Yoshiaki Izaike

Factors affecting the survivability of bovine oocytes vitrified in droplets

Vitrification of bovine oocytes performed using the traditional, in straw system has not given satisfactory results. Although an alternative approach based on minimizing the volume of the vitrified sample has recently resulted in a much more promising survival rate of vitrified oocytes, we attempted to examine some additional factors influencing the survival and subsequent fertilization and development rates of bovine oocytes subjected to vitrification according to the minimum drop size approach. In total, 748 bovine, in vitro matured oocytes were vitrified using VS14 vitrification solution, containing 5.5-M ethylene glycol and 1.0-M sucrose after different pre-equilibration and equilibration protocols performed at 35 degrees to 37 degrees C. Experiment 1 showed no significant toxic effect during pre-equilibration treatments of oocytes in 2%, 4% or 6% ethylene glycol solutions, except the lower cleavage rate of oocytes exposed to 6% ethylene glycol (77.2% vs. 93.9% in control, P< 0.05). In Experiment 2, 12 to 15 min of pre-equilibration treatments in 0%, 1% or 2% ethylene glycol solutions were tested, followed by 30 or 45 sec of equilibration in VS 14 solution and vitrification in droplets of medium dropped directly into liquid nitrogen. The development rate of vitrified oocytes to the blastocyst stage tended to be higher after 30-sec equilibration treatment (9.5%, 13.9% and 13.8% in groups of oocytes pre-equilibrated in 0%, 1% or 2% ethylene glycol solutions, respectively). Experiment 3 tested pre-equilibration treatments in 0%, 1%, 2%, 3%, 4%, 5% or 6% ethylene glycol solutions, followed by 30-sec equilibration and vitrification in droplets. The highest cleavage, blastocyst and hatched blastocyst rates, which were not significantly different from control, were achieved in a group of oocytes pre-equilibrated in 3% ethylene glycol solution (76%, 30% and 15% vs. 89%, 42% and 21% in control, respectively). A healthy calf was born on Feb 22 1999, after transfer of 4 morula/blastocyst stage embryos developed from oocytes vitrified in droplets after pre-equilibration in 3% ethylene glycol solution. We conclude that gentle pre-equilibration of bovine oocytes in diluted, 3% ethylene glycol solution is an important factor improving the effectiveness of vitrification in droplets of bovine oocytes.

Implantation and placental development in somatic cell clone recipient cows.

Successful somatic cloned animal production has been reported in various domesticated species, including cattle; however, it is associated with a high rate of pregnancy failure. The low cloning yield could possibly arise from either an abnormal and/or poorly developed placenta. In comparison to control cows, fewer placentomes were found in somatic cell nuclear recipient (NT) cows at day 60 of gestation, suggesting a retardation of fetal/placental growth in these animals. NT cows not only had fewer numbers of chorionic villi but also had poorly developed caruncles. Macroscopic examination revealed atypical development of the placentome in terms of shape and size. Histological disruption of chorionic villi and caruncular septum was found in NT cows. Of particular interest was that the expression of genes, as well as proteins in the placentome, was disparate between NT and artificially inseminated cows, especially placental lactogen (PL) and pregnancy-associated glycoprotein (PAG). In contrast, prolactin-related protein-1 (PRP-1) signals were comparable across cows, including NT cows carrying immotile fetuses. The expression of extracellular matrix degrading molecule, heparanase (HPA), in NT cows was divergent from that of control cows. Microarray data suggest that gene expression was disorientated in early stages of implantation in NT cows, but this was eliminated with progression of gestation. These findings strongly support a delay in trophoblast development during early stages of placentation in NT cows, and suggest that placental specific proteins, including PLs, PAGs, and HPA, are key indicators for the aberration of gestation and placental function in cows.

In Vitro and In Vivo Developmental Potential of Nuclear Transfer Embryos Using Bovine Cumulus Cells Prepared in Four Different Conditions

We examined the effect of culture of donor cells on nuclear transfer efficiency using bovine cumulus cells treated with four different conditions: (1). group A, the cells removed from cumulus-oocyte complexes (COC) after aspiration of ovarian follicles; (2). group B, the cells removed from COC after in vitro maturation; (3). group C, the cells cultured in Dulbeccos Modified Eagles Medium (DMEM) with 10% fetal bovine serum (FBS) for 3 days after some subculture; and (4). group D, the cells cultured in DMEM with 0.5% FBS for an additional 5 days. Analysis of cell cycle using flow cytometry revealed that the relative proportion of donor cells at G0/G1 phase of cell cycle was 89.7% in group A, 89.5% in group B, 76.0% in group C, and 90.6% in group D. The developmental rates to blastocyst stage in groups C (45.3%) and D (46.4%) were significantly (p < 0.05) higher than in groups A (17.5%) and B (31.9%). After transfer of blastocysts produced in each group, nine of 24 recipients became pregnant on day 30. A total of five live calves were obtained from cumulus cells in all groups (group A [n = 1], group B [n = 1], group C [n = 2], and group D [n = 1]).

Expression of Trophoblast Cell-Specific Pregnancy-Related Genes in Somatic-Cell-Cloned Bovine Pregnancies

Abstract We compared the expression of bovine prolactin-related protein-1 (bPRP-1), placental lactogen (bPL), and pregnancy-associated glycoproteins-1 (bPAG-1) and -9 (bPAG-9) genes in artificially inseminated (AI) and nuclear transferred (NT) cows during the first trimester of gestation using real-time reverse transcription-polymerase chain reaction and in situ hybridization. Placentomal (cotyledonary, caruncular) and interplacentomal (intercotyledonary, intercaruncular) tissues of AI and NT cows carrying either motile (M) or immotile (IM) fetuses were examined. Transcripts for bPL and bPAG-9 were lower (P < 0.01) in the fetal membranes of NT (n = 4) cows at Day 30 of gestation, compared with AI (n = 4) cows. There was no difference in the mean (± SEM) levels of expressions of bPRP-1, bPL, and PAG-1 in the placentomal and interplacentomal tissues of AI (n = 5) and NT (M, n = 4) cows at Day 60 of gestation. The mRNAs for bPRP-1, bPL, bPAG-1, and bPAG-9 genes were higher (P < 0.01) in the caruncular tissue of AI cows, compared with NT (IM, n = 4) cows at Day 60 of gestation. Expression of bPRP-1, bPL, bPAG-1, and bPAG-9 in the placentomal and interplacentomal tissues of the NT (n = 3) group varied considerably more, compared with the AI (n = 4) group at Day 100 of gestation. These findings suggest defective binucleate cell-specific gene transcriptional commands in NT cows.

THE EFFECT OF GENTLE PRE-EQUILIBRATION ON SURVIVAL AND DEVELOPMENT RATES OF BOVINE IN VITRO MATURED OOCYTES VITRIFIED IN DROPLETS

Vitrification of bovine oocytes performed using traditional straw systems, have not given satisfactory results. Some alternative methods (Biol. Reprod. 54:1059-1069, 1996; Theriogenology 49:176, 1998 ) were described recently with relatively much higher survival and subsequent fertilization and development rates of vitrified oocytes. We report here a simple and efficient method for vitrification of bovine oocytes. Abattoir derived, in vitro matured and denuded oocytes were subjected to equilibration in 2 ml of 0, 1, 2, 3, 4, 5 or 6% ethylene glycol (EG) solution in TCM 199 medium supplemented with 20% FCS at 32-35°C. After 12-15 min of equilibration, groups of 5 to 8 oocytes were transferred within 30 sec through 3 changes of VS14 vitrification solution (5.5 M E G and 1.0 M sucrose, J. Reprod. Fertil. 98:459-465, 1993) and dropped directly into LN2 in droplets containing about 6 ~1 of vitrification medium. Droplets were collected to cryotubes using fine forceps and kept in LN2 1 to 4 h. Thawing was performed by means of direct immersing and stirring of each droplet in 2 ml of warm TCM 199/20% FCS medium. After washing in fresh medium, oocytes were subjected to IVF (Day 0) and IVC procedures (Theriogenology 47:357, 1997) and cultured up to Day 10. Data representing 3 or 4 replicates were analysed using Chisquare test. The cleavage, blastocyst and hatched blastocyst rates are presented in Table 1. The highest cleavage and blastocyst rates were obtained after preequilibration of oocytes

Association between evaluation of the reproductive tract by various diagnostic tests and restoration of ovarian cyclicity in high-producing dairy cows

The uterine condition of clinically normal postpartum Holstein-Friesian dairy cows (n = 45) was evaluated once weekly (Weeks 3 to 7) by endometrial cytology, vaginal mucus collection device (VMCD), vaginoscopy, and ultrasonography to establish a relationship with postpartum resumption of ovulatory cycles. The time of first detection of the corpus luteum (CL) by ultrasonography and plasma progesterone concentration >or=1 ng/mL was recorded. By 49 d postpartum, 78% of the cows (n=35) had resumed ovarian function (CL group), whereas the remainder (n=10) had no CL (NCL group). There was a positive correlation between VMCD score and presence of fluid in the uterus in cows with a CL (P<0.01) during Week 3 postpartum but no significant correlation in cows without a CL. Percentage of polymorphonuclear leukocytes (PMN%) was higher in the NCL group (mean+/-SEM, 24.6+/-9.4%) than in the CL group (11.7+/-2.2%) during Week 5 postpartum (P<0.05). The PMN% (4.5+/-6.5%) and VMCD (0.5+/-0.5) scores during Week 5 in cows ovulating by Day 28 were lower (P<0.01) than the PMN% (15.0+/-14.3%) and VMCD (1.1+/-0.9) scores in those ovulating by Day 49. In conclusion, higher PMN% at 5 wk postpartum was associated with delayed resumption of ovarian cyclicity in high-producing dairy cows.

Observation by ultrasonography of embryonic loss following the transfer of two or three embryos in beef cows

Abstract Ultrasonographic observations were carried out at 10-day intervals from 27 to 107 days of gestation, to monitor embryonic losses in Japanese Black cows which received two or three embryos that had been transferred nonsurgically on Day 7 (Day 0 = estrus). Group I cows (n=60) received two embryos, one in each uterine horn. Group II cows (n=31) received three embryos, two embryos in the uterine horn ipsilateral to the corpus luteum and one embryo in the uterine horn contralateral to the corpus luteum. The Group II pregnancy rate was maintained above 83.9% until Day 37, but it decreased significantly (P

In vitro oocyte culture and somatic cell nuclear transfer used to produce a live-born cloned goat.

The use of an in vitro culture system was examined for production of somatic cells suitable for nuclear transfer in the goat. Goat cumulus-oocyte complexes were incubated in tissue culture medium TCM-199 supplemented with 10% fetal bovine serum (FBS) for 20 h. In vitro matured (IVM) oocytes were enucleated and used as karyoplast recipients. Donor cells obtained from the anterior pituitary of an adult male were introduced into the perivitelline space of enucleated IVM oocytes and fused by an electrical pulse. Reconstituted oocytes were cultured in chemically defined medium for 9 days. Two hundred and twenty-eight oocytes (70%) were fused with donor cells. After in vitro culture, seven somatic cell nuclear transfer (SCNT) oocytes (3%) developed to the blastocyst stage. SCNT embryos were transferred to the oviducts of recipient females (four 8-cell embryos per female) or uterine horn (two blastocysts per female). One male clone (NT1) was produced at day 153 from an SCNT blastocyst and died 16 days after birth. This study demonstrates that nuclear transferred goat oocytes produced using an in vitro culture system could develop to term and that donor anterior pituitary cells have the developmental potential to produce term offspring. In this study, it suggested that the artificial control of endocrine system in domestic animal might become possible by the genetic modification to anterior pituitary cells.

Influences of Metabolic Traits on Subclinical Endometritis at Different Intervals Postpartum in High Milking Cows

Seventy pluriparous high-yielding cows were used to investigate the impact of metabolic traits and body condition score (BCS) during early lactation on subclinical endometritis diagnosed at weeks 5, 6 and 7 postpartum (pp). Blood samples were collected from animals with no peripartum problems from the second (W2) to seventh (W7) weeks pp to estimate some blood metabolites including non-esterified fatty acids (NEFA), β-hydroxybutyric acid (BHBA), blood glucose, total cholesterol (T-chol) and blood urea nitrogen (BUN). Reproductive tract examination was carried out at weeks 5, 6 and 7 pp by endometrial cytology (percentage of polymorphonuclear cells; PMN%). Based on PMN%, animals having <5% were defined as subclinical endometritis group (ENDM group) while animals unaffected by endometritis were defined as no subclinical endometritis group (NOENDM group). Animals with endometritis during week 5 were identified as ENDM5, during week 6 identified as ENDM6 and during week 7 identified as ENDM7 or animals with no endometritis during weeks 5 (NOENDM5), 6 (NOENDM6) and 7 (NOENDM7) pp. Animals diagnosed at week 5; BUN and BCS were lower p < 0.05 in ENDM 5 than NOENDM5 group at W2, W4, W6 and W7. Cows diagnosed at week 6; T-chol was significantly higher (p = 0.05) in ENDM6 group (279.2 ± 12.5 mg/dl) than NOENDM6 group (246 ± 9.5 mg/dl) at W7. Moreover, blood glucose was significantly low (p < 0.05) in ENDM6 group when compared to NOENDM group at W4 pp (49.2 ± 1.8 vs 53.8 ± 1.3 mg/dl). BCS was significantly lower (p < 0.001) in animals suffered from endometritis during week 7 when compared to NOENDM7 cows at W3, W4, W5, W6 and W7. In conclusion, lower blood glucose, BUN and BCS could be a risk factor for cytologically diagnosed endometritis at weeks 5, 6 and 7 pp.

Impact of Ovarian and Uterine Conditions on Some Diagnostic Tests Output of Endometritis in Postpartum High-Yielding Dairy Cows

The effect of ovarian predominating structures and uterine condition on the result of some diagnostic tools for the evaluation of endometritis was studied in postpartum (pp) Holstein-Friesian dairy cows (n = 58). Endometrial cytology (EC) and the evaluation of vaginal mucus by vaginoscopy or Metricheck were performed weekly from week 3 to 7 pp. The ovarian studies involved the predominating structures including cystic follicles with plasma progesterone (P(4) ; more or <1 ng/ml; >23 mm), corpus luteum (CL), pre-ovulatory follicles (10-23 mm) and small follicles (<10 mm). The uterine conditions comprised uterine involution, tonicity and fluid in uterus (FIU) regarding echogenicity extent. During week 5, the percentage of polymorphonuclear neutrophils (PMN%) was higher (p < 0.05) in animals with pre-ovulatory follicles (mean ± SEM, 26.3 ± 7.6%) than animals having CL (11.0 ± 3.6%). In cystic ovaries, during week 5, PMN% was higher (p < 0.05) in follicular cysts with low progesterone (P(4) < 1 ng/ml; 9.3 ± 2.6%) than those with high P(4) (P(4) ≥ 1 ng/ml; 1.5 ± 1.1%). Moreover, PMN% was higher (p < 0.01) in animals with non-involuted uterus (11.5 ± 7.4%) than those with involuted uterus (2.7 ± 0.6%) during week 7 pp. The animals that had abnormal mucus determined by Metricheck was higher in animals with atonic uterus than those with tonic uterus during week 6 (82.6% vs 51.5%; p < 0.05) and 7 (71.4% vs 25.7%; p < 0.01) pp. In addition, by vaginoscopy, the prevalence of animals with abnormal discharge showing small follicles (100%, 5/5) during week 3 pp and pre-ovulatory follicles (40.0%, 8/20) during week 5 pp was higher (p < 0.05) when compared to those having CL during week 3 (33.3%; 1/3) and week 5 pp (7.7%; 2/26), respectively. In conclusion, endometrial cytology, Metricheck and vaginoscopy were influenced by the predominating various ovarian structures and uterine condition in early pp high-yielding dairy cows.