Bovine in vitro fertilization with frozen-thawed semen

This research was carried out to define in vitro conditions that would enable ejaculated rabbit sperm to fertilize ova in vitro. During 20 mm exposure of sperm cells to defined media of increasing NaCI concentrations, progressive removal or alteration of sperm surface seminal plasma antigenic components was initiated and completed as reflected by an immunological assay at approximately 300 and 380 mOsm/kg, respectively. Hypertonic (380 mOsm/kg) medium effected complete removal or alteration of these components as determined by the immunological assay during the first 5 mm of sperm treatment. Isotonic (305 mOsm/kg) treatment was shown to have effected removal or alteration of some but not all of the antigenic sperm coating seminal plasma components detectable by this means during 20 mm of exposure. Sperm within the perivitelline apace, presence of pronuclei. and 2- and 4-cell cleavage stages were taken as evidence for fertilization. None of 34 ova incubated for 27 h in vitro with once-washed sperm showed evidence of fertilization. Following treatment of sperm with hypertonic (380 mOsm/kg) medium prior to in vitro insemination, proportions of ova showing evidence of fertilization ranged from 8.1 percent (12 to 148 ova) to 72.2 percent (52 of 72 ova); and, following treatment of sperm with isotonic (305 mOsm/kg) medium, fertilization of ova ranged from 0 percent (0 to 19 ova) to 73.9 percent (SI of 69 ova) when results were considered according to individual male sperm donors. No large differences were found by the immunological assay that could be linked to variability of fertilizing ability of sperm from different bucks. Differences in metabolic characteristics of sperm cells was suggested as a likely reason for differences in fertilizing ability of sperm from different bucks. In experiments using sperm and ova from the same sources, no differences in fertilization results were apparent following 305 and 380 mOsm/kg sperm treatments, Initiation, but not completion, of removal or alteration of sperm surface seminal plasma antigenic components, then, was a necessary prerequisite to in vitro insemination for successful achievement of fertilization in vitro. Sperm penetration into ova occurred in some cases before 7-10 h after insemination (4 of 54 ova penetrated), but actively motile sperm were found within the perivitelline apace of other ova as late as 25 h after insemination. One hundred and seventy-six 2- and 4-cell stage embryos were transferred to 14 recipient does. Twenty-four embryos (13.1 percent) implanted in 6 recipients but most were resorbed. Three embryos resulting from in vitro fertilization with in vitro capacitated ejaculated rabbit sperm were carried successfully to term by 2 recipients and 2 of the offspring were males. This provides documentation for the authenticity of in vitro capacitation of ejaculated sperm of a mammalian species and represents the initial successful combination of in vitro sperm capacitation with in vitro fertilization and embryo transfer in the rabbit.

Capacitation of Rabbit Spermatozoa in vitro

The zona-free hamster ovum was evaluated as a substitute for human o va in the assessment of the fertilizing capacity of human spermatozoa. Zona-intact ova completely resisted sperm penetration. Using nonpreincu bated spermatozoa sperm penetration of zona-free ova began 4-5 hours after insemination. However when spermatozoa were preincubated in a modified Krebs-Ringer solution for 4 hours sperm penetration began within 1 hour. There is some evidence that this is associated with the completion of sperm capacitation and the acrosome reaction. The results suggest that zona-free hamster ova can be substituted for human ova in the preliminary assessment of the fertilizing capacity of human spermatozoa.

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The Use of Zona-Free Animal Ova as a Test-System for the Assessment of the Fertilizing Capacity of Human Spermatozoa

The spermatozoa of most invertebrates (e.g., sea urchins) and nonmammalian vertebrates (e.g., fishes and amphibians) have full capacity to fertilize eggs upon leaving the testis. Testicular spermatozoa of mammals, on the other hand, do not possess the ability to do so. Their fertilizing capacity develops as they pass through the epididymis (Young, 1931; Nishikawa and Waide, 1952; Blandau and Rumery, 1964; Bedford, 1966; Orgebin-Crist, 1967). This process, apparently “unique” to mammals, is referred to as the epididymal maturation of spermatozoa. Even after their maturation, however, spermatozoa require an additional phase of maturation or capacitation within the female genital tract before they are able to fertilize eggs (Austin, 1951, 1952; Chang, 1951a). Thus, epididymal maturation and capacitation are two extra steps that mammaliam spermatozoa must take before they can effect fertilization. In this chapter, I will discuss how mammalian spermatozoa prepare themselves for fertilization and how the spermatozoa and eggs interact during fertilization. The process and mechanisms of sperm transport in the female genital tract will not be dealt with extensively here. Readers are referred to Bishop (1961, 1969), Blandau (1969), Bedford (1970b, 1972b), Thibault (1972, 1973a), Zamboni (1972), Blandau and Gaddum-Rosse (1974), Hafez and Thibault (1975), Overstreet and Katz (1977), Overstreet and Cooper (1978, 1979b), Overstreet et al. (1978), Shalgi and Kraicer(1978), Cooper et al. (1979) and Hunter (1975, 1980). The rejection or elimination of extra spermatozoa by the fertilized egg, one of the most fascinating events in fertilization, will not be discussed here. Instead, readers are referred to the chapter by Dr. Wolf in this volume. The physiology of egg activation has been described and discussed to some extent by Gwatkin (1977) and Yanagimachi (1978a). There are numerous reviews dealing with general aspects of mammalian fertilization. The following are recommended to aid in grasping the outline of mammalian fertilization: Austin and Bishop (1957), Austin and Walton (1960), Austin (1961, 1968), Blandau (1961), Piko (1969), Thibault (1969), Bedford (1970a,b, 1972b), Gwatkin (1976, 1977), Yanagimachi (1977, 1978a), Bedford and Cooper (1978) and Hunter (1980).

Mechanisms of Fertilization in Mammals

The mammalian spermatozoon

A repeatable procedure for fertilization of bovine ova in vitro is described. Oocytes were recovered from ovarian follicles or from oviducts near the time of ovulation following treatment of donors with pregnant mare's serum gonadotropin (PMSG) and prostaglandin F2 alpha (PGF2 alpha). For in vitro capacitation semen was incubated, then high ionic strength treated and subsequently incubated in defined medium prior to insemination of oocytes. In one experiment frozen bull semen was successfully used. In experiments with 4 bulls (B, C, D, F), 34 (43.6%) of 78 ova and 13 (19.7%) of 66 follicular oocytes were fertilized in vitro. In the last series (spermatozoa from Bull F) the fertilization of 22 (62.9%) of 35 tubal ova was achieved. In vitro development proceeded to the 8-cell stage. No fertilization in vitro followed use of one male (Bull E), even though his spermatozoa could penetrate zona-free hamster ova in vitro, and higher than usual bacterial contamination of his semen was implicated as the probable cause. Findings suggested vigorous progressive sperm motility and acrosome integrity to be important features of good sperm samples. In one experiment a 4-cell stage embryo was transferred with the result that the recipient gave birth to a normal bull calf on June 9, 1981. The first calf resulting from in vitro fertilization has been found to be completely normal.

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Normal development following in vitro fertilization in the cow.

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Fertilization and Early Development of Cow Ova

The inhibition of crystalline tyrpsin and chymotrypsin and the acrosomal trypsin-like enzyme by soybean and ovomucoid trypsin inhibitors was ivestigated. A concentration of 1.0mg/ml of soybean trypsin inhibitor was necessary for good inhibition of in vitro fertilization. There was a close correlation between inhibition of the acrosomal trypsin-like enzyme and inhibition of in vitro fertilization. This indicated that the acrosomal enzyme is the essential enzyme for penetration of the zona pellicida of the rabbit ovum.

Inhibition of in Vitro Fertilization of Rabbit Ova by Trypsin Inhibitors

Sperm capacitation may involve the removal of seminal plasma factor(s) from the surface of the sperm. To determine the presence of seminal plasma components and in order to follow their release or alteration, antibodies to rabbit seminal plasma were produced in guinea pigs. The agglutinating ability of rabbit spermatozoa when mixed with antiserum was followed after washing the sperm cells for extensive periods. Eighteen hours of washing did not diminish the agglutination reaction. However, when ejaculated spermatozoa were incubated in utero, the agglutination of the sperm cells induced by the antiserum to seminal plasma diminished with time in utero. Additional data indicated that a higher percentage of cleaved ova resulted following insemination in vitro with uterine spermatozoa recovered at increasing intervals after coitus (9, 44, 55, and 74% at 3, 6, 12, and 18 h, respectively). Furthermore, utilizing the binding of ‘4C-labeled antibodies against rabbit seminal plasma, the sperm-bound seminal plasma components were shown to be bound tightly to the sperm and were not removed during 24 h of incubation in vitro in a defined medium. However, when washed ejaculated sperm are incubated in uterine fluid, the uptake of 24C-antibodies falls off with increasing time of incubation, up to 65% decrease with 6 h of incubation. These findings may provide a basis for a rapid quantitative assay for sperm capacitation.

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Benjamin G. Brackett

Papers

Capacitation of Rabbit Spermatozoa in vitro

Normal development following in vitro fertilization in the cow.

Fertilization and Early Development of Cow Ova

Inhibition of in Vitro Fertilization of Rabbit Ova by Trypsin Inhibitors

Immunological Assessment of Surface Changes of Rabbit Sperm Undergoing Capacitation