Prevention of in vitro fertilization of canine oocytes by anti-ovary antisera: a potential approach to fertility control in the bitch.

Antisera raised against canine ovaries were found to induce light scattering of the surface of the egg zona pellucida even when diluted 10,000 times, and to delay digestion of the zona by pronase. High concentrations of antiserum were required, however, to inhibit in vitro fertilization of the oocytes. Absorption of the antisera with canine ovaries removed these effects, whereas absorption with liver, uterus and serum did not. These results demonstrate the antigenicity of the canine ovary and suggest the plausibility of an anti-zona pellucida vaccine for bith control in the bitch.

Retention of biologic characteristics of zona pellucida in highly concentrated salt solution: the use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa.

When unfertilized hamster eggs are placed in highly concentrated solutions of neutral salts (e.g., 2 M ammonium sulfate), the egg proper, or vitellus, shrinks, creating a large perivitelline space; the zona pellucida remains unchanged in appearance under the light microscope. When these eggs are inseminated, many spermatozoa attach to and penetrate the zona. The specificity as well as several physical and chemical characteristics of the zona seem to remain unchanged during storage of the eggs in salt solutions. The properties of the human zona pellucida which allow the attachment and penetration of human spermatozoa are also retained in concentrated salt solutions. Shipment of salt-stored human eggs at ambient temperature to any part of the world is feasible and inexpensive. The present study suggests that salt-stored eggs can be used as substitutes for fresh living eggs in the preliminary assessment of fertilizing capacity of spermatozoa when collection of a large number of fresh unfertilized eggs, particularly in humans, is not practical.

Maturation and sperm penetration of canine ovarian oocytes in vitro.

Canine ovarian oocytes were cultured in a medium consisting of TC medium 199, fetal calf serum and antibiotics. Ninety-nine percent of the apparently healthy oocytes were in the germinal vesicle (dictyate) stage when recovered from the ovaries; 25% of them reached metaphase I or II by 72 hours of culture. Washed ejaculated spermatozoa were added to BWW medium containing oocytes which had either been removed directly from the follicles or which had been cultured for 24--72 hours. The earliest acrosome reaction and zona penetration by spermatozoa were seen at seven hours after insemination. Seventy-four percent of the oocytes examined between 11 and 24 hours after insemination showed evidence of zona penetration by spermatozoa. Neither the condition of the oocyte vitellus nor the stage of nuclear maturation influenced the incidence of zona penetration. Decondensing sperm nuclei were found in the vitellus of 27% of the oocytes which had not been cultured and in the vitellus of 20% of those which had been cultured for 24--72 hours and were in various stages of maturation. These results indicate that (1) canine ovarian oocytes can be matured in vitro, (2) the spermatozoa require capacitation which takes approximately seven hours in vitro and (3) maturation of the oocytes is not required for sperm passage through the zona pellucida or entry into the vitellus nor for sperm nuclear decondensation.

The sperm acrosome reaction and fertilization in the guinea-pig: a study in vivo.

Female guinea-pigs were naturally or artificially inseminated before or after ovulation and the distribution of spermatozoa in the oviducts and the time of sperm penetration into the eggs were determined. When animals were inseminated before ovulation, the spermatozoa stayed in the distal half of the oviduct until about the time of ovulation. Only a very few spermatozoa were present in the proximal half of the oviduct at the time of ovulation, but these were sufficient to effect fertilization. When animals were inseminated after ovulation, the spermatozoa ascended the oviduct faster than when animals were inseminated before ovulation, and fertilization commenced in 4 hr. Regardless of the time of insemination, the spermatozoa participating in fertilization appeared to undergo the acrosome reaction after they reached the proximal part of the oviduct or when they were very near the eggs.