Comparing follicle stimulating hormone from two commercial sources for oocyte production from out-of-season dairy goats.

Until recently, two sources of follicle stimulating hormone (FSH-P; Schering-Plough; Kenilworth, NJ and Super-Ov; FSH-SOV; AUSA International, Tyler, TX) have been commercially available in the United States and routinely used for superovulation of ruminants. Because there have been no comparative follicle stimulating hormone studies on small ruminants, we determined the difference between the number of follicles induced and the number of oocytes that can subsequently be harvested from goats stimulated with either of these two follicle stimulating hormone products. Anestrous Saanen does were fitted with a progestin implant then randomly assigned to one of two ovarian stimulation groups. Starting 4 d after introducing the progestin implant, donors in treatment 1 were administered daily injections of FSH-P for 4 d. Does in treatment 2 were similarly treated but were administered FSH-SOV for 4 d. Follicle aspirations were performed by laparotomy in the morning of treatment d 8. In summary, no difference was detected between the two stimulatory agents for the number of follicles and quality of oocytes harvested from stimulated does, indicating that these two commercial FSH products could be used successfully for ovarian stimulation of anestrous dairy goats.

Production of live foals from sperm-injected oocytes harvested from pregnant mares.

In vitro fertilization in horses has been less successful than anticipated owing to: (i) the inability to collect large numbers of good quality oocytes; (ii) alterations in the zona pellucida that occur during in vitro maturation of equine oocytes; and (iii) inadequate preparation of equine sperm cells. In addition, studies in humans, mice and cattle have indicated that high concentrations of glucose in culture media may inhibit embryonic development in vitro and this may also be a problem for development of equine embryos in vitro. The aims of the present study were: (i) to achieve fertilization of equine oocytes by sperm injection; and (ii) to determine whether culture media containing low concentrations of glucose are beneficial for the development of early stage equine IVF-derived embryos. In Expt 1, in vitro matured oocytes obtained from pregnant mares were subjected to intracytoplasmic sperm injection (ICSI), subzonal sperm injection (SUZI) or one of three control treatments. The cleavage rates were greater for oocytes subjected to ICSI (39%) than for oocytes subjected to SUZI (6%) (P < 0.05). The transfer of two embryos into one recipient mare resulted in the presence of an embryonic vesicle in the uterine body at day 14 after ICSI, but it was lost subsequently between days 16 and 18 after ICSI. In Expt 2, oocytes were subjected to ICSI and cultured for 48 h in either TCM-199 or P-1(TM) medium (glucose- and phosphate-free) supplemented with 15% fetal bovine serum. The cleavage rates for embryos cultured in the two culture media were different (47% and 63% in TCM-199 and P-1(TM), respectively; P < 0.10). In addition, four grade 1 embryos were transferred surgically into the oviducts of four recipient mares 48 h after ICSI. Three pregnancies were identified by ultrasonography by the presence of an embryonic vesicle in the uterine body by day 16 after ICSI. Two of these pregnancies proceeded to term, resulting in the birth of two healthy fillies, one at day 319 and the other at day 328 of gestation.

Transvaginal ultrasound-guided oocyte retrieval following FSH stimulation of domestic goats.

The objectives of this study were to evaluate different ovarian stimulation protocols on donor goats and to develop a safe, repeatable method for harvesting oocytes from FSH-treated does (Experiment I). Based on the preliminary findings of the first experiment, 32 crossbred does were used in a second experiment (Experiment II), 16 that had not been previously aspirated and 16 that had undergone one previous aspiration, were used to fine tune the procedure. Females were randomly subjected to 1 of the 2 ovarian stimulation protocols: Treatment (A) does were implanted with a norgestomet ear implant. Starting 10 d post-implantation, does were administered FSH daily for 4 d. Does in Treatment (B) were treated similarly to those in (A) but were implanted for only 3 d before starting the FSH injections and implants were not removed prior to aspiration. Using a 2 x 2 factorial arrangement, fresh does (n=16), not previously aspirated, were then further randomly assigned to either a laparoscopic aspiration procedure (LAP) or a transvaginal ultrasound-guided aspiration procedure (TUGA). The LAP procedure was performed using a fiber optics. For the TUGA, the doe was placed in dorsal recumbency, and a 5 MHz human transvaginal transducer, attached to the ultrasound unit, was positioned vaginally for oocyte aspiration. In summary, there was no significant difference among treatment groups for parameters evaluated, with the exception of methods for oocyte collection. The number of follicles detected and oocytes harvested using TUGA (9.5 and 4.3, respectively) was less than for females obtained by LAP (17.4 and 14.4, respectfully). The percentage of oocytes recovered from does subjected to the TUGA (68%), however, was similar to those subjected to the LAP (69%). Unlike donor does subjected to a repeated LAP, there was no evidence of adhesions in donor does from the repeated TUGA group. The TUGA approach to oocyte collection should not be overlooked in an effort to decrease the chances of adhesions in valuable donor goats.

In vitro maturation and fertilization of oocytes recovered from free-ranging Burchell's zebra (Equus burchelli) and Hartmann's zebra (Equus zebra hartmannae).

A noninvasive repeatable method to harvest oocytes for in vitro fertilization (IVF) could potentially be used to assist reproduction in endangered equid species. The objectives of this study were to evaluate a specific transvaginal ultrasound-guided oocyte recovery procedure for use in zebra mares and the general applicability of IVF procedures in zebra. Ovaries were collected from Burchell's zebra (Equus burchelli) and Hartmann's zebra (Equus zebra hartmannae) mares at routine culling for Expt. I. Of the 144 oocytes recovered from these ovaries, 70% were of excellent quality. No significant difference in oocyte quality was found between the two zebra species. Zona drilling was performed on in vitro-matured oocytes prior to IVF. Epididymal sperm from culled Burchell's zebra stallions were used for IVF. The sperm either were exposed to calcium ionophore or were not treated and served as a control. In vitro fertilized oocytes were then co-cultured with zebra granulosa cells (ZGC) or with bovine oviduct cells (BOC) for up to 8 days. Overall, a 38% cleavage rate was obtained with 16% of sperm-exposed oocytes developing to the morula or blastocyst stage. All of the embryos that developed to at least the morula stage were cultured on BOC; whereas, none of those cultured on ZGC reached the morula stage during the same interval. Cleavage rates of oocytes inseminated with ionophore-treated or with control sperm were not significantly different, suggesting that ionophore treatment of epididymal sperm for IVF in these zebra species may be redundant. In Expt. II, 10 transvaginal ultrasound-guided oocyte aspiration procedures on five captive Burchell's zebra mares recovered a total of 33 oocytes (6.6 oocytes/female) of which 94% were considered viable. This approach may be an attractive means of producing gametes for assisted reproduction in endangered species. Furthermore, results from this study indicate that IVF may become a means of producing offspring from zebra and other equid species in the future.

Effects of follicular aspiration and flushing, and the genotype of the fetus on circulating progesterone levels during pregnancy in the mare.

When aspirating ovarian follicles in pregnant mares to obtain oocytes for in vitro fertilisation (IVF), the effect of the manipulation on circulating concentrations of progesterone may be an important consideration in terms of the maintenance of pregnancy. The object of this study was to compare the effects of 3 different forms of transvaginal ultrasound-guided follicle aspiration (Treatment 1, no aspiration, n = 4; Treatment 2, aspirate only follicles > or =20 mm in diameter, n = 7; Treatment 3, aspirate all visible follicles, n = 7) on peripheral plasma progesterone concentrations between Days 21 and 150 of gestation in 9 mares carrying intraspecies horse and 9 mares carrying interspecies mule conceptuses. The 3 follicle aspiration treatments were applied at the peak of each follicular wave as determined by follicular mapping by means of transrectal ultrasonography on alternate days. The plasma progesterone profile in mares undergoing Treatment 1 was in close agreement with those reported previously in pregnant mares. A decline in plasma progesterone levels occurred after Day 53 of gestation in Treatments 2 and 3 mares, indicating that the follicular aspiration procedures did interfere with the formation of secondary corpora lutea. However, the levels in individual mares never dropped low enough to endanger the pregnancy. Mares carrying mule pregnancies exhibited higher mean plasma progesterone concentrations between Days 39 and 45 of gestation than mares carrying horse pregnancies, equivalent levels between Days 46 and 66 despite the lower circulating concentrations of chorionic gonadotrophin (mule CG) in their blood during this period and lower progesterone levels between Days 67 and 150 of gestation. The results indicate that the primary corpus luteum in the pregnant mare may be more sensitive to mule CG than horse CG. Furthermore, the earlier disappearance of CG from the circulation in mares carrying mule fetuses is reflected by an earlier decline in plasma progesterone concentrations in this type of equine pregnancy.

Effect of unilateral ovariectomy, gonadotropin stimulation and immunization against a synthetic peptide of the inhibin alpha-subunit on follicular development and oocyte recovery in cattle.

Nulliparous Holstein cows were randomly distributed among 4 treatment groups to test the effects of treatments, including unilateral ovariectomy, anti-inhibin immunization and gonadotropin stimulation on ovarian follicle population and oocyte recovery. The Control treatment consisted of intact cows (I-Control). Unilaterally ovariectomized cows were included in the 3 remaining treatments consisting of ovariectomy alone (U-Control), cows immunized against a synthetic peptide of the alpha(c)-subunit of bovine inhibin (alpha(c)I; U-IH), and cows stimulated with FSH (Super-Ov; 75 units/female/week) and also immunized with alpha(c)I as in the previous treatment (U-IH/FSH). Oocytes were collected by transvaginal ultrasound-guided aspiration on a weekly basis from cows in each treatment for 5 consecutive weeks. Intact Control cows had a greater (P<0.05) number of follicles > or = 3 mm per female (4.7) than the U-Control and U-IH cows (2.6 and 2.9, respectively), and had a similar number of follicles as the U-IH/FSH treatment group (3.5). The numbers of follicles aspirated (2.7 to 3.6) and oocytes recovered/cow (1.6 to 2.6) were similar for cows in the I-Control, U-IH and U-IH/FSH treatment groups. Cows in the U-Control treatment group had a lower (P<0.05) number of aspirated follicles (2.0) and recovered oocytes (1.1) than the I-Control cows. Cows in the U-IH/FSH and U-IH treatments had follicles with larger (P<0.01) diameters (8.7 and 8.2 mm, respectively) than cows in the I-Control (6.6 mm) and U-Control (5.7 mm) treatments. In conclusion, unilateral ovariectomy did not result in compensatory increase of follicle number or size in the intact ovary; cows in the U-IH/FSH treatment group had a greater number of follicles aspirated than the U-Control cows. In addition, the anti-alpha(c)I immunization may have played a role in increasing the number and diameter of the follicles. None of the treatments evaluated in this study improved oocyte retrieval over that of the intact, nontreated cows.

Transvaginal aspiration of oocytes from hormone-treated pregnant beef cattle for in vitro fertilization.

The ability to produce oocytes from genetically valuable, pregnant donors in a safe, repeatable manner would broaden the application of in vitro fertilization (IVF) procedures for beef and dairy cattle. The objectives of this study were to evaluate two gonadotropin treatment schedules for follicle stimulation of pregnant donor cattle and to determine the efficacy and safety of the repeated oocyte aspiration procedure from pregnant cattle. In Exp. 1, pregnant donors at 60 to 90 d of gestation were randomly allotted to three treatment groups. Cows in Treatment A received a total dose of 40 mg of FSH. Cows in Treatment B were administered a total of 20 mg of FSH, and females in Treatment C served as pregnant vehicle-treated controls. A group of luteal phase cows received a total of 40 mg of FSH and served as nonpregnant controls (Treatment D). Ultrasound-guided transvaginal oocyte aspiration was performed 12 h following the last FSH or saline injection. Following follicle aspiration, oocytes were matured for 24 h and then entered a standard bovine IVF procedure. Experiment 2 was conducted to determine the repeatability of this procedure on first trimester cows. Cows in Exp. 2 were selected (after a 20-d recovery period) from each of the three pregnant treatment groups in Exp. 1 and each given 40 mg of FSH before a second oocyte aspiration procedure. The number of follicles aspirated per cow in treatment groups receiving the high FSH dose treatment (40 mg of FSH total dose) was not different (Treatment A, Treatment D, and cows in Exp. 2).(ABSTRACT TRUNCATED AT 250 WORDS)