THESIS

This is to certify that the ,

thesis entitled

XENOGENOUS FERTILIZATION OF BOVINE AND PORCINE OOCYTES

presented by

Patricio Jorge Hirst

has been accepted towards fulfillment
of the requirements for

Master of Science Animal Sciences

 

degree in

 

 

Major professor

26 November 1980

 

Date

0-7639

 

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XENOGENOUS FERTILIZATION 0F BOVINE AND PORCINE OOCYTES

BY

Patricio Jorge Hirst

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Animal Sciences

1980

ABSTRACT
XENOGENOUS FERTILIZATION 0F BOVINE AND PORCINE OOCYTES
By

Patricio Jorge Hirst

Fertilization of bovine and porcine follicular oocytes and
hamster oviductal oocytes in the oviduct of the pseudopregnant rabbit
(xenogenous fertilization) was studied.

When oviductal recovery of hamster oocytes from the rabbit
oviduct is 32 hr or earlier after insemination, no tubal ligation is
necessary to avoid ovum loss.

The 2-ce11 stage block observed with hamster xenogenously
fertilized embryos cannot be overcome with either Fetal Calf Serum
(FCS) nor sheep serum heat inactivated at 56°C for 30 minutes.

Bovine and porcine follicular oocytes were capable of being
fertilized xenogenously as shown by overall fertilization and recovery
rates of 13.4% and 44.8%, respectively, for bovine, and 2.0% and 36.1%,
respectively, for porcine oocytes. A significant decrease in recovery
(P<<0.0l) and fertilization rates (P<:0.001) for porcine oocytes with
respect to bovine oocytes was observed.

Transport in 0.15 M NaCl solution of ovaries at 0° vs 37°C
had no effect on either recovery or fertilization rates for bovine and

porcine oocytes.

Patricio Jorge Hirst

Several recovery times were used for bovine oocytes. Recovery
times from 50 to 70 hr significantly differed (P< 0.01) from earlier
(40-45 hr) or later (70-75 hr) times.

Fertilization was not affected by recovery time, although an
increased number of 2-ce11 embryos was recovered at 50 hr after
insemination.

12;vitrg_culture of oocytes in medium supplemented with 5 ug/ml
of follicular stimulating hormone (FSH) decreased (P<=0.1) the recovery
rate from the rabbit oviduct (91.9% of control vs. 76.7% when treated
with FSH). No effect was observed on fertilization. Cleavage to the
2-cell stage was achieved when oocytes were preincubated with FSH.

ln_vitrg_culture of bovine follicular oocytes in medium for
25 hr prior to xenogenous fertilization significantly increased the
recovery from the rabbit oviduct (P‘<0.01). No effect was observed

on the fertilization rate.

To my gnandpanenté

Rodoifio Schwunz and HLch K. dc Schwanz

ii

ACKNOWLEDGMENTS

I would like to express my gratitude to my advisor, Dr. W.
Richard Dukelow, not only for his assistance and sound advice but
also for his friendship.

I wish to thank Dr. R. H. Nelson, Chairman of the Department
of Animal Sciences and to the members of my committee, Drs. D. Hawkins,
D. Eversole, and J. Ireland for their assistance and advice.

I also want to express my appreciation to Francesco J. DeMayo,
Bonnie Cleeves, and everybody else at the Endocrine Research Unit that
made this work possible.

Finally, I want to express my greatest appreciation to my
grandparents, Mr. and Mrs. Rodolfo Schwarz and to my parents Mr. and
Mrs. Guillermo A. Hirst, without whose assistance and dedication this

study would not have been possible.

*****

iii

TABLE OF CONTENTS

 

Page

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . vi
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW 3
Bovine ovum development in culture . 3
Porcine ovum development in culture 6

In vitro fertilization of cattle ova . 8

In vitro fertilization of swine ova . . 9

Ovum culture in the rabbit reproductive tract 11
Xenogenous fertilization . 15

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . 18

Animals . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Hamsters . . . . . . . . . . . . . . . . . . . . . . . . l8
Bovine . . . . . . . . . . . . . . . . . . . . . . . . . l8
Porcine . . . . . . . . . . . . . . . . . . . . . . . . 19
Rabbits . . . . . . . . . . . . . . . . . . . . . . . . 19

Experimental Design . . . . . . . . . . 19
Xenogenous fertilization of hamsters . . . . . . . . . . 19
Xenogenous fertilization of bovine oocytes . . . . . . . 21
Xenogenous fertilization of porcine oocytes . . . . . . 23

Procedures Used . . . . . . . . . . . 24
Ovulation induction in hamsters and rabbits . . . . . . 24
Bovine and porcine follicular oocyte aspiration,

medium used for ova handling . . . . . . . . . . . . . 25

Sperm handling . . . . . . . . . . . . . . . . . . . . . 27
Anesthesia and surgery . . . . . . . . . . . . . . . . 28

Ovum recovery from the rabbit . . . . . . . . . . . . . 29

Criteria of fertilization . . . . . . . . . . . . . . . 30

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . 43

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

iv

APPENDIX Page
A. PUBLICATIONS BY THE AUTHOR . . . . . . . . . . . . . . . 53

B. VITA . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Table

LIST OF TABLES

Timed stages of development of bovine oocytes in vivo
and 111-. Vitro O O O O O C O C O O O O O O O 0

Medium used for ovum handling and sperm dilutions

The effects of ligation at the uterotubal junction,
on recovery and xenogenous fertilization of hamster
embryo rates . . . . . . . . . . . . . . . . . . .

The recovery and xenogenous fertilization rates of
bovine and porcine follicular oocytes in the oviduct
of the pseudopregnant rabbit . . . . . . . . . .

Temperature effects on ovary transport on recovery
and xenogenous fertilization rates of bovine oocytes

Temperature effects on ovary transport, recovery,
and xenogenous fertilization rates of porcine oocytes

The recovery and xenogenous fertilization rates of
bovine oocytes recovered at varying times after
insemination . . . . . . . . . . . . . . . .

The effect of culture in_vitro for 20 hours in medium
supplemented with S ug/ml FSH, on recovery and
xenogenous fertilization rates of bovine oocytes

 

Recovery and fertilization rates of oocytes matured

in_vitro for 25 hours prior to xenogenous fertilization .

vi

Page

26

32

33

33

34

35

35

37

INTRODUCTION

Xenogenous fertilization is the fertilization of an oocyte of
one species in the oviduct of another species using homologous sperm.

There are several ways of increasing the number of embryos
available for research. Superovulation techniques (with gonadotropins)
to increase ovulation rates are widely used. Follicular oocytes have
been shown to be capable of maturation in culture (Pincus and Enzman,
1935; Chang, 1955a, 1955b; Edwards, 1962, 1965, 1966; Quirke, 1969;
Sreenan, 1970; Cross and Brinster, 1970; Mukherjee, 1972; Foote and
Thibault, 1969; Hunter et a1., 1972; Jagiello et a1., 1974, 1975;
Shea et a1., 1976) and furthermore, can be fertilized in vitrg_and
reach different stages of development depending on the species (Suzuki
and Mastroiani, 1968; Edwards et a1., 1969; Sreenan, 1970; Bedirian et
a1., 1975; Edwards, 1966; Hunter et a1., 1972)- Xenogenous fertiliza-
tion coupled with superovulation offers a means of increasing the number
of embryos available for research. When superovulation techniques are
used an increased number of follicles grow to the preovulatory stages.
Aspiration of oocytes from these follicles and xenogenous fertilization
provides the possibility of studying time development, of different
stages of embryos at early times of their development.

Explanation of the occurrence of events at early times after
fertilization can be more easily approached by using this technique.
Early embryos obtained from the rabbit oviduct can be examined further

knowing their developmental stage.

With the present status of embryo transfer and embryo freezing
technology, all the transferred embryos are produced by in vivg_ferti-
lization of gonadotropin-induced ovulated oocytes. These oocytes are
fertilized naturally or with frozen semen with artificial insemination
techniques. These embryos must then be flushed from the donor's repro-
ductive tract and they must be transferred to a synchronized recipient
to produce a pregnancy. However, in the case of tubal or ovarian
abnormalities, ovulation or fertilization are impaired. In this case,
laparoscopic follicular aspiration of oocytes with further xenogenous
fertilization of these oocytes can increase the percentage of embryos
recovered. Follicular aspirations of ovaries obtained at slaughter
or by other surgical procedures allow fertilization and the increased
availability of embryos for further teratological or morfological
analysis. In the present study, a review on the current literature
of xenogenous fertilization, in_vitrg_fertilization, ova culture in.
vitro, and embryo culture in the rabbit reproductive tract is presented.
The main objective of the present work was to determine the possibility
of fertilizing bovine and porcine oocytes in the pseudopregnant rabbit
reproductive tract. Variables such as the effect of ligation of the
oviduct, temperature of transport of the ovaries, different times of
recovery after insemination from the rabbit oviduct, in_vitrg_culture
with medium supplemented with FSH and in_vitrg_culture for 25 hr prior
to xenogenous fertilization were investigated in relation to recovery

and xenogenous fertilization rates.

LITERATURE REVIEW

Bovine ovum development in culture

 

Hamilton and Laing (1946) timed the developmental stages of
bovine oocytes as shown in Table 1. They reported that the egg enters
the uterus 96 hr after ovulation in the 8- to 16-ce11 stage. Ovulation
occurred 10 to 15.5 hr after the end of estrus. Hunter et al. (1972)

indicated that follicular oocytes matured in_vitro and transferred to

 

previously inseminated heifers are capable of fertilization and devel—
opment. Oocytes that had been cultured for 24 to 26 hr were transferred.
Recipients were slaughtered 21 to 23 hr after transfer and 29 of 55
(52.3%) tubal eggs were recovered. Sixteen (55.2%) of these oocytes
had been penetrated by sperm. Eleven were at the pronuclear stage,
six of which had the opposing pronuclei located eccentrically. The
male pronucleus was larger in most cases. Three eggs were recently
penetrated and were in the anaphase of the second meiotic division.
The sperm head was swollen. Two 2-ce11 embryos were recovered.
Assuming that 6 to 8 hr were required to complete oocyte maturation,
cleavage was believed to have occurred 16 hr after sperm penetration.
Recently fertilized bovine oocyte would be expected to develop
in_vivg_to a late morula or early blastocyst over six days (Table l).
Eight-cell ova developed in_vivg_to the late morula and blastocyst in
a period of four days. However, most cultured ova are retarded in this

development (Tervit et al., 1972).

Cleavage of in_vivo_fertilized eggs in a chemically defined
medium having pyruvate as the only source of energy was achieved
48 to 72 hr after hCG or LH injection (McKenzie and Kenney, 1973).
Cleavage from 4 to 8 cells occurred between 48 and 72 hr of culture.
The rate of cleavage was accelerated in the donor animals when corpus
luteum regression was hastened by gonadotropin injections than when

manually enucleated. The rate of cleavage was similar to the in_vivo

 

cleavage rate reported by Hamilton and Laing (1947).

Whenever follicular oocytes are matured in_vi£rg, they undergo
germinal vesicle breakdown and progress to metaphase II within 19 to
31 hr (Shea et a1., 1976; Edwards, 1965; Newcomb et al., 1978; Jagiello
et a1., 1974). Ovulation occurs approximately 28 hr after the LH peak
in heifers (Christenson et a1., 1974), 27 hr from onset of estrus
(Hammond, 1927) or 10 to 18 hr after the end of estrus (Nalbandov and
Casilda, 1942). Thus, maturation in_vivg_requires less than 28 hours.
Undoubtedly, some oocytes commence atresia within the ovarian follicle
(Edwards, 1966). This may account for the failure of some individual
oocytes to mature or be fertilized in_vitrg,

Brackett et al. (1977, 1978) observed cleavage of the 2-cell
stage within 24 hr of culture of gonadotropin-induced ovulated oocytes
recovered 30 hr after onset of estrus. Further cleavage to the 4-cell
stage required an additional 24 hr of culture (Table l). Newcomb et al.
(1978) showed that follicular oocytes matured in_vitrg_are capable of
being fertilized. Two bull calves were born from these follicular

oocytes that, after maturation, were transferred to inseminated

 

 

 

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recipients. Seven days after transfer the heifers were sacrificed
and ova recovered. Of the recovered ova 23.3% cleaved at least to
the 8-cell stage. Two blastocysts were transferred to another recipient
and two normal bull calves were born.

In gonadotropin treated cows, pronuclear ova are found 5 to
12 hr after ovulation (Thibault, 1967; Brackett et a1., 1980), and the
8-cell stage is reached at 44 hr after ovulation (Thibault, 1966;

Brackett et al., 1980) (Table l).

Porcine ovum development in culture

 

With the report of Leman and Dziuk (1971) indicating that

porcine follicular oocytes are capable of being fertilized in_vivo

 

and develop to a 25 day embryo, a new area of research was opened.

Ova develop to the 4-cell stage in the oviduct. The uterine
environment is required for further normal cleavage (Rundell and
Vincent, 1969).

It was shown by Rundell and Vincent (1969) that ova cultured
in_vitrg_can develop up to the 4-cell stage. However, when ova were
transferred without culture, development to 105 days of pregnancy
was achieved.

Ovulation occurs 24 to 48 hr after injection of hCG (Hunter
and Polge, 1966; Baker et al., 1967; Hunter 1972) or at 30 to 36 hr
after the onset of estrus (Lewis, 1911; Burger, 1952; Pitkjamen, 1958).
When ova were recovered from 3 to 6 hr after mating, 83% of the animals
had fertilized ova. Sperm penetration was observed as early as 3 hr
after mating. Mature male and female pronuclei in syngamy were seen

6 hr after mating (Hunter, 1972).

The evolution of the sperm head to an early pronucleus required
1% to 2 hr whereas the first cleavage of pig ova was not observed until
12 to 14 hr after penetration (Hunter, 1972).

Davis and Day (1978) reported recovery of 4-cell embryos on the
fourth day after the onset of estrus, and recovery of 2-cell embryos
two to three days after first detection of estrus. When 1- and 2-cell
embryos were cultured for 72 hr in_vitrg, only 16 of 29 2-cell eggs
cleaved to the 4-cell stage and only 3 more than doubled their cell
number. None completed two cleavage divisions (l6-cell stage). How-
ever, when 4-cell embryos or later stages were cultured in_vitrg,
blastocysts were formed. Early 4-cell embryos should reach the blas-
tocyst stage in three days (Davis and Day, 1978). Morulae recovered
on day 5 or 6 from the uterus of the sow will develop to blastocysts
in_vitrg_and undergo some expansion (Davis and Day, 1978).

One-cell fertilized ova will cleave to the 2- and 3-cell stage
by 48 hr of culture. Two—cell embryos cleave to 4- to 8-cells and
4-cell cleave to 13- to 16-cells by 48 hr of culture. When 72 hr
culture was used, the l-cell ova cleaved to 3-cell, the 2-cell ova
cleaved to 4- to 8-cell and the 4-cell cleaved from 4- to more than
20-cells in_vitrg_(Davis and Day, 1978).

Cleavage of 4-cell embryos to morula was achieved by 24 hr of
culture (8- to 16-cells). This appears to be near the in_vivg_rate of
cleavage. Although some 4-cell embryos did not cleave during the first
24 hr, they eventually developed to blastocysts. This is not neces-

sarily abnormal since Hunter (1974) observed that the 4-cell stage

lasted 20 to 24 hr in_vivg, By 48 hr all embryos destined to become
blastocysts had assumed the characteristics of morulae and some had
developed a blastocoel. By 72 hr most embryos were blastocysts (Davis
and Day, 1978). From Hunter's (1974) report, it appears that early
4-cell embryos require 72 hr to form blastocysts in_vivg, This
coincides with the findings of Davis and Day (1978) in vitro,

Loss of the zona pellucida is reported to occur in_vivg_on
the sixth day after ovulation (Hunter, 1974). Loss of zona pellucida
in_vitrg_(starting from 4-cell embryos) occurs six to eight days after

ovulation (Davis and Day, 1978).

In vitro fertilization of cattle ova

 

Several attempts to fertilize bovine oocytes in_vitrg_have been
made. Von Bregulla et al. (1974) used ovaries from slaughtered cows.
The oocytes were held in TC 199 containing FCS and antibiotics. Sperm
capacitation was both in_vivg_and in_vitrg, Of 1977 oocytes, they
recovered 12 (0.6%) in the first polar body stage; 13 (0.7%) in the
second polar body stage and 3 (0.2%) at later development stages. A
total of 6 (0.3%) cells divided. Of these, one was 2-cells, two were
4-cells, two were at the 5-cell stage and one was a 9-ce11 embryo.

Edwards (1973) reported evidence of in_vitrg_fertilization
of bovine oocytes without giving details.

Iritani and Niwa (1977) used oocytes from ovaries of slaughtered
cows. Capacitation of the sperm was in the reproductive tract of an
estrous cow, estrous doe, or in a Kreb's-Ringer bicarbonate (KRB)

solution. After capacitation, the sperm was washed and added to the

oocyte culture. They reported that 60% of the follicular oocytes
matured to the second metaphase. No fertilization was observed when
KRB was used for capacitation. However, 6 of 48 (20.7%) oocytes showed
evidence of sperm penetration when the oviduct of the estrous cow was
used for sperm capacitation. When the uterus of an estrous cow was
used for capacitation, a total of 5 of 44 (18.5%) oocytes were pene-
trated. When the uterus of an estrous doe was used for capacitation,
10 of 78 (21.2%) of the oocytes were penetrated.

Brackett et al. (1978) reported in_vitrg_fertilization for
bovine at a rate of 56% when ejaculated sperm capacitated in_vitrg_
were used. These scientists also reported no difference in fertili-
zation of follicular or oviductal oocytes. Furthermore, they reported
that 71.4% of the oocytes matured to the 2-cell stage within 24 hr.

Of these, 57.1% reached the 4-cell stage within 48 hr of culture
(Brackett et al., 1977, 1978). The donor cows were primed with PMS

and ovulation induced with prostaglandin F2a (PGF2a)'

In vitro fertilization of swine ova

 

Iritani et al. (1975) determined the rate of in_vitrg_ferti-
lization and cleavage rates in swine. When using spermatozoa pre-
incubated in modified Kreb's-Ringer bicarbonate (KRB), oviduct, and
uterus of an estrous sow, rates of fertilization were of 10/137 (7.3%);
8/128 (5.2%); and 54/469 (11.5%), respectively. They also reported
cleavage rates of in_vitrg_fertilized porcine eggs of: 0/128 (0%),
42/469 (9.0%), and 12/137 (8.8%) using sperm capacitated in KRB,

oviduct, and uterus, respectively. Their report also demonstrated

10

that none of 263 eggs cultured without sperm cleaved. In these
experiments, ovaries were obtained at slaughter and follicular

oocytes aspirated and cultured for 26 to 30 hr to reach metaphase II.
Collected semen was pretreated in one of three ways: (a) modified
KRB, (b) in the oviduct, or (c) in the uterus of an estrous sow.
Fertilization was determined 6 to 16 hr after addition of sperm.

The criteria used for fertilization was sperm penetration through the
vitellus, evidence of second meiosis and cleavage of ova. Cleaved ova
were noted between 16 and 40 hr after insemination. Sperm penetration
into periviteline space was observed 3 to 5 hr after insemination.
Sperm penetration into the vitellus was observed from 5 to 10 hr after
insemination. Second meiotic division was observed at 10 to 16 hr.
Two pronuclei and syngamy were seen at 16 to 24 hr and cleavage at

16 to 30 hr.

Iritani et al. (1978) reported improved in_vitrg_fertilization
of pig oocytes. Ovarian oocytes from slaughter house and ejaculated
and epididimal sperm were used. The oocytes were cultured for 24 hr
and sperm were capacitated in modified KRB, oviduct, and uterus for
4.5 to 5 hr. Oocytes and sperm were cultured together for 17 to 20
hr. Again 60.6% of oocytes matured to at least metaphase after the
maturation period of 24 hr. A total of 12/120 (10.0%) of the oocytes
were fertilized when ejaculated sperm were used. None of 400 oocytes
were fertilized when the sperm were preincubated in modified KRB,
whereas 4/40 (10.0%) and 8/48 (16.7%) were fertilized when the sperm
were preincubated in the oviduct and uterus of an estrous sow,

respectively. l

11

When epididimal sperm were preincubated in KRB, oviduct, or
uterus of estrous sow, the fertilization rates were of 1/30 (3.3%),
9/46 (19.6%) and 14/53 (26.4%) with a total of 24 fertilized eggs
from 129 cultured (18.6%).

Ovum culture in the rabbit reproductive
tract

 

Work by Harper (1965) demonstrated that there is a decrease
in the rate of ovum transport in the reproductive tract of estrus,

1 day and 2 day pseudopregnant rabbits. Rates of transport are 11.4,
8.9, and 5.7 mm/min in estrus, ovulation, and on day 2.5 of pseudo—
pregnancy, respectively. No further change was noted between day 2.5
and 15 of pseudopregnancy.

Rabbit ova held in the oviduct by ligation develop into early
blastocysts, and this development appears to be faster in the luteal
phase than in estrous does, however, rabbit eggs cannot develop beyond
the early blastocyst stage in the oviduct (Adams, 1973).

Averill, Adams, and Rowson (1955) reported that following
transfer to the genital tract of the rabbit, fertilized sheep ova can
survive for at least 5 days (120 hr) and develop to the early blastocyst
stage.

Hunter et al. (1962) obtained six pregnancies and four live
lambs after aerial transport of sheep embryos in the ligated oviduct
of the pseudopregnant doe. A total of 17 sheep blastocysts or morulae
were recovered after transport and storage in the rabbit oviducts for

101 to 128 hr. Sixteen of these were transferred.

12

Longer storage periods were obtained by Adams et al. (1968)
when, of 20 sheep morulae that were transferred to the oviducts of
follicular phase rabbits for 4-7 days, 14 developed into blastocysts.
Of these, five had lost the zona pellucida. Two of the latter blasto-
cysts, that were in the rabbit oviduct for 6 days, developed to term
after re-transfer to a synchronized ewe. Of 20 cow morula cultured
3 to 4 days, all but one showed further development; however, no
pregnancies resulted when some of these blastocysts were transferred
to synchronized cows (Adams et al., 1968).

The maximum bovine embryo cleavage obtained in the ligated
oviduct of the pseudopregnant rabbit (Sreenan et a1., 1968) was of
64 blastomeres. This followed a storage of an 8-cell embryo for a
period of 4 days. A total of 138/189 (73%) of the embryo transferred
to the rabbit were recovered and 71/138 (51.4%) continued to cleave in
the rabbit oviduct (Sreenan et al., 1968).

Sreenan and Scanlon (1968), using follicular fluid as the
transfer medium and pseudopregnant does as recipients, obtained
development to early blastocysts. When they used homologous serum
as the transfer medium, the success was very limited (development to
the 19-cell stage, maximum). The pseudopregnant ligated oviduct appears
to be a very suitable environment for embryos (Sreenan and Scanlon,
1968; Averill et a1., 1955; Hunter et a1., 1962; Sreenan et a1., 1968;
Seidel et al., 1971; Lawson et al., 1972b). The follicular phase rabbit
oviduct appears to sustain development of embryos as does the pseudo—
pregnant doe (Adams et a1., 1968; Lawson et al., 1972a, 1972b; Trounson

et al., 1976a; Allen et al., 1976).

13

Cleavage of fertilized ova to the 2-, 8—, and lO-cell stages
and cleavage of a 2-ce11 to the 8-cell stage, occurred in the pseudo-
pregnant rabbit oviduct after two days of culture; however, no further
cleavage was obtained when ova with more than two balstomeres were
transferred (Seidel et al., 1971).

Neither the initial cleavage of the sheep embryos transferred
to the rabbit nor the condition of the rabbit (estrus or pseudopregnant)
appears to affect the development of sheep embryos. It has been shown
that 93% of all sheep embryos continue to develop normally when trans-
ferred to the rabbit oviduct (Lawson et al., 1972b). It was also found
that uncleaved sheep ova could develop to apparently normal blastocysts
in the oviducts of estrous rabbits. This differs from the findings of
Brinster and Tenbroeck (1969) who showed that mouse ova are unable to
develop in the rabbit oviduct.

The rate of cleavage of sheep ova in the oviducts of estrous
rabbits appears to be slightly retarded in comparison to natural in_
vivg_conditions (Lawson et al., 1972b). Such retardation could be
caused by effects of ovum manipulation (Tarkowski, 1959). However,

2- and 4-cell eggs appear to develop more rapidly in the oviducts of
pseudopregnant rabbits. This could imply that the oviduct of the
pseudopregnant rabbit may provide a better environment for development
(Lawson et al., 1972b).

Sheep ova appear to remain viable in the rabbit oviduct for
up to 5 days. Storage for more than 5 days decreased significantly

the viability (Lawson et al., 1972b). These results agree with those

14

of Witenberger-Torres (1956) who found that sheep ova can remain in
the oviduct of the ewe until day 7 without developing a retardation
in their development.

When follicular phase rabbit oviducts are used to culture
bovine eggs, all of the l-cell eggs develop into morula and the
majority of the 2- and 4-cell egg develop into l6—cell egg by
recovery 3 days later (Lawson et al., 1972a).

The transfer of 15 ova cultured three or four days in the
rabbit oviduct resulted in 11 (73%) calves (Lawson et al., 1972a).

Trounson et al. (1976a) used the rabbit follicular phase -
oviduct as an environment, to study development of ova subjected to
different temperatures of storage in_vitrg, Day 5 ova were left in
the rabbits' oviducts for 48 hr and day 3 ova were left in the oviduct
for 96 hr. The day 3 ova were considered normal after 96 hr incubation
and had reached the early blastocyst stage with little expansion of the
blastocoele. Whereas blastocysts derived from day S ova were generally
well expanded, with detectable embryonic discs after 48 hr of incuba-
tion. This suggests a retardation in development of the eggs incubated
for 96 hr in the rabbit oviduct with respect to those incubated for
only 48 hr (Trounson et al., 1976a).

It has also been shown that horse embryos remain viable for
at least 2 days in the ligated oviduct of the estrous doe. Pregnancy
in three mares of four receiving embryos transported in the oviducts
of rabbits demonstrate the viability of these embryos (Allen et al.,

1976).

15

Xenogenous fertilization

 

Several attempts (Sreenan, 1970; Bedirian et al., 1975;
Trounson et al., 1977; Shea et al., 1976) have been made to fertilize
oocytes from one species in the reproductive tract of another (xeno-
genous fertilization). Follicular oocytes from various species will
resume meiosis up to extrusion of first polar body (Pincus and Enzmann,
1935; Chang, 1955a, 1955b; Edwards, 1962, 1965, 1966; Quirke, 1969;
Sreenan, 1970; Cross and Brinster, 1970; Mukherjee, 1972; Foote and
Thibault, 1969; Hunter et al., 1972; Jagiello et al., 1974; Jagiello
et al., 1975; Shea et al., 1976). These matured oocytes can be ferti-
lized in_vitrg_or in_vivg_and are physiologically normal (Suzuki and
Mastroianni, 1968; Edwards et a1, 1969; Sreenan, 1970; Bedirian et al.,
1975; Edwards, 1966; Hunter et al., 1972).

Sreenan (1970) reported successful fertilization of cattle
oocytes in the oviduct of estrous ewes. He used non-gonadotropin
stimulated follicular oocytes cultured in_vitrg_for 27-32 hr to obtain
maturation to the metaphase II stage. Of 375 bovine oocytes transferred
to the oviducts of previously inseminated (whole fresh semen) estrous
ewes, 15% showed evidence of fertilization. When extended semen
collected 24 hr earlier was used, no fertilization occurred. Par-
thenogenic cleavage was ruled out in these studies, since only 1 of
28 noninseminated control oocytes fragmented to a 2-cell. This
fragmented ova upon staining showed no evidence of nuclei in one
blastomere and only scattered chromatin in the other blastomere.

An overall fertilization rate of 8.6% was obtained when ewes

were used as recipients and inseminated with either fresh or extended

16

semen. Cleavage to the 8-cell stage occurred. When estrous rabbits
were used as hosts, no fertilization occurred.

Bedirian et al. (1975) used oocytes from gonadotropin primed
cows and transferred them to the oviduct of ovariectomized heifers
that were inseminated. They showed that fertilization in_vivg_can
occur under these conditions. However, this is not xenogenous fer-
tilization since only a single species was used. They did, however,
transfer bovine oocytes to the oviduct of prepuberal gilts. Six of
nine oocytes showed evidence of fertilization.

Follicular oocytes from prepuberal heifers superovulated with
PMS were another source of oocytes used for transfer. These were
recovered from the uterine horn of ovarectomized cows after l6-l8 hr
and the results suggested that development to the pronuclear stage
occurred in about 20 hr (Bedirian et al., 1975).

Trounson et al. (1977) used bovine oocytes matured in_vitro_
or in_vivg_for fertilization trials. An estrous or pseudopregnant
rabbit was used as the host recipient. A total of 633 oocytes were
cultured for 24 hr in Fetal Calf Serum (FCS) and were transferred to
does that previously had been inseminated. They found no evidence of
fertilization with recovery ranging from 12 hr to 7 days. Although
24 hr culture resulted in 73% of the oocytes reaching the metaphase II
stage, there was no fertilization. Control does receiving oocytes but
no sperm had 8% cleaved oocytes indicating possible fragmentation. The

authors suggested this might be parthenogenic activation.

17

When in_vivg_matured oocytes were transferred to oviducts of
does which had been inseminated 2 hr previously, 3 of 50 had developed
to the 2- to 4-cell stages but there was no evidence of sperm
penetration.

As it has been reported for other species (hamster: Barros
and Austin, 1967; rabbit: Chang, 1955b; Brackett et al., 1972; rat:
Noyes, 1952; pig: Leman and Dziuk, 1971; and man: Edwards, 1973),
oocytes from cows can develop normally after transplantation provided
they have undergone the first meiotic division in vivg_(Trounson et al.,
1977).

Further attempts at fertilization of bovine follicular oocytes
in the pig and sheep oviducts were reported by Shea et a1. (1976).
Oocytes were matured for 30 hr, then placed in the oviducts of immature
pigs previously injectedvfiifliPMS and hCG, or cycling ewes in estrus.
Transfer was approximately 20 hr before ovulation. Bull semen was
used for insemination. Recovery rates of 67 and 43% from the oviducts
of the sheep and pig were obtained, respectively. After recovery, 88%
of the oocytes from the sheep and 69% from the pig had extruded a polar
body. Sperm penetration was not observed in the oocytes recovered from
sheep oviducts. In the pig, 17% of the oocytes recovered appeared to

be penetrated by spermatozoa and had normal appearing pronuclei.

MATERIALS AND METHODS

Animals
Four species of animals were used. The oocytes donor animals

used were: cattle (Bos taurus), swine (Sus scrofa), and hamsters

 

 

(Mesocricetus auratus). The host animal for the xenogenous

 

fertilization was the rabbit (Oryctolagus cuniculus).

 

Hamsters. Immature hamsters (two to three weeks old) were
obtained from the Health Laboratory of the State of Michigan (Lansing,
Michigan). Hamsters, not exceeding six per cage, were kept in plastic
cages with metal tops. Water was supplied ad_libitum and the hamsters
were fed a commercially prepared diet (Wayne Laboratory animal diets,
Lab-Blox, Allied Mills Inc., Chicago, Illinois). The cages were washed
weekly and the bedding was replaced (San-I-Cel, Paxton Processing Co.,
Inc., Paxton, Illinois). These animals were kept in a 14 hr light,

10 hr dark cycle. Temperature was maintained between 21° and 26°C
with ambient relative humidity.

Bovine. Bovine ovaries from mixed breeds were obtained from
Van Alstine Packing Co. (Okemos, Michigan). Ovary transport was in
plastic bags containing 0.15 M NaCl. These bags were held in either:
(a) an ice bucket at 0°C or (b) in a dewar flask (thermos) at 37°C.

In other instances ovaries from surgical ovariectomies at the M.S.U.
Dairy Cattle Research and Training Center were used. These ovaries

were transported in the same manner as previously described. The

18

19

donor animals received no type of gonadotropin injection. Upon arrival
at the laboratory, the ovaries were placed in an incubator at 37°C or
held in the ice bath.

Porcine. Mature sow ovaries were obtained at Milligan's
slaughter plant (Rives Junction, Michigan) or at the.Michigan State
University Meat Laboratory. The porcine ovaries were handled in a
similar manner as the bovine ovaries.

Rabbits. Adult New Zealand female rabbits were used for
xenogenous fertilization. These animals were selected because of
previous work showing that the oviduct of the pseudopregnant rabbit
is an excellent embryo incubation environment (Adams et al., 1961).
Other factors affecting this decision were the large size of the oviduct
in comparison to other laboratory animals and the fact that rabbits have
a 17 to 18 day pseudopregnancy period after nonfertile ovulation.

Rabbits were isolated in individual stainless steel cages. They
were maintained at a temperature of 20°C during the fall and winter and
at ambient temperature during spring and summer. Water was administered
ad libitum and they were fed Lab Rabbit Chow #5321 (Ralston Purina Co.,

St. Louis, Missouri).

Experimental Design

 

Xenogenous fertilization of hamsters. Because of past success

 

with xenogenous fertilization in hamsters, this species was used to
examine two procedures involved in xenogenous fertilization prior to
studies with cattle and swine. The following questions were set forth

to be answered.

20

1. What is the effect of ligation of the tubal uterine

 

junction? Adult female hamsters were given the superovulatory regime
(page 24) and one-day pseudopregnant rabbits were used as hosts for
xenogenous fertilization. The ova were deposited into the ampulla
following the described techniques (page 28). At the time of tubal
ligation, one of the oviducts was. ligated and the other was not.
Oocytes were recovered (page 29) 32 hr later. This recovery time

is optimal for recovery of 2-ce11 hamster embryos derived from
xenogenous fertilization (DeMayo, personal communication).

2. What is the effect of sheep serum on cleavage rate of

 

xenogenously fertilized hamster eggs? One problem with xenogenously

 

fertilized hamster ova is that they will not develop beyond the 2-cell
stage (DeMayo et al., 1980). This series of experiments conducted to
determine if the ip_vi££g_culture with sheep serum instead of Fetal
Bovine Serum can reverse the block to cleavage at the 2-cell stage.

Adult sheep at the Endocrine Research Unit were bled to provide
serum. Approximately 50 ml of blood were drawn from three adult sheep.
This blood was allowed to coagulate and was centrifuged at 1058 Xg for
15 min. The serum was separated from the blood clot and filtered
through a 0.45 pm Millex R-HA filter (Millipore Corp., Bedford,
Massachusetts).

The serum was heat inactivated at 56°C for 30 min and then
cooled at 4°C. The medium was mixed keeping the same proportions
as in Table 2, but replacing the 66 Free Fetal Bovine Serum with

the sheep serum.

21

Xenogenously fertilized hamster ova that were recovered from
rabbit oviducts were placed in this medium. Further observations of
l-cell ova and 2-cell embryos for development was done.

Xenogenous fertilization of bovine oocytes. Eggs that were

 

held in the 37°C incubator were transferred to the ampullae of
pseudopregnant rabbits and inseminated with homologous sperm.
Several hours after insemination these oocytes were recovered.
Recovery and fertilization rates were calculated.

The variables studied included temperature of ovary transport,
time of recovery from the rabbit oviduct, culture previous to transfer,
and the addition of exogenous FSH for in vi££p_culture of the oocytes
prior to xenogenous fertilization.

1. What is the effect of temperature of ovary transport on

 

recovery and xenogenous fertilization rates from the oviduct of the

 

pseudopregnant rabbit? Bovine ovaries were transported in plastic

 

bags containing 0.5 M NaCl solution. These bags were put in ice
buckets so as to maintain the temperature at 0°C.

Control ovaries were transported in a 37°C, 0.15 M NaCl solution
kept in a Dewar flask (thermos). Upon arrival to the laboratory, the
ovaries transported at 0°C were kept on ice and the ovaries transported
at 37°C were transferred to a 37° water bath.

2. What is the effect of time of recovery from the rabbit

 

oviduct on xenogenous fertilization and recovery rate? Experiments

 

were conducted to determine the stages of maturation, fertilization,

and recovery rates of bovine and porcine ova from the pseudopregnant

22

rabbit oviduct. Recovery times were calculated from the time of
insemination to the time of cervical dislocation.

In some instances the oocytes were stained immediately, but
in other cases a further ip_vi££9_culture was carried out.

3. What is the effect of in vitro culture after recovery

 

from the rabbit oviduct? Morphologically normal ova or ova that

 

seemed to be near cleavage were cultured ip_yi££g, Ova recovered
at early stages of development were nearly all tested for their
ability to develop ig_vi£393

Ova culture was carried out for not more than 25 hr due to
the need of staining ova in good conditions prior to possible
degeneration.

4. What is the effect of culture of oocytes in medium

 

containing 5 ul/ml of FSH? The cattle and swine used in these studies

 

were not treated with gonadotropins prior to oocyte aspiration. Oocytes
aspirated from the follicle were generally immature with no polar bodies
and tight cumulus cells surrounding the oocyte. In lieu of gonadotropin
priming, a series of experiments involving attempts at 12.21339.
maturation of the oocytes with FSH (Burns-Biotic Laboratory, Oakland,
California) were done (Eppig, 1980).

Dilutions were made from a commercial FSH-P (Burns-Biotic) as
to get a final concentration of 5 ug/ml of FSH in the culture medium
(Table 2). This medium with 5 ug/ml of FSH was used to aspirate the
follicular oocytes and was used throughout the culture period. The

time of culture (37°C) in the FSH-media ranged from 19 to 20.5 hr.

23

Incubations were performed in the chamber/slides as will
be described (page 29). For a control treatment an equal number of
oocytes were ip_zi££g_cultured with the normal culture medium (Table 2).
Upon completion of the maturation period, as described (page 28),
oocytes were transferred.

Xenogenous fertilization of porcine oocytes. Porcine ova

 

were handled in a similar manner to those of cattle. Aspiration of
follicular oocytes was done as described (page 25) and the oocytes
maintained in a 37°C, 5% CO2 in air incubator. Oocytes were trans-
ferred to the ampulla of the pseudopregnant rabbit using a 5 ul
micropettor. Insemination was with 0.05 ml of diluted sperm of
concentrations of 1.35 x 107 and 4.5 x 107 sperm/ml. Oviducts were
ligated at the utero-tubal junction. At various times after insemi-
nation, oocytes were recovered. The same general criteria were used

for both bovine and porcine xenogenous fertilizations.

1. What is the effect of temperature duripg transport?

 

Porcine ovaries were transported in Dewar flask (thermos) that con-
tained 37°C 0.15 M NaCl solution. The time period from slaughter to
aspiration of the oocytes from the follicles, was from two to four hrs.
This time is considerably greater than the transport time of bovine
ovaries due to the longer distance to the slaughter plant.

In other instances porcine ovaries were transported in 0°C
0.15 M NaCl solution in plastic bags in an ice bucket. The transport

was similar to that for bovine ovaries.

24

2. What is the effect of time of recovery from the rabbit

 

oviduct on xenogenous fertilization? Standard procedures as described

 

(page 29) were followed to recover the porcine oocytes from the rabbit
oviduct at various times after insemination.

After recovery, oocytes were placed in a tissue culture chamber
(Lab-Tek products) for further examination under a phase contrast
microscope. Criteria for fertilization were as described (page 30).
Differences between means were examined by chi square (Gil, personal

communication).

Procedures Used

 

Ovulation induction in hamsters and rabbits. Hamsters were

 

induced to ovulate with an i.p. injection of 30 IU of pregnant mare
serum gonadotropin (PMSG, Biological Specialties, Middleton, Wisconsin)
between 9:00 and 12:00 hr. An i.p. injection of hCG (A.P.L§:)Ayerst,
New York, New York) followed 52 to 64 hr later. The hamsters were
killed by cervical dislocation 14 hr after hCG injection. The oviducts
were dissected from the rest of the reproductive tract. Using a dis-
secting microscope (30X), the oviducts were flushed from the fimbrial
end towards the utero—tubal junction. For this purpose, a 1 m1
tuberculin syringe with 30 gauge %" blunt needle was introduced

into the oviduct through the infUndibulum. A total volume of 0.3-

0.5 ml of medium was flushed through the oviduct. The medium was
recovered in a watch glass and the same microscope was used to locate
and aspirate the oocytes. From this point, the oocytes were handled

in the same way as the bovine or porcine oocytes.

Pseudopregnancies were induced in rabbits one day prior to
xenogenous fertilization. An i.v. injection of 100 IU of hCG (A.P.L.C>
Ayerst Laboratories, Montreal) in the marginal ear vein was given in
the morning of the day prior to surgery. As ovulation occurs 10 hr
after hCG injection (Harper, 1963), rabbits were considered to be
one day pseudopregnant at the time of oocyte transfer.

Preliminary experiments with hamsters have demonstrated that
the day of pseudopregnancy does not effect the xenogenous fertility
rate (DeMayo, personal communication).

Bovine and porcine follicular oocyte aspiration, medium used

 

for ova handling. All visible follicles on the ovarian surface were

 

aspirated regardless of size. Follicles were aspirated using a 3 ml
Luer-Lok tip plastic syringe with a 25 gauge, 5/8" needle. This syringe
contained 0.2-0.3 ml of 37°C medium (Table 2). This medium was used for
ovum handling at all times. All equipment was kept at 37°C.

After aspiration, the contents of the syringe were transferred
to a prewarmed watchglass. The aspirated contents were observed under
a dissecting microscope (30X) for the presence of oocytes.

The oocytes were picked from the watchglass using a micro/
pettor A (Scientific Manufacturing Industries, SMI) of 4 to 5 ul
volume, and transferred to a spot plate depression chamber that
contained 0.4 ml of medium. The chamber was covered with a glass

slide and placed in a 37°C incubator under 5% COz/air.

26

Table 2. Medium used for ovum handling and sperm dilutionsa

 

 

 

Quantity Source
Medium 199b 80% Gibcod Laboratories,
Grand Island, New
York
GG Free Fetal 20% Gibcod Laboratories,

Bovine Serum
Pyruvic acid

Gentomicin reagent
solution

Penicillin-streptomycin

Hyaluronidasef

Heparin sodiumg

115.2 ug/ml
0.1 mg/ml
100 units and
100 ug/ml

1 mg/ml

l unit/m1

Grand Island, New
York

Sigma Chemical Co.,
St. Louis, Missouri

Schering Corp.,
Kenilworth, New Jersey

North American Biol.,
Miami, Florida

Sigma Chemical Co.,
St. Louis, Missouri

Upjohn Co.,
Kalamazoo, Michigan

 

8Medium was filtered through a 0.45 um millipore filter into

sterile vacutainer. Medium was stored at 4 C.

b

With 25 mM HEPES Buffer, Earle's Salt and L. Glutamine.

cMycoplasma tested and virus screened, heat inactivated at 56°C

for 30 minutes.

dGrand Island Biological Company

eSodium Salt Crystalline Type II.

fFrom bovine testes, 1yophilized powder type 1-5.

gFrom beef lung.

27

Sperm handling. Different sources of sperm were used based

 

on availability.

1. Fresh semen. This was collected from mature bulls or boars

 

of proven fertility using an artificial vagina and standard collection
procedures. The sperm sample was then transported at 37°C to the
laboratory. The sperm were washed by dilution of 1 ml of semen with

9 m1 of medium and centrifuged at 1058 x g for five minutes. The
pellet was resuspended and washed again. Finally, the sperm were
diluted in 9 m1 (If 37°C medium and the motility visually estimated.
The sperm suspension was held at 37°C until insemination. Whenever
freshly ejaculated boar semen was collected, only the sperm-rich
fraction was used.

2. Frozen semen. Bull semen ampules and straws were provided

 

by MABC (Michigan Animal Breeders' Corporation, East Lansing, Michigan).
Boar pellets were provided by International Boar Semen (United Suppliers,
Eldora, Iowa). Boar semen was frozen using the technique of Pursel and
Johnson (1975). Frozen semen was thawed and washed following the same
procedure described above, but in same experiments nonwashed frozen
semen was used.

Boar semen pellets or bull semen, frozen in straws and ampules,
were held in liquid nitrogen. These were thawed in a 37°C water bath
and diluted with medium (approximately 3 m1 of medium). At this point
they were either washed or stored at 37°C until ready for use. In
preliminary experiments using hamster oocytes, the source of sperm

was the cauda epididymis.

28

Mature male hamsters were killed by cervical dislocation and
the testes removed. The cauda epididymis was minced on a watch glass
and diluted with medium. A visual estimate of motility was made. The
sperm suspension was then transferred to a test tube and held at 37°C
until insemination.

The inseminating device consisted of a 0.25 ml glass tuberculin
syringe, with a 20 gauge 1%" needle that had a 4 cm polyethylene tube
attached (I.D. 0.034"; O.D., 0.050"; PE 90 Intramedic®, Clay Adams,
Parsippany, New Jersey). The polyethylene tubing was inserted 1 to
2 cm into the ampulla.

Sperm concentrations were determined using a hemocytometer and
motility was based on visual estimation.

Anesthesia and surgery. Rabbits were anesthetized with a

 

sodium pentobarbital (Nembutal, Sodium, Abbot Laboratories, North
Chicago, Illinois) intravenous injection at a level of 60 mg per 5 lbs
of body weight. A surgical plane of anesthesia was maintained by ether
inhalation.

While under anesthesia, the ventral hair was clipped and the
surgical area cleaned with BetadineC) surgical scrub (The Purdue
Frederick Co., Norwalk, Connecticut).

The anesthetized rabbit was restrained on a surgery table
tilted at a 20° angle. A 7 cm ventral incision was made to expose
the reproductive tract. All instruments used were cold sterilized
in a 1:750 aqueous solution of Zephiran Chloride® (benzalkonium

chloride, Winthrop Laboratories, New York, New York).

29

Oocytes were removed from the 3-chamber culture dishes with
the micropipetter and immediately transferred to the oviduct of the
pseudopregnant rabbit in 4-5 ul aliquots. Twenty-two to forty-one ova
were deposited 2 cm through the fimbriated end of the ampulla into each
oviduct. Homologous sperm was then deposited in the oviducts as pre-
viously described in aliquots of 0.05 ml. The tubal-uterine junction
was ligated with 00 chromic gut to prevent oocyte loss, and the
incision closed.

Furacin soluble powder (Eaton Veterinary Laboratories, Norwich,
New York) was applied on the wound before closing. Procaine Penicillin-
G (300,000 IU) was injected intramuscularly to prevent infections.

Ovum recovery from the rabbit. At varying times after

 

insemination, the oocytes were recovered from the oviducts. The
rabbits were killed by cervical dislocation. A ventral incision was
made and the reproductive tract removed. The tubal uterine ligation
was removed and the oviducts flushed with medium from the uterine end.
A 5 ml plastic syringe with a 25 gauge 5/8" blunt needle was used for
this purpose. The syringe contained 2 ml of medium and 3 ml of air.
The flushing medium was collected in a watch glass at 37°C.
A dissecting microscope (30X) was used to locate all oocytes. These
were transferred to a tissue culture chamber/slideqp (Lab-Tek Products,
#4838 Miles Laboratories Inc., Naperville, Illinois). The chambers
each contained 0.25 ml of 37°C medium. Fertilization was assessed
as described in the next section.
In some cases further ip_!i££9_culture was done to determine

if further development would occur.

30

Criteria of fertilization. Oocytes with two pronuclei and

 

two polar bodies or cleaved ova were considered to be fertilized.
These observations were confirmed by staining with giemsa stain
(T. Asakawa, personal communication). This procedure consisted
of gradual fixation using three fixatives and warm moist-air

drying (Mizoguchi and Dukelow, 1981).

RESULTS

Preliminary studies were for technique development. One of
these studies consisted in studying the effects of ligation of the
oviduct at the tubo-uterine junction on the rates of recovery and
xenogenous fertilization. Hamster oocytes were used for these
studies. Table 3 shows the results for these experiments. No
significant differences were observed at 32 hr recovery times from
the rabbit oviduct. The criterion for fertilization was 2-ce11
embryos. It was concluded that it is not necessary to ligate the
oviduct of the rabbit when recovery is less than 32 hr post imsem-
ination. The need for ligation for longer periods of oviductal
incubation is yet to be determined.

The effect of sheep serum vs Fetal Calf Serum (FCS) on ham-
ster xenogenously fertilized embryos was studied. A total of 46 ova
recovered from the rabbit oviduct were cultured using the standard
medium with sheep serum instead of FCS. Of these, 7 were in the 2-cell
stage. Ova were ip_viE£p_cultured for 11 hr. No further development
was observed in either the l-cell or 2-cell embryos by the end of the
culture. Most of the embryos were either in early or late phase of
degeneration by this time.

An experiment was undertaken to test the fertilizability and
recovery rates of bovine and porcine ova xenogenously fertilized in

the oviduct of the pseudopregnant rabbit. The results are shown in

31

32

Table 3. The effects of ligation at the uterotubal junction, on
recovery and xenogenous fertilization of hamster embryo rates
(recovery 32 hours after insemination)

 

 

 

Oocytes Oocytesa
Recovered/Transferred (%) Fertilized/Ova Recovered (%)
Nonligated
oviduct 24/41 (58.5) 4/24 (16.7)
Ligated
oviduct 22/41 (53.7) 3/22 (13.6)

 

aCriterion of fertilization, 2-cell.

Table 4. The recovery rate for bovine was higher than that of swine
(P< 0.01). Fertilization rates were also significantly different
(P < 0.001) .

Bovine ovaries were transported from the slaughter house to the
laboratories at either 0° or 37°C in physiological saline. Table 5
shows the effect of the two transport temperatures on the recovery
and xenogenous fertilization rates.

The number of oocytes recovered was higher when the ovaries were
transported at 0°C, but this increase was not significantly different
from the rate when ovaries were transported at 37°C (x2: 1.12 for
recovery and 1.64 for fertilization). Fertilization rates were not
statistically significant between the two treatments.

Table 6 shows the effect of two different temperatures of
transport on recovery and fertilization rates of porcine oocytes.

No significant differences between treatments were found for either

33

Table 4. The recovery and xenogenous fertilization rates of bovine
and porcine follicular oocytes in the oviduct of the
pseudopregnant rabbit

 

 

No. of
Oocytes Rabbits Recovered/Transferred (%) Fertilized/Recovered (%)

 

Bovine 10 261/582 (44.8) 35/261 (13.4)

Porcine 6 148/410 (36.1)8 3/148 (2.0)b

 

aSignificantly different from bovine (P<=0.01).

bSignificantly different from bovine (P<‘0.001).

Table 5. Temperature effects on ovary transport on recovery and
xenogenous fertilization rates of bovine oocytes

 

 

 

No. of Oocytes Recovered/ Oocytes Fertilized/
Temperature Rabbits Transferred (%) Recovered (%)
0°C 4 88/228 (38.6) 10/88 (11.4)

37°C 4 88/259 (34.0) 16/88 (18.2)

 

34

Table 6. Temperature effects on ovary transport, recovery, and
xenogenous fertilization rates of porcine oocytes

 

 

 

No. of Oocytes Recovered/ Oocytes Fertilized/
Temperature Rabbits Transferred (%) Recovered (%)
0°C 3 83/241 (34.4) 1/83 (1.2)
37°C 3 65/169 (38.5) 2/65 (3.1)

 

recovery or fertilization rates. The recovery times for these
experiments were 22.3 to 70 hr after insemination.

When bovine oocytes were xenogenously fertilized, several
times of recovery were used. Table 7 shows four time intervals with
their respective recovery and fertilization rates.

Intervals of 50-55 and 65-70 hr are significantly greater for
recovery rate (49.8 and 39.6%, respectively) than intervals of 40-45
and 70-75 hr (11.4 and 8.5%, respectively) (P< 0.01). There were no
significant differences between intervals for the fertilization rates
(X2==5.595). Ova cleavage was observed starting at times of recovery
of 50 hr and beyond.

The effect of ip_vi££g_culture of bovine follicular oocytes
in medium supplemented with 5 ug/ml FSH is shown in Table 8. The time
of culture in the supplemented and control media was of 20 hrs. Ovi-
ductal recovery rates of 76.7 and 91.9% for the treated and control
oocytes were observed, respectively. There was a tendency for oocytes

treated with FSH to have lowered recovery compared with controls

35

Table 7. The recovery and xenogenous fertilization rates of bovine
oocytes recovered at varying times after insemination

 

 

 

Recovery Time (Hr' No. of' Oocytes Recovered/ Oocytes Fertilized/

Post Insemination) Rabbits Transferred (%) Recovered (%)
40-45 1 5/44 (11.4)3 1/5 (20.0)
50-55 3 103/207 (49.8)b 10/103 (9.7)
65-70 3 61/154 (39.6)b 13/61 (21.3)
70-75 1 7/82 (8.5)3 2/7 (28.6)

 

aDifferent superscripts indicate significant difference
(P< 0.01).

Table 8. The effect of culture ip_vitro for 20 hours in medium
supplemented with 5 ug/ml FSH, on recovery and xenogenous
fertilization rates of bovine oocytes

 

 

 

Oocytes Recovered/ Oocytes Fertilized/ Stage of
Transferred (%) Recovered (%) Fertilization
FSH 23/30 (76.7)a 2/23 (8.7) Z-cell, 2pbb
Control 34/37 (91.9) 1/34 (2.9) 2pb

 

aDifferent from control (P<<0.l).

b2 pb = 2 polar body stage.

36

(P<<0.10). There was no difference between treated and control
oocyte fertility.

Further trials to obtain ip_vi££p_maturation of the follicular
oocytes prior to sperm exposure were performed. In the rabbit oviduct
a mucin coat is deposited around the oocyte which limits the ferti-
lizable life (of rabbit oocytes) to six hours. To determine if mucin
deposition was effecting xenogenous fertilization, bovine follicular
oocytes were cultured in_vi£rg_for 25 hr prior to deposition in the
rabbit oviduct. Results indicate that (Table 9) of 28 oocytes incu-
bated ip_vi££p_and transferred to the rabbit oviduct, 28 (100%) were
recovered. Of these 6/28 (21.4%) showed evidence of fertilization.
Five of the ova were at the 2 polar body stage and showed condensed
cromatin when stained. The other ovum was recovered as a 2-cell
embryo. The ip_vit£g_culture was carried out for a period of 25 hr
and the oocytes were further cultured in the oviduct for 47 hr (72 hr
total). Control I oocytes were of the same age (70 hr) that had been
in the rabbit oviduct for the entire culture period. Control 11 oocytes
were not cultured ip_vi££p_but were in the oviduct approximately the
same time as the treated oocytes (50 hr). These oocytes (Control 11)

were 25 hr younger than the ip_vitro cultured oocytes. Table 9 shows

 

that there was a significant increase of the recovery rates when
oocytes were cultured ip_vitro for 25 hr (P< 0.01). No significant

effect in fertilization was noted.

37

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DISCUSSION

The xenogenous fertilization technique using the pseudopregnant
rabbit as host was developed using hamster oviductal oocytes that were
placed in the rabbit oviduct. It was demonstrated that ligation, which
is primarily performed to avoid oocyte loss, is not necessary when
recovery times are 32 hr or less. This recovery time is the ideal
to recover 2-cell hamster embryos (F. J. DeMayo, personal communica-
tion). Ligation does not interfere with fertilization. This confirms
the findings of Adams (1973) and Sharp and Black (1975) who studied the
effects of ligation on rabbit ovum fertility. The ease of xenogenous
fertilization of hamster oocytes to the 2-cell stage has been reported
(DeMayo et al., 1980). In the present work, similar cleavage rates were
obtained. DeMayo et al. (1980) reported cleavage rates of 14%, whereas,
in the present studies, a cleavage rate of 16.7% was found with the
unligated oviduct and 13.6% with ligation. It still must be determined
if recovery rates decrease once the oocytes enter the uterus, or if
longer recovery times are used as is the case with cattle or swine
follicular oocytes.

Hamster embryos fertilized xenogenously do not develop beyond
the 2-cell stage in the rabbit oviduct and this could not be altered by
substituting sheep serum for FCS. It appears that there is a "Z-cell
block" that cannot presently be overcome in either the oviduct of the

pseudopregnant rabbit or ip_vitro.

38

39

Successful xenogenous fertilization of bovine oocytes had been
reported in estrous ewes (Sreenan, 1970) and prepuberal gilts (Bedirian
et al., 1975; Shea et al., 1976). However, Trounson et a1. (1977)
reported negative results when using the pseudopregnant and estrous
rabbit as host for the transfer. In the present study, both bovine
and porcine follicular oocytes can be fertilized in the oviduct of
the one-day pseudopregnant rabbit. Parthenogenic activation has been
ruled out in the present study by staining and observation of normal
pronuclei, sperm tail remanants in the cytoplasm and normal nuclei
after cleavage to the 2-cell stage.

A difference in the present technique and that reported by
Trounson et a1. (1977) is the site of insemination. Their work was
done with does previously vaginally inseminated. The present work
was done by oviductal insemination to the ampulla immediately after
to oocyte deposition. They held oocytes matured ip_vivg_(after gona-
dotropin treatment of heifers) in Phosphate Buffered Saline (PBS) at
room temperature, whereas, in the present study, oocytes were held at
all times after aspiration at 37°C.

Both bovine and porcine oocytes are capable of being fertilized
in the reproductive tract of the pseudopregnant doe as shown in the
present study. There was a significant difference between species with
respect to fertilization (P< 0.001). The difference was not as signif-
icant (P<<0.01) for recovery rates. The longer transport times involved
in the transport of swine ovaries could be a limiting factor, as the
bovine ovaries were brought to the laboratory at times varying from 2

to 4 hrs and the porcine ovaries transport time was greater (6 to 7 hr).

40

No effect of temperature on recovery or xenogenous fertilization
rates for both bovine nor swine ovaries was observed. Trounson et al.
(1976b) reported tolerance of bovine blastocysts to cooling to 0°C,
but it appears that at the 8-cell stage cooling to 0°C produces
irreversible damage of the embryo (Wilmut et al., 1975). When bovine
morulae are cooled at 0°C, only a small percentage of the embryos
survive cooling (Trounson et al., 1976a, 1976b). In the present study,
no apparent effect was observed by cooling the ovary to 0°C compared
with 37°C.

Different times of recovery were used to determine stages and
rates of fertilization at time intervals of 50 through 70 hr after
insemination. There was a significant increase in recovery rates.

The decrease of recovery after 70 hr probably reflects a degeneration
and reabsorption of nonfertilized oocytes. This same loss of nonfer-
tilized oocytes could result in an apparent increase in the fertili-

zation rate (28.6% fertilization for 70-75 hr vs 21.3% for 65-70 hr).

Bovine follicular oocytes mature ip_yi££g_from germinal vesicle
to metaphase II (GVBD) by as early as 19 hr (Jagiello et al., 1974) to
as late as 31 hr (Edwards, 1965). The maturation period is from 19 to
31 hr for the follicular oocytes and 24 hr for ip_vit£g_cleavage to the
2-cell (Brackett et al., 1977, 1978). Therefore, ip_vi££p_cleavage
would be expected 43-56 hr from initiation of culture. In the present
studies, cleavage to the 2-cell stage was observed from 50 to 70 hr
after insemination. This represents a delay from the cleavage

observed ip_vivo by Hamilton and Laing (1946).

41

In the present work, pronuclear stages were observed as early
as 40 hr. This can be compared with the ip_vivp_pronuclear stage 5
to 12 hr after ovulation (Thibault, 1967; Brackett, 1980) considering
maturation periods of 19-31 hr for follicular oocytes (Edwards, 1965;
Jagiello et a1., 1974; Shea et a1., 1976; Newcomb et al., 1978).

Eppig (1980) reported cumulus cell expansion hyaluronic acid
synthesis and hastened maturation of mouse follicular oocytes when
they were cultured in medium containing FSH and FCS. In the present
work, ip_vi££g_incubation with FSH had a deleterious effect on recovery
from the rabbit oviduct. It is suggested that if maturation is hastened
degeneration might occur at earlier stages. Thus, reabsorption or
complete degeneration of nonfertilized oocyte devoid of protective
cumulus cells could occur in the rabbit oviduct. Fertilization rate
was not significantly affected by ip_!i££p_culture with FSH; however,
2-cell embryos were recovered from the oviduct after 54 hr incubation.
Ip_viyp_rates of development show 8-cell stages at 44 hr after ovulation
(Thibault, 1966; Brackett, 1980) or 2- to 4—cell (Hamilton and Laing,
1946). This shows that it appears to be a delay in the xenogenous
fertilized embryo development when compared with ip_vivp_fertilized
embryos.

The mucin coat built up in the rabbit impairs fertilization
of rabbit oocytes after six hr. An attempt to increase maturation of
oocytes before transfer to the rabbit oviduct did significantly increase
oocyte recovery. This appears to be due to loss of degenerated oocytes.

Edwards (1966) reported the start of atresia within the ovarian follicle

42

but it appears that there is no readily discerned morphological
difference between a degenerative oocyte and a viable one imme-
diately after aspiration from the follicle. After 25 hr in culture,
degenerative oocytes can be easily observed. Degenerated oocytes
were not knowingly transferred.

Fertilization rates did not increase when oocytes were
incubated ip_vi££g, but a highly significant increase in recovery
rates was observed. It would be advantageous to mature oocytes ip_
viE£g_to obtain higher recovery rates from the rabbit oviduct. When
oocytes were previously cultured, two of six cleaved to the 2-cell
stage upon recovery (47 hr). In this case cleavage appears to be
similar to that obtained ip_vi££p_(Brackett, 1977, 1978) and ip_!ivp_

(Hamilton and Laing, 1946).

SUMMARY AND CONCLUSIONS

Fertilization of bovine and porcine follicular oocytes and
hamster oviductal oocytes, in the oviduct of the pseudopregnant rabbit
(xenogenous fertilization) was studied.

When oviductal recovery of hamster oocytes from the rabbit
oviduct is 32 hr or earlier after insemination, no tubal ligation is
necessary to avoid ovum loss.

The 2-cell stage block observed with hamster xenogenously
fertilized embryos cannot be overcome with either Fetal Calf Serum
(FCS) nor sheep serum heat inactivated at 56°C for 30 min.

Bovine and porcine follicular oocytes were capable of being
fertilized xenogenously as shown by overall fertilization and recovery
rates of 13.4% and 44.8%, respectively, for the bovine, and 2.0% and
36.1%, respectively, for the porcine oocytes. A significant decrease
in recovery (P< 0.01) and fertilization rates (P< 0.001) for porcine
oocytes with respect to bovine oocytes was observed.

Transport of ovaries at 0°C vs 37°C had no effect on either
recovery or fertilization rates for both bovine and porcine oocytes.

Several recovery times were used for bovine oocytes. Recovery
times from 50 to 70 hr significantly differed (P<:0.01) from earlier
(40-45 hr) or later (70-75 hr) times.

Fertilization was not affected by recovery time, although
an increased number of 2-cell embryos were recovered at 50 hr after
insemination.

43

44

Ip_vit£g_culture of oocytes in medium supplemented with 5 ug/ml
of follicular stimulating hormone (FSH) decreased (P< 0.1) the recovery
rate from the rabbit oviduct (76.7% vs 91.9% of control). No effect was
observed on fertilization. Cleavage to the 2-cell stage was achieved
when oocytes were preincubated with FSH.

Ip_vi££9_culture of bovine follicular oocytes in medium for
25 hr prior to xenogenous fertilization increased significantly the
recovery from the rabbit oviduct (P< 0.01). No effect was observed

on the fertilization rate.

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of cooling to 20°, 0° and -196°C. J. Reprod. Fert. 45, 409-411
(1975).

52

Witemberger-torres, S. Les rapports entre l'oeuf en segmentation at
1e tractus maternal chez la brebis. 3rd Int. Congr. Anim. Reprod.
and A. I. Cambr. 1, 62-64 (1956).

Wright, R. W., Jr., Anderson, G. B., Cupps, P. T., and Drost, M.
Successful culture ip_vitro of bovine embryos to the blastocyst
stage. Biol. Reprod. 14, 157-162 (1976a).

Wright, R. W., Jr., Anderson, G. B., Cupps, P. T., and Drost, M.
Blastocyst expansion and hatching of bovine and cultured ip_
vitro. J. Anim. Sci. 43, 170-174 (1976b).

APPENDIX A

PUBLICATIONS BY THE AUTHOR

Papers:

Abstracts:

APPENDIX A

PUBLICATIONS BY THE AUTHOR

Alternative methods of fertilization for reproductive
toxicology. W. R. Dukelow, P. J. Hirst, T. Asakawa,
F. J. DeMayo, and P. J. Chan. Proc. 8th European
Teratology Conference (in press, 1981).

Xenogenous fertilization of laboratory and domestic
animals in the oviduct of the pseudopregnant rabbit.
P. J. Hirst, F. J. DeMayo, and W. R. Dukelow.
Theriogenology (in press, 1981).

Xenogenous fertilization of nonhuman primate, laboratory
and domestic animals. P. J. Hirst, F. J. DeMayo and

W. R. Dukelow. Proc. 3lst Annual Session of American
Association for Laboratory Animal Science. October 7,
1980. Indianapolis, Indiana.

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APPENDIX B

VITA

NAME:

Born:

Place of Birth:

Formal Education:

Degrees Received:

APPENDIX B

VITA

PATRICIO JORGE HIRST
October 8, 1956

Buenos Aires, Argentina

Instituto-Cardenal Newman 1970-1973

Buenos Aires, Argentina

Pontificia Universidad Catélica Santa Maria
de Los Buenos Aires 1974-1978

Buenos Aires, Argentina

Bachiller 1973
Ingeniero en Produccién Agropecuaria 1977
Licenciado en Ciencias Agrarias

(Especialidad Zootecnia) 1978

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