Benjamin G. Brackett

Penetration of zona-free hamster ova as a test to assess fertilizing ability of bull sperm after frozen storage.

Frozen stored sperm samples of two Holstein bulls (A and B) were compared for their abilities to interact with zona-free hamster ova. The percentage of hamster vitelli interacting with sperm from Bull A was significantly higher than that interacting with sperm from Bull B (94.5% vs. 68.2%, P<0.01), and these results were in good agreement with 60 day non-return data for the same months (68.2% for Bull A vs. 64.3% for Bull B). Sperm from Bull A also excelled in average numbers attached to vitelli, and in average numbers of sperm penetrated into the zona-free hamster ova. However, of the penetrated vitelli, sperm of Bull B resulted in more pronuclei. In these experiments the percentage of progressively motile sperm at insemination was highly correlated with the percentage of vitelli interacting with sperm. The percentages and numbers of sperm cells with intact acrosomes were significantly correlated with the average number of sperm attached per vitellus. These observations encourage further development of this test for assessing sperm fertilizing ability.

Partial Purification and Identification of a Reversible Decapacitation Factor from Rabbit Seminal Plasma

Efforts have been directed toward partial purification and identification of a reversible decapacitation factor from rabbit seminal plasma. Five fractions were consistently obtained following fractionation of rabbit seminal plasma on a Sephadex G-200 column. Only the first two fractions had decapacitation factor (DF) activity. On SDS polyacrylamide gel electrophoresis, several carbohydrate-and protein-containing components were found in each fraction from the Sephadex column. Further, DF activity could be obtained in the precipitate resulting from cetyl pyridinium chloride (CPC) treatment of the active fractions from the Sephadex G-200 column. In the comparison of the components observed with SDS gel electrophoresis throughout these fractionation procedures, the reversible decapacitation activity appeared to be associated with a component containing protein and carbohydrate. From these studies, then, reversible decapacitation activity of rabbit seminal plasma appears to be associated with a glycoprotein of an approximate molecular weight of 115,000.

In Vitro Fertilizing Ability of Testicular, Epididymal, and Ejaculated Rabbit Spermatozoa

The fertilizing ability of testicular, epididymal, and ejaculated rabbit spermatozoa was evaluated in vitro following in vitro capacitation by high ionic strength treatment. Fewer than 11% of inseminated ova were apparently fertilized (i.e., in pronuclear, two-, and four-cell stages as determined by light microscopy) when testicular sperm treated with caffeine, caput epididymal, or corpus epididymal sperm samples were tested. A greater fertilizing ability, reflected by the percentage of ova fertilized and more normal progression of embryonic development, was exhibited by cauda epididymal sperm. Of 93 ova, 68 (73.1%) were fertilized by cauda sperm, whereas ejaculated sperm from the same 10 bucks fertilized 34 (36.6%) of 93 ova (P is less than 0.005). Ultrastructural examination of selected ova apparently fertilized by sperm from levels of the male reproductive tract proximal to the cauda epididymidis revealed abnormal activation. Authentic fertilization occurred when ova were inseminated with cauda epididymal and ejaculated sperm. An unusual and infrequent form of activation involving failure of cortical granule breakdown in ova penetrated by cauda epididymal and ejaculated sperm was seen. A comparison of fertilizing ability of sperm from first, second, and third ejaculates revealed a significant decrease with the third ejaculate (P is less than 0.01).

Effects of Microsurgical Removal of the Rabbit Uterotubal Junction

25 mature New Zealand white rabbit does (weighing between 3500 and 4500 grams) were used in an investigation to determine whether resection of the uterotubal junction (UTJ) alters the postovulatory functional blocking mechanism which keeps newly fertilized ova in the oviduct from 72-96 hours. A unilateral resection of the UTJ was performed in each animal. 2 of the 25 rabbits died 1-3 days following the anastomosis. 2 other rabbits refused the male although isolated on 2 occasions for a period of 21 days. In these 2 rabbits laparotomy disclosed a paucity of follicles in either ovary; no corpora lutea or adhesions were found and the anastomosis was patent. 10 additional rabbits failed to have pregnancies in either side despite many corpora lutea present in their ovaries. Many adhesions had formed in these 10 animals immobilizing their oviducts. Dissection of these adhesions around the oviducts revealed that the fallopian tubes were not distended with accumulated fluid and the anastomosed area was patent. 11 of the rabbits were pregnant on both sides. 8 of these animals were in good condition. In 2 there were fine adhesions between omentum and bladder and between intestines. In all instances the oviducts and uterine horns were free and mobile. A good correlation between the number of corpora lutea and the number of implantations of the control side was found.

A review of bovine fertilization in vitro

Abstract Progress has been made toward developing conditions to enable bull sperm to penetrate and fertilize ova in vitro. Efforts toward understanding oocyte maturation also hold promise. A procedure for bovine in vitro fertilization (IVF) involving recovery of oocytes near the time of ovulation, fertilization with in vitro capacitated sperm, embryo culture and transfer has led to development of normal offspring. Additional research is needed to facilitate recovery of mature oocytes and to improve in vitro conditions to insure development of viable embryos that can continue normal development following non-surgical uterine transfer. Bovine IVF promises a means to overcome certain types of infertility, to produce large numbers of half-siblings simultaneously, to greatly extend valuable semen, a better way to assess functional performance of male and female gametes, to provide synchronously developing pronuclear stage ova for nuclear transfer and/or gene injection, and many possibilities in research and for future applications.

Ultrastructure of Rabbit Ova Recovered from Ovarian Follicles and Inseminated in Vitro

Rabbit ova were obtained from presumably preovulatory ovarian follicles of gonadotropin-treated does and inseminated with uterine sperm in vitro. Ova were serially sectioned for light and electron microscopy 0, 1 1/2, 3, 6, and 9 hours after in vitro insemination. Control ova were maintained in culture for 25 hours and examined by light microscopy for cleavage. Little difference in fertilization rate (i.e., ova fertilized/ova inseminated) was observed when criteria were based on ultrastructural evidence (56.5%) and on light microscopic evidence 25 hours post-insemination provided by cleavage of control ova (54.8%). Therefore, observations of cleavage (as in controls here) can be an accurate criterion for fertilization in the rabbit, at least under the in vitro conditions used in this study. After insemination of ova recovered from preovulatory follicles, the developmental sequence was as follows: after 1 1/2 hours, sperm penetration into the vitellus; after 3 hours, male pronuclear formation and second polar body extrusion; after 6 hours, male and female pronuclear enlargement; and after 9 hours, pronuclei in apposition. This sequence compares favorably with the normal temporal sequence of events already well known for fertilization of ovulated ova in the rabbit. Penetrating sperm cells within the matrix of the zona pellucida and the supplementary sperm cells lying in the perivitelline space had undergone the acrosome reaction. Changes associated with the acrosome reaction took place before the penetration of the zona pellucida. The presence of a number of supplementary sperm, usually five to six per ovum, within the perivitelline space of the already activated ovum indicated that the block to polyspermy in these experiments operated mainly at the level of the vitelline membrane, as is the case in the normal fertilization process. The vitelline membrane block to polyspermy occurred rapidly after penetration of the fertilizing sperm into the vitellus and was associated with cortical granule breakdown. Ultrastructural details of rabbit ova recovered from ovarian follicles and inseminated in vitro revealed no distinguishable characteristics when compared with reported observations of ovulated rabbit ova undergoing fertilization in vivo.

Ovulation induction in rhesus monkeys by treatment with gonadotropins and prostaglandins.

Abstract In rhesus monkeys ( Macaca mulatta ), superovulation has been successfully induced using a combined regimen of PMS, HCG and PG E1 or E2. The ovulation was confirmed by a number of ova recovered either from the tubes or from the uterine flushings at various intervals after ovulation. The time of ovulation in this study under the conditions cited appears to be around 60 hrs after HCG, 36 hrs after PG administration.

Efforts to correlate laboratory with field observations on bull sperm fertility.

This work represents efforts towards development of the zona-free hamster ovum sperm penetration assay for predicting relative levels of fertility for semen from individual bulls. Results reported here followed insemination of hamster vitelli with bull sperm, after frozen storage, with observations of sperm acrosomes and parallel inseminations of more than 1000 cows with semen from each bull. The average 75-day non-return rate for the four bulls was 74.0% (range 71.6 to 75.6). Laboratory studies indicated the following: the percentage of sperm with intact acrosomes varied from 55 to 73 between bulls, the percentage of motile sperm varied from 41 to 64, the percentage of sperm with progressive motility ranged from 24 to 40, the number of sperm interacting per (zona-free hamster) ovum ranged from 1.6 to 3.8, the number of sperm attached per ovum ranged from 1.4 to 2.9, the number of sperm within each penetrated ovum ranged from 1.5 to 1.8, the percentage of ova interacting with sperm ranged from 76 to 92, the percentage of ova penetrated ranged from 62 to 85, and the percentage of ova with male pronuclei ranged from 33 to 49. Although predictive ranking in the laboratory of these bulls with less than 4% variation in fertility levels was not possible, the zone-free hamster ovum test could be useful in identifying potentially subfertile bulls before they enter a young sire-sampling program.

In Vitro Culture of the Zygote and Embryo

Scientific investigations during the last three decades have led to the present capability of nurturing the embryonic development from zygote to blastocyst stages in vitro for several mammalian species including cow (Wright et al., 1976b), ferrett (Whittingham, 1975), man (Edwards et al., 1970), mouse (Whitten and Biggers, 1968; Mukherjee and Cohen, 1970), rabbit (Maurer et al., 1969; Kane and Foote, 1971; Ogawa et al., 1971; Kane, 1972), and sheep (Tervit et al., 1972). As ova of several species (man, rabbit, sheep, cow, and ferret) are relatively large and thus have greater endogenous reserves, it was suggested by Biggers (1979) that they might be more independent of the oviductal and uterine environments before implantation than the relatively smaller ova of other species (rat, hamster, and vole). Successful culture to blastocysts of ferret, mouse, rabbit, and human zygotes has followed in vitro fertilization. Various aspects of preimplantational development of mammalian embryos in culture have been well reviewed previously (see Biggers et al., 1971; Whittingham, 1971, 1975; Brinster, 1972, 1973; Seidel, 1977; Anderson, 1978; Maurer, 1978; Biggers, 1979; Brackett, 1979b; Brinster and Troike, 1979).

Elevation of rhesus monkey plasma luteinizing hormone levels in response to E series prostaglandins

Abstract Experiments were carried out to assess the influence of prostaglandins (viz. PGE 1 , PGE 2 and PGF 2α ) on plasma concentrations of FSH and LH in the female rhesus monkey. Monkeys were ovariectomized and treated with estradiol benzoate to suppress endogenous gonadotropin levels prior to these experiments. Femoral venous blood was taken at intervals following a single carotid arterial injection of the PG in anesthetized monkeys. FSH and LH concentrations, determined by radioimmunoassay, were not significantly altered in 4 control animals receiving saline (2) or ethanol-saline (2), the vehicles for PGF 2α and for the E series PGs, respectively. PGE 1 (5mg) effected dramatic elevations of LH within 5 min in 3 animals and the high plasma concentrations were maintained at least for 60 min. Similarly, 5.0 mg of PGE 2 effected rapid elevation of LH concentrations, from 2- to 7-fold pre-injection levels in 3 animals. In contrast, FSH levels were not so markedly altered by PGE 1 and PGE 2 , but in general, appeared to be somewhat decreased by these treatments. PGF 2α had no effect on plasma FSH and LH concentrations. These data demonstrate the ability of PGs of the E series to elevate plasma LH concentrations in the rhesus monkey and support studies in other species suggesting a modulating role for PGs on gonadotropin secretion or release.