.7 zygotes. Successful transgenesis was accomplished with SALT and PRE procedures for estrus synchronization and superovulation.     

In vitro development of bovine morulae in bovine serum albumin, normal steer serum and uterine flushings

One hundred fifty-three excellent and good bovine morulae were cultured in Ham's F-10, supplemented with 10 % steer serum, bovine serum albumin, or uterine flushings (64 mg protein/ml) to compare embryo development. Embryos were observed every 12 h in culture. Treatment differences were evaluated by assigning a numerical value of 0 to 4 to each embryo representing the stage of development reached in vitro. The final morphological developmental score of the embryos was comparable for steer serum (2.66) and bovine serum albumin (2.50), but it differed significantly for uterine flushings obtained from ovariectomized, steroid-supplemented cows (< 0.1) or heat-treated uterine flushings (0.07). Since albumin alone was able to support development, it suggests that the albumin component of steer serum may be responsible for the observed development. Uterine fluids were unable to support growth of embryos, suggesting that incompatibility may be due to asynchrony between the early bovine embryo and uterine constituents, or a concentration of uterine components may exacerbate actions of inhibitory substances.     

Successful co-culture of 1-4-cell cattle ova to the morula or blastocyst stage

Bovine ova (n = 326) collected at the 1-4-cell stage were cultured in TCM-199 + 10% foetal calf serum with or without oviducal cells. The bovine oviducal cells were collected and seeded either on the day of ovum recovery (BOC-0) or 3 days earlier (BOC-3). In Exp. 1, the effect of age of oviducal cells in co-culture on ovum development was examined. In the BOC-0 and BOC-3 treatments, respectively, 36/46 (78%) and 30/37 (81%) of ova developed to morulae or blastocysts, while no ova developed past the 8-16-cell stage in the absence of oviducal cells. In Exp. 2, the effect of age of oviducal cells and of physical contact between the oviducal cells and ova on ovum development was examined. In the BOC-0 and BOC-3 treatments, respectively, 29/42 (69%) and 23/43 (53%) of the ova developed to morulae or blastocysts, while 1/42 (2%) developed to the morula stage in the absence of oviducal cells. Physical separation of the ova using a microporous membrane inserted between the oviducal cells and the ova did not affect ovum development, with 26/42 (62%) and 22/42 (52%) of ova developing to morulae or blastocysts in the BOC-0 and BOC-3 treatments, respectively. A high proportion of the morulae and blastocysts in Exp. 1 (57/66, 86%) and Exp. 2 (67/100, 67%) were of quality grades 1 or 2, with mean nuclei counts of 85 for morulae and 111 for blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro development of bovine embryos cultured with stem cell factor or insulin-like growth factor-I following IVF with semen of two bulls having different field fertility

The usefulness of IVF as a potential tool to evaluate the field fertility of bulls is equivocal and growth factor addition to culture media research is needed to delineate components needed for providing defined environments for embryos. The overall aim was to evaluate the in vitro development of embryos derived using a serum supplemented and serum-free production systems and semen from two bulls of different field fertility. The study was conducted to determine the combinatorial effect of stem cell factor (SCF) and/or insulin-like growth factor-I (IGF-I) in culture on subsequent embryo development in cattle. Oocytes were aspirated separately from ≥3 to <3 mm follicles to test different follicle size populations and were matured in TCM-199 supplemented with LH, FSH, estradiol and BSA (Fraction V). Matured oocytes were fertilized in BSA supplemented synthetic oviductal fluid (SOF)-IVF medium. Presumptive zygotes were cultured for 8 d (in humidified 5% CO₂ at 38.5 °C) in BSA supplemented SOF-in vitro culture (IVC) medium. SOF-IVC medium was supplemented with fetal bovine serum (4%), IGF-I (100 ng/mL), SCF (50 ng/mL) or IGF-I (100 ng/mL) + SCF (50 ng/mL). The development competence of embryos did not differ between the bulls and among the culture environments. Nevertheless, there was an effect of follicle size on cleavage rate (P < 0.05) and a greater cleavage rate resulted from oocytes aspirated from ≥3 mm follicles (71.0 ± 1.5%) compared to those collected from <3 mm follicles (64.8 ± 1.6%). The overall cleavage rate (%); blastocyst formation (%); and expanded/hatched blastocyst formation (%) were 68.2 ± 1.5 and 67.7 ± 1.7; 29.4 ± 1.4 and 28.6 ± 1.5; and 18.6 ± 1.2 and 18.5 ± 1.1, respectively, for the bull of above and below average field fertility. The results indicate that follicle size for oocyte aspiration is effective for determining IVC success and that IVF may not discriminate among bulls of different field fertility.