Rev. sci. tech. Off. int. Epiz.,
1985, 4 (4), 859-866.
The potential of bovine embryo transfer
for infectious disease control*
D . A . STRINGFELLOW**
Summary: The capability to collect preimplantation embryos, store them in
the frozen state for extended periods of time, and subsequently thaw and suc­
cessfully transfer them provides a new alternative to traditional means of
improving blood lines. Research to date involving specific bovine pathogens
has shown that this method of moving genetic material can be a valuable tool
to those concerned with preventing the introduction of infectious disease into
previously uninfected populations of cattle. In addition, an assessment of
basic epidemiological factors involved in the process of embryo transfer leads
to the conclusion that the movement of embryos is an innately safer process.
The embryo is an unlikely host for many pathogens because of primary resis­
tance provided by the zona pellucida (ZP) and possibly the trophectoderm and
secondary resistance factors such as young age, small size, and limited mobility.
The fact that many of the bacterial and viral pathogens have been shown
to be too large to penetrate the ZP and that some do not survive well in
embryo support media further reduces the possibility of an infectious agent
being spread by embryo transfer.
Environmental factors involved in embryo transfer procedures are perhaps
the most important to consider. The donor environment limits exposure to
infectious agents and provides testing opportunities before, during, and after
collection of embryos.
Dilution factors, mechanical washing, antimicrobial agents, and enzyme
treatments may be provided to the embryo in its in vitro environment. Addi­
tionally, there is the possibility for visualizing the embryo in its entirety under
the microscope and exerting control over the sterility of its surroundings.
The recipient animal provides the last safeguard since she represents a con­
tainment facility for the newly transferred embryo and can serve as a sentinel
for the presence of any infectious agent.
K E Y - W O R D S : Bacteria - Cow - Disease control - Disease resistance E m b r y o - Environment - Testing procedures - Transplantation Viruses.
INTRODUCTION
Bovine embryos can now be collected nonsurgically from donor females, stored
in the frozen state for extended periods, and transferred surgically or nonsurgically
* Report presented at the O I E / I E T S Round Table Meeting on sanitary problems related to embryo
transfers. Paris, 9 December 1985.
** Department of Microbiology, Auburn University College of Veterinary Medicine, Auburn Uni­
versity, Alabama 36849, U S A .
— 860 —
to recipients resulting in the birth of normal calves with a high degree of success (1).
When this technology became available, it was suggested that the transport of
embryos might be used as an alternative method to shipping livestock internatio­
nally (2). The economic and h u m a n e advantages of shipping flasks of frozen pre­
implantation embryos as opposed to truck-, airplane-, or boat-loads of m a t u r e ani­
mals are obvious; however, early studies with laboratory animal species created
concerns about the possibility of embryo transfer being a m o d e of transmission for
livestock pathogens (3). With these concerns as the impetus, n u m e r o u s studies have
been conducted in recent years to investigate the potential for transmission of
disease producing agents through embryo transfer. The results of these studies,
involving specific agents have been summarized recently (4, 5) and lead to the con­
clusion that embryo transfer can be safer than traditional means of shipment of
genetic material.
In addition to these research findings involving specific agent-embryo inter­
actions there are sound epidemiological factors which indicate that the movement
of embryos is innately safer than the movement of cattle. The purpose of this paper
is to describe those agent, host and environmental factors which cumulatively sup­
port the use of embryo transfer as a means of controlling the spread of infectious
diseases in national and international livestock populations.
HOST FACTORS
Host determinants of infectious diseases are categorized as intrinsic and extrin­
sic factors (6). In a consideration of the epidemiological aspects of bovine embryo
transfer, the intrinsic and extrinsic factors for three potential hosts of disease, the
donor, the embryo, and the recipient are of significance, but should be considered
separately. For our purposes the embryo is considered the host of primary concern.
The donor and recipient animals will be considered under environmental factors
and testing possibilities. Determinants of disease of primary importance in the
embryonic host are intrinsic and include primary resistance factors such as the zona
pellucida (ZP) and trophectoderm and secondary resistance factors such as age,
mobility, and conformation. The size of the embryo also has epidemiologic signifi­
cance.
The Z P is a glycoprotein shell which is secreted by the oocyte and the granulosa
cells immediately surrounding it during the development of the secondary follicle
(7, 8). Most of the functional and conformational information about this structure
has been obtained through studies with murine embryos. It has been shown that the
Z P is permeable to macromolecules (9) and even viruses (10) in mice. However, in
the bovine embryo this protective shell resists adherence and penetration by many
pathogens including A k a b a n e virus, bovine leukemia virus (BLV), bluetongue virus
(BTV), bovine viral diarrhea virus (BVDV), foot and m o u t h disease virus (FMDV)
and Brucella abortus (11-15). In addition, it has been shown that the Z P resists
penetration although not adherence by infectious bovine rhinotracheitis virus
(IBRV) (16).
Attempts to demonstrate infection of zona pellucida-free (ZP-F) embryos after
in vitro exposure with BLV, BVDV, bovine parvovirus (BPV) and B. abortus (12,
15, 17, 18) have failed, leading to the speculation that blastocysts possess the ability
to resist infection by certain agents even without the protection of the intact ZP.
— 861
The demonstration of lysosome-like structures in ultrastructural studies of bovine
blastocysts provides additional support for this speculation (19).
Young age, limited mobility, and proper conformation are important secondary
resistance factors possessed by the bovine embryo. The majority of embryos for
transfer are collected on day 7 (1). The embryo at this stage is approximately
160 microns in diameter and contains 80-120 cells surrounded by the Z P (20). In
considering infectious diseases in which transovarian transmission does not occur,
infection of the embryo would have to take place between ovulation and recovery
of the embryo for transfer. This limits the opportunity for exposure to a 7-day time
frame. The limited mobility of the embryo is also a consideration, since it remains
relatively isolated in the oviduct and uterus until recovery. These factors serve to
reduce the potential for exposure to a given etiologic agent. Conformation is the
final important secondary resistance factor. Embryos that are better able to resist
infection or adherence of pathogens will have perfectly symmetrical Z P of uniform
thickness, will be free of adherent matter and will contain little or no extruded
material.
The size of an embryo is not a resistance factor, but it is epidemiologically signi­
ficant. At the stage in which it is recovered, the embryo can be visually examined in
its entirety, enabling the integrity of its defence barrier (ZP) to be examined and the
quality of its cells to be evaluated. Based on information available on many bovine
pathogens, the presence of an intact Z P and viable healthy cells is indicative of
noninfection.
AGENT FACTORS
The size and ability of living agents to survive in the environment are of epide­
miological significance. Other properties of living agents related to their action in
the host such as infectivity, pathogenicity, virulence, and immunogenicity are also
pertinent in an assessment of the hazards of causing a diseased state in the host (6).
Penetration of the intact Z P by infectious agents which are not known to be
present on or in the gametes must occur along channels left when follicular cell p r o ­
cesses are withdrawn as the corona cells are shed shortly after ovulation. The pene­
tration of mengovirus t h r o u g h the mouse Z P by this latter route has been demons­
trated (10). Mengovirus is an extremely small R N A virus (picorna virus). Larger
viruses and certainly bacteria will be prevented from passing through the intact Z P
because of their physical size.
Survivability of aetiologic agents in the artificial environments of the embryo
created by embryo collection and storage techniques is also important. For exam­
ple, free BVD virus (i.e. not cell associated) has been shown to lose viability with
time when held in embryo support media at room or incubator temperatures (15)
and suspensions of B. abortus subjected to embryo freezing and thawing proce­
dures have been shown to undergo a 6 4 % reduction in viability (21).
Infectivity refers to the ability of an agent to lodge itself in the host, pathogeni­
city refers to an agent's ability to produce disease, and virulence is the property of
the agent which determines the severity of the disease produced (6). Published
reports have only shown that one bovine agent, IBRV, is capable of attaching itself
on the bovine Z P . Penetration of the intact bovine Z P has not been demonstrated
— 862 —
for any agent. If future studies were to show that penetration of the Z P by an infec­
tious agent is possible, then the pathogenicity and virulence of that agent for the
embryo would have epidemiological significance. If penetration and infection
occurred, and the embryo was rapidly destroyed, the hazards of transmitting the
agent via embryo transfer techniques would be minimal since the degenerate
embryo would not be transferred.
Immunogenicity is an agent factor which is not of importance in its direct rela­
tionship to the embryo since a day 7 embryo has not developed an immune system.
However, the immune responses elicited in the donor and recipient animals are per­
tinent and will be discussed under environmental factors.
ENVIRONMENTAL FACTORS
Environmental determinants of the potential for disease transmission or nontransmission t h r o u g h embryo transfer are important factors to those concerned
with preventing the spread of disease since they are most subject to inspection and
control. Transferred embryos are exposed to three separate microenvironments, the
donor environment, the in vitro environment and the recipient environment.
Disease control provisions occur naturally or can be provided in each of these envi­
ronments.
In the short time from ovulation until recovery for transfer the ova are afforded
a degree of isolation and protection in the oviduct and uterus of the donor. The
timing of this stay in the reproductive tract has epidemiological significance for it
occurs during the first week of the oestrus cycle. At this time, the uterus has just
experienced an immunological purging. The enhanced immune response in the ute­
rus at the time of oestrus has long been recognized. The high blood levels of oestro­
gen achieved at oestrus are associated with a leukocytic infiltration and increased
blood supply to the uterus with associated higher levels of immunoglobulins secret­
ed and a relaxation of the cervix with retrograde flow of uterine content (22, 23).
All of these activities serve to purge the uterus of any existing infections such as has
been shown for B. abortus (24, 25) and to protect against introduction of infection
at the time of breeding (22). Furthermore, there is some indication that hormones
injected in superovulatory treatments may serve to enhance the humoral immune
response in some donor animals (26). In addition to these natural provisions for a
clean environment, freedom of the d o n o r ' s reproductive tract from infectious
agents can be ensured by traditional means of isolation, testing, a n d / o r vaccination
of the donor animal.
The in vitro environment of the embryo in normal transfer procedures provides
multiple opportunities for elimination of pathogens which may be present. These
include the dilution factor of recovery medium used, mechanical washing, anti­
microbial agents in recovery and wash media, and enzyme treatments (27). There is
also the opportunity to observe the embryo in its entirety under the microscope
and, in addition, the embryo can be completely isolated and control exerted over
the sterility of its environment.
The third microenvironment of the embryo is the uterus of the recipient. As
with the donor, the recipient's uterus can be assured to be free of infectious agents
by traditional means of isolation, testing, a n d / o r vaccination and these precautions
should be taken before recipients receive embryos.
— 863 —
TESTING
POSSIBILITIES
The use of bovine embryo transfer techniques to replace traditional means of
shipment of livestock permits unique testing opportunities which are associated
with each of the microenvironments of the embryo. The donor female represents a
quarantine facility for the embryo which is to be collected. For an agent-embryo
encounter to occur, the d o n o r must first be exposed to the agent. To detect the presence of agents for which a reliable serological test exists, paired sera collected at
the time of embryo recovery and approximately two weeks after collection can be
used to provide assurance that the embryo was free from exposure. Embryos can be
held in the frozen state until serological evidence is provided. It should be noted
that, while the absence of seroconversions could ensure that exposure did not
occur, the presence of seroconversion does not automatically mean that exposure
did occur.
Available techniques for testing embryos are destructive and they are not presently a viable option (5). Test procedures now include attempts to isolate agents
from (i) flush fluids, (ii) embryo washes a n d / o r (iii) selected embryos or nonfertilized or degenerate ova from a group recovered at a single collection.
The recipient can also be tested after receiving an embryo when a reliable serological test exists for detecting the presence of specific pathogens. However, good
disease control practices dictate that the embryo's health status should be known
before transfer if possible. In selected situations the recipient could be held in isolation as a sentinel animal and tested for seroconversion following a certain period of
time.
*
* *
L'INTÉRÊT POTENTIEL D U T R A N S F E R T D ' E M B R Y O N S BOVINS
PROPHYLAXIE D E S M A L A D I E S INFECTIEUSES. — D . A . Stringfellow.
POUR
Résumé : La possibilité de récolter des embryons transférables, de les conserver sous forme congelée pendant une période prolongée, puis de les décongeler
pour les transplanter avec succès représente une alternative intéressante aux
méthodes traditionnelles de l'amélioration génétique (importation d'animaux
vivants, insémination artificielle).
Jusqu'à présent, les recherches sur les agents pathogènes spécifiques des
bovins ont montré que ce mode de transport du matériel génétique est très
utile pour éviter l'introduction de maladies infectieuses dans des populations
bovines précédemment indemnes. En outre, l'évaluation des facteurs épidémiologiques fondamentaux impliqués dans le transfert d'embryons a permis
de constater que ce procédé était par lui-même moins dangereux.
L'embryon est un hôte peu vraisemblable pour beaucoup d'agents pathogènes, à cause de la résistance primaire conférée par la zone pellucide et peutêtre le trophectoderme, et de facteurs de résistance secondaires tels que son
jeune âge, sa petite taille et sa mobilité limitée.
Beaucoup de virus et de bactéries sont trop volumineux pour traverser la
zone pellucide et certains survivent mal dans le milieu de l'embryon, ce qui
réduit encore le risque de propagation d'agents infectieux par le transfert
d'embryons.
LA
— 864 —
Les facteurs d'environnement impliqués dans cette méthode sont peut-être
les plus importants à considérer. L'environnement de la donneuse limite les
contacts avec les agents infectieux et fournit des possibilités de contrôle avant,
pendant et après la collecte des embryons.
L'embryon peut être soumis à des dilutions successives, des lavages mécaniques, à l'action d'agents antimicrobiens et des traitements enzymatiques
dans son milieu in vitro. De plus, on peut le visualiser entièrement sous le
microscope et s'assurer qu'il est maintenu dans une ambiance stérile.
La vache receveuse représente la dernière garantie puisqu'elle est le réceptacle de l'embryon nouvellement transféré et peut être utilisée comme moyen
d'alerte en cas de présence d'un agent infectieux.
M O T S - C L É S : Bactéries - E m b r y o n - Environnement - Méthodes d'analyse Prophylaxie sanitaire - Résistance aux maladies - Transplantation - Vache - Virus.
*
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INTERÉS P O T E N C I A L D E L A TRANSFERENCIA D E EMBRIONES BOVINOS EN EL
CONTROL D E L A S E N F E R M E D A D E S INFECCIOSAS. — D . A . Stringfellow.
Resumen : La posibilidad de recoger embriones transferibles, conservarlos en
forma congelada durante un período prolongado, y descongelarlos después
para transplantarlos con éxito constituye una opción interesante frente a los
métodos tradicionales de mejoramiento genético (importación de animales
vivos, inseminación artificial).
Hasta ahora, las investigaciones de los agentes patógenos específicos de los
bovinos han venido probando que esta forma de transporte del material genético es de suma utilidad para evitar que se introduzcan enfermedades infecciosas en las poblaciones bovinas que estaban libres de enfermedades. Además, la
evaluación de los factores epidemiológicos fundamentales implicados en la
transferencia de embriones permitió comprobar que este procedimiento era de
por sí menos peligroso.
El embrión es un huésped poco probable para muchos agentes patógenos,
a causa de la resistencia primaria que confiere la zona pelúcida y acaso la trofectodermis, y de factores secundarios de resistencia tales como su joven edad,
pequeño tamaño y movilidad limitada.
Muchos virus y bacterias son demasiado voluminosos para atravesar la
zona pelúcida y algunos sobreviven mal en el medio del embrión, con lo que se
aminora aún más el riesgo de que se propaguen agentes infecciosos con la
transferencia de embriones.
Acaso los factores ambientales implicados en este método sean los más
importantes que se han de considerar. El medio ambiente de la donante limita
los contactos con los agentes infecciosos y proporciona posibilidades de control antes, en y después de la recolección de embriones.
Se puede someter el embrión a diluciones sucesivas, lavados mecánicos, a
la acción de agentes antimicrobianos y tratamientos enzimáticos en su medio
in vitro. Se le puede visualizar completamente además en el microscopio, verificando que se mantiene en un ambiente estéril.
La vaca receptora representa la última garantía por cuanto es el receptáculo del embrión recién transferido, pudiéndose utilizar como medio de alerta
en el supuesto de que existan agentes infecciosos.
— 865 —
P A L A B R A S C L A V E : Bacterias - Control sanitario - E m b r i ó n - Medio
ambiente - Métodos de análisis - Resistencia a las
enfermedades - Transplante - Vaca - Virus.
*
* *
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— 866 —
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