The use of synthetic gonadotropin releasing hormone treatment in the collection of sheep embryos

Gonadotropin releasing hormone (GnRH) treatment was examined as a means of improving the efficacy of embryo collection in the sheep following intrauterine insemination of frozen-thawed semen. In summary, treatment consistently improved fertilization rates and the number of fertilized ova collected per ewe was enhanced compared with untreated ewes. The yield of fertilized ova in ewes treated with follicle stimulating hormone (FSH) was maximized by administering GnRH 36 h after progestagen treatment; 24 h was the preferred time in ewes treated with pregnant mare serum gonadotropin (PMSG). There was a significant (P < 0.001) increase in the percentage of unfertilized ova in the former treatment when GnRH was given at 24 h. An examination of the time of insemination (0, 6, 12 and 18 h before the median time of ovulation) indicated that fertilization rates were highest when insemination occurred at 6 h in both GnRH-treated ewes and in untreated ewes. In GnRH-treated ewes, the recovery of ova was lowest when insemination occurred at the time of ovulation. The number of motile frozen-thawed spermatozoa required for fertilization following treatment was estimated to be approximately 20 x 10(6) per uterine horn. GnRH-treatment also improved the yield of fertilized ova in sheep that were naturally mated, although this yield was lower than that obtained with intrauterine insemination of frozen-thawed semen. It is concluded that fertilization failure, a major problem in sheep embryo collection, can be eliminated through judicious use of GnRH treatment and properly timed intrauterine insemination.     

Time of ovulation in the South Australian Merino ewe following synchronization of estrus. 2. Efficacy of GnRH treatment and its relevance to insemination programs utilizing frozen-thawed semen

In a study of the time of ovulation following synchronization of estrus in the ewe, the effect of time of treatment with GnRH (24 vs 36 h after pessary removal) and dosage (6.25 to 100 ug per ewe) were examined. All treatments synchronized the time of ovulation irrespective of when untreated ewes commenced to ovulate. As part of an evaluation of GnRH treatment in artificial insemination programs, an assessment was made of the quality of eggs obtained from control ewes and ewes treated with GnRH at either 24 or 36 h after pessary removal. Treatment at 24 h increased the number of retarded embryos (P < 0.01) and unfertilized ova (P < 0.01) collected per ewe, reduced the number of embryos collected per ewe (P < 0.01), and reduced (P < 0.05) the percentage of pregnant ewes compared with other groups. However, there were no differences between control ewes and ewes treated with GnRH at 36 h. GnRH treatment at 36 h was consequently examined as a means of improving conception rates following the intrauterine insemination of frozen-thawed semen. Insemination of GnRH-treated ewes 8 to 12 h before the median time of ovulation resulted in a nonsignificant increase (range 5.7 to 7.3%) in the percentage of ewes of mature age which became pregnant. Insemination 0 to 4 h before the median time of ovulation resulted in a nonsignificant decrease in the percentage of pregnant ewes. GnRH treatment did not influence the number of fetuses per ewe. Reasons for the failure of this treatment to significantly improve ewe fertility are discussed.     

Comparison of zona cutting and zona drilling as techniques for assisted fertilization in the mouse

Zona cutting and zona drilling of the mouse oocyte significantly increased the fertilization rate (3.8-90%) at low sperm concentrations (less than 200,000/ml) compared with zona-intact controls (0-45%). More oocytes were fertilized after zona drilling. Zona cutting was associated with a low loss of oocytes (less than 1%), no increase in polyspermy and normal development in vitro and in vivo after fertilization. There was a 4% oocyte loss rate after zona drilling, mostly due to extrusion of the oocyte from the zona during the procedure. Hatching of blastocysts occurred about 12 h earlier for zona-drilled than for zona-cut and zona-intact control oocytes. Zona drilling was associated with a higher, but not statistically significant, rate of polyspermy at all sperm concentrations tested. The proportion of zygotes developing to the blastocyst stage was not different between the techniques (zona cut, 77%; zona drilled, 66%; control, 71%). Similarly, no difference was found in the percentage of embryos implanting after blastocyst transfer to the uterine horns of pseudopregnant female mice (zona cut, 67%; zona drilled, 68%; control, 77%). Transmission electron microscopy demonstrated the induced defects in the zona with no damage to the oocyte or oolemma. Parthenogenetic activation was not seen after either of the micromanipulative techniques. Both techniques have promise for application to the human.     

Production of steroids and release of prostaglandins by spherical pig blastocysts in vitro

Levels of pregnenolone and progesterone in spherical pig blastocysts (near 4 and 15 microM respectively) exceeded respective levels in histotroph by about 400-fold. When blastocysts were cultured for 5 days in a synthetic medium containing pregnenolone sulfate (1 microM), daily rates of release of pregnenolone, progesterone, androstenedione, testosterone, oestrone and oestradiol were determined to be near 320, 45, 26, 27, 0.8 and 9.2 fmol per blastocyst respectively. Daily outputs of progesterone and testosterone (fmol per blastocyst) diminished (P less than 0.05) to 1.3 and undetectable levels (less than 2) respectively in the presence of Trilostane (94 microM). Increasing the content of pregnenolone sulfate in the culture medium (to 4.5 microM) resulted in higher daily rates of release of pregnenolone and progesterone (to near 1740 and 380 fmol per blastocyst respectively), verifying activity of 3 beta-hydroxy-delta 5-steroid dehydrogenase, and of arylsulfatase, in tissues of intact spherical pig blastocysts. Prostaglandin E2 was the predominant prostaglandin (PG) released by cultured blastocysts (about 1 fmol per blastocyst per hour), hourly rates of release of PGH2 (derived) and PGF2 alpha being near 0.1 and less than 0.06 fmol per blastocyst respectively. The data establish a capacity for spherical pig blastocysts to release a range of steroids and PGs of possible significance to embryonic growth and development in vivo.     

Fertilization and embryo loss in Booroola Merino x South Australian Merino ewes: Effect of the F gene

The effects of Booroola genotype (F+, ++); the number of ovulations per ewe (one, two or three); and the age of a ewe (2.5 yr vs 3.5 to 6.5 yr) on the percentage of ova fertilized, embryo loss and fetal loss were examined in Booroola x South Australian Merino ewes slaughtered on Days 4, 21 and 90 after insemination. Ewes slaughtered on Day 90 were examined by real-time ultrasound imaging (RUI) on Day 45. Fertilization failure was independent of ewe genotype, ovulation rate and age of ewe, and it was not an important source of wastage (F+, 9.4%; ++, 6.7%). Most embryo loss occurred during the first 21 d (F+, 54.7%; ++, 40.3%). Interpretation of the effects of genotype and ovulation rate on embryo wastage measured on Days 21, 45 and 90 was obscured by significant (P < 0.05) genotype and ovulation rate interactions with the day of slaughter/RUI. The effect of age on embryo loss was not significant (P > 0.05). Reasons for the high rate of wastage observed in this experiment require further study.     

Effect of sheep and human follicular fluid on the maturation of sheep oocytes in vitro

This study examines the effect of sheep and human follicular fluid on the in vitro maturation (IVM) of sheep follicular oocytes. Oocyte cumulus complexes recovered post mortem were matured for 24 to 26 h at 38.6 degrees C, 5% CO(2) in air, in TCM-199 bicarbonate medium supplemented with 20% fetal calf serum (FCS) and, where stated, with maturation hormones, including FSH (5.0 ug/ml), LH (5.0 ug/ml) and estradiol (1 ug/ml), or with sheep follicular fluid recovered from large (>5mm) or small (2 to 5mm) ovarian follicles post mortem, or with human periovular follicular fluid obtained during routine IVF procedures. The matured oocytes were then denuded, and their maturation stage and developmental capacity were assessed by in vitro fertilization (IVF) and culture (IVC). It was found that inclusion of sheep or human follicular fluid or hormone supplements in the IVM media more than doubled the number of oocytes completing maturation (FCS alone 33%, compared with 76.2% for maturation hormones, 84.2% for fluid from large and 69.6% for fluid from small sheep follicles and 82.6% for human follicular fluid), and significantly increased fertilization rates (FCS alone 51.6%, compared with 71.9% for maturation hormones, 78.4% for fluid from the large and 75.7% for fluid from small sheep follicles and 73.1% for human follicular fluid) without discernible adverse effects on the development of the cleaving embryos to the morula or blastocyst stage in culture. Omission of FCS and supplements from the IVM medium resulted in a marked reduction (56%) in the number of oocytes maturing. This reduction could be offset to a large part, but not completely, by inclusion of human follicular fluid or human follicular fluid plus LH (5 ug/ml) in the medium. The results of this study show that addition of sheep or human follicular fluid to maturation medium can enhance rather than inhibit the maturation and fertilizability of sheep follicular oocytes in vitro.     

Measurement of blood volume in fetal and neonatal sheep using red blood cells labelled with 99m technetium

Blood volume has been measured in fetal and neonatal sheep using red blood cells labelled with 99mTc. The calculated volumes were highly correlated with simultaneous measurements made using the standard 51Cr labelled red cell method, although in absolute terms the 99m Tc method provided volumes which on average exceeded by a small percentage those determined with the 51Cr method. Measurements using the 99mTc method were also made at different ages in fetal and neonatal sheep and, while no correlation could be demonstrated between blood volume and either fetal or neonatal age, neonatal blood volumes were highly correlated with body weight. The 99mTc method is considered to be a reliable technique for measuring perinatal blood volumes in sheep with the short half-life of the isotope offering additional advantages.