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Commentary
Recommendations for Ensuring Good Welfare of Horses Used
for Industrial Blood, Serum, or Urine Production
Xavier Manteca Vilanova 1, Bonnie Beaver 2, Mette Uldahl 3and Patricia V. Turner 4,5 ,*
Citation: Manteca Vilanova, X.;
Beaver, B.; Uldahl, M.; Turner, P.V.
Recommendations for Ensuring Good
Welfare of Horses Used for Industrial
Blood, Serum, or Urine Production.
Animals 2021,11, 1466. https://
doi.org/10.3390/ani11051466
Academic Editor: John Madigan
Received: 18 April 2021
Accepted: 18 May 2021
Published: 20 May 2021
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1School of Veterinary Science, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain;
xavier.manteca@uab.es
2College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4474, USA;
bbeaver@cvm.tamu.edu
3Vejle Equine Practice, 7120 Vejle, Denmark; mette@vejlehestepraksis.dk
4Global Animal Welfare & Training, Charles River, Wilmington, MA 01887, USA
5Department of Pathobiology, University of Guelph, Guelph, ON N1G 2W1, Canada
*Correspondence: pvturner@uoguelph.ca
Simple Summary:
Because of their large size, blood, serum, or other substances are often collected
from horses for production of biologics and therapeutics used in humans and other animals. There
are few international guidelines that provide recommendations for caring for horses kept for these
purposes. In this paper, general guidelines are provided to ensure well-being of horses kept for
production of biologics.
Abstract:
Various pharmaceutical products have been derived from horse blood and urine for over a
century. Production of biologics and therapeutics from these samples is a niche industry and often
occurs in regions with little regulation or veterinary oversight. To ensure good welfare of horses
maintained for these purposes, guidance has been developed to support the industry.
Keywords: PMSG; equine chorionic gonadotropin; animal welfare; horse
1. Introduction
Horses are frequently used for producing medical substances such as hormones,
antibodies, immune serum, and other substances. Except for pregnant mare urine and
snake antivenom production, there are no international or industry guidelines for much
of the work conducted to obtain medical substances from horses. The use of horses
for producing therapeutics is likely to continue as long as nonanimal alternatives are
unavailable or are significantly more expensive to produce. Thus, the objective of this
paper is to establish recommendations to guide industry and ensure good welfare of horses
used for producing human and animal biologics and therapeutics.
Horses have been deemed particularly useful for producing therapeutics for human
use because of their relatively large blood (or urine) volume, which can be collected
repeatedly and extracted for antibody, hormone, or other protein isolation, and because of
their general ease in handling and maintenance [
1
]. As a result, pharmaceutical products
have been derived from horses for over a century for use in human diseases and other
conditions. The development of equine neutralizing polyclonal antibodies (‘antitoxins’)
to treat diphtheria and tetanus was first described in 1890 by von Behring and Kitasato,
and equine antibodies were first used successfully to treat a child sick with diphtheria in
1891 [
2
]. Prior to the use of equine hyperimmune serum there were no effective treatments
for the condition, caused by an exotoxin produced by the bacterium Corynebacterium
diphtheriae, and it was the leading cause of death in children up to the age of 14 [
2
,
3
].
By 1895, facilities for largescale production of diphtheria antitoxin from horses had been
established by pharmaceutical companies in Germany and were soon to be established
in other countries [
4
]. Tetanus, caused by a toxin produced by the bacterium Clostridium
Animals 2021,11, 1466. https://doi.org/10.3390/ani11051466 https://www.mdpi.com/journal/animals
Animals 2021,11, 1466 2 of 11
tetani, was a similarly devastating and untreatable disease of horses and humans until the
tetanus antitoxin (derived from horses) became widely available [5].
Given the tremendous success of equine-derived therapies against two particularly
devastating conditions, neutralizing antibodies from horse serum were subsequently devel-
oped for passive immunization or therapeutic treatment of humans against a wide variety
of diseases or conditions, including botulism (Clostridium botulinum), gas gangrene (C. novyi,
C. perfringens, and C. septicum), tularemia (Francisella tularensis), Streptococcus pneumoniae,
Hemophilus influenzae, meningitis (Meningococcus spp.), anthrax (Bacillus anthracis), and
endotoxim (lipopolysaccharide) [
4
]. Equine neutralizing antibodies are still used today for
passive immunization of humans following rabies virus exposure in parts of the world [
6
–
8
]
and recently have been recommended for treatment of Ebola virus [
9
], as well as Junin virus
infections, the causative agent of Argentine haemorrhagic fever [
10
]. Equine hyperimmune
serum is used as antivenom to treat poisonous bites or stings from various snakes, spiders,
jellyfish, stonefish, and scorpions [
11
]. Horse anti-thymocyte globulin is also used as a first
line therapy to treat acquired aplastic anemia, a severe immune-mediated hematopoietic
and stem cell precursor disorder that results in pancytopenia [
12
,
13
]. More recently, equine
hyperimmune serum has been advocated for treating SARS-CoV2-infected patients [14].
In addition to these uses for immunotherapy, large volumes of blood are collected
from pregnant horses for production of equine chorionic gonadotropin (eCG, also known
as pregnant mare serum gonadotropin or PMSG) for managing fertility in pigs, cattle, small
ruminants and other animals destined for human meat consumption [
1
], and large volumes
of urine are collected from pregnant mares to extract estrogen for treating menopausal
symptoms in women [
15
]. In both industries, mares may be intentionally bred to ex-
tract hormones that would not otherwise be available in such high concentrations in
nonpregnant animals.
2. Existing Guidance for the Care of Horses Used for Industrial Blood or Urine Production
Except for pregnant mare urine collection and snake antivenom production, there are
no international or industry guidelines for much of the work conducted to produce and
extract therapeutic substances from horses, including guidance for oversight, care, and
well-being of the horses as well as any foals produced as a result of pregnancy [1].
The World Health Organization has produced general guidelines for snake antivenom
production; however, the emphasis of the document is on the safety of substances being pro-
duced for use in human use rather than on animal welfare [
16
]. In the course of producing
polyclonal antibodies, horses receive multiple injections, for example,
50–200 uL/site
over
multiple sites [
16
], and low-grade fevers, abscesses, and local injection site inflammation
may result because of the necessary use of adjuvants, in addition to direct side effects from
venom injections [
17
]. More refined adjuvants might reduce these side effects, but because
these are low volume industries there is no current incentive to implement refinements [
18
].
For other horse serum industries, such as diphtheria antitoxin production, development
of potent, efficacious, and widely used vaccines has led to a decreased need for antitoxin
and a marked decline in global availability of the product. Some diphtheria antitoxin
is still made in Russia, Brazil, and India, but the quality is variable, and it is difficult to
import into the USA and the EU. This resulted in the tragic deaths of two children suffering
from diphtheria in 2017 in the EU [
19
]. The global shortage triggered a search for and
discovery of a nonanimal treatment for diphtheria toxemia that uses recombinant human
antibodies [
20
]. Hopefully, these will gain widespread acceptance as a replacement therapy
for equine diphtheria antibodies.
The use of pregnant mares for urine collection and estrogen production has received
animal activist attention in the past because of concerns about insufficient attention to
horse well-being [
15
]. Subsequently, the industry revised practices and expectations for
farms managing horses, and pregnant mare urine (PMU) production is currently overseen
by the Equine Ranching Advisory Board (ERAB) in Canada. The ERAB, which includes
a veterinary behaviorist and veterinary specialists from the American Association of
Animals 2021,11, 1466 3 of 11
Equine Practitioners (AAEP), has worked together with the Manitoba and Saskatchewan
provincial governments in Canada to develop a recommended industry code of practice
for the care and handling of horses on PMU ranches [
21
]. The code sets forth requirements
for veterinary care of horses and foals as well as general expectations for maintenance of
facilities and management of animals. Adherence to the code and participation in periodic
audits are mandatory for participating horse ranches [
22
]. The industry is reviewed
regularly by the Canadian Veterinary Medical Association, the American Association of
Equine Practitioners [
23
], and the American Veterinary Medical Association [
24
], amongst
other groups. This partnership between industry, government, and relevant veterinary
associations could serve as a model for how oversight could be managed for other industry
sectors that use horse blood or serum for therapeutic products.
In general, Western society, and in particular, veterinary practitioners, remain largely
unaware of the use of horses for extraction of eCG or other substances. These are niche
industries involving relatively small numbers of horses (tens of thousands) compared
to the millions of horses that exist within the multi-billion-dollar global equine industry
(https://www.equinebusinessassociation.com/equine-industry-statistics/, accessed on 18
May 2021). In the distant past, pharmaceutical companies normalized the use of horses
for serum collection by releasing movies or images of hygienic conditions on farms or in
research facilities to the public [
5
,
25
]. Attention from animal activist groups has made
farms and various industries reluctant to discuss their challenges more broadly. Regardless
of the numbers of horses involved, it is essential that guidelines be in place to ensure the
care and well-being of these animals that are so essential to human health and animal
production industries.
3. Animal Welfare and Ethical Considerations
Although there are many definitions for animal welfare, the basic concept of each
relates to how well an animal is doing physically and mentally within its environment. The
most recognized definition is that of the World Organisation for Animal Health: “animal
welfare means the physical and mental state of an animal in relation to the conditions in
which it lives and dies” [26].
Definitions have evolved over time and across cultures. Originally, the concept of
good welfare consisted of good health and the general care that was necessary to maintain
it. Animals were expected to have access to food, water, shelter, and health care. More
recently, a behavioral component was added to those things considered necessary for good
welfare. This highlighted the animal’s need to show species-typical behaviors. Gradually,
the concept of animal sentience has been appreciated, and with it, the recognition that the
animal’s perceptions really determine the difference between good and bad welfare. As a
result, the view of assessing welfare is shifting from the often cited five freedoms to the
currently most popular five domains.
The five freedoms (freedom to express normal behavior and from hunger and thirst;
discomfort; pain, injury or disease; fear and distress) are an input-based listing of what
animals should have for good welfare [
27
]. Inputs cover basic things that affect welfare,
such as genetics, housing, diet, veterinary care, and training of and handling by the
stockperson [
28
–
31
]. Because inputs are relatively easy to measure, they tend to be favored
in formal welfare assessments [
32
]. Alone, however, input-based parameters do not ensure
that the animal is receiving good welfare.
Unlike the input-based criteria, outcome-based parameters quantify specific animal
responses, such as health, production, behavior, and physiological measurements, and
are indicators of how well the management systems (inputs) are working [
29
,
31
,
33
,
34
].
Although outcome-based parameters are often difficult to measure, they are considered
to be better indicators of an animal’s welfare and sentience [
35
–
38
]. They are also more
consistent with the revised five domains model, which emphasizes animal perceptions [
39
].
This model recognizes four physical and functional domains intended to draw attention
to areas relevant to welfare assessments. First, the nutritional domain emphasizes the
Animals 2021,11, 1466 4 of 11
importance of not just food and water but that they be available in adequate amounts and
quality. The environmental domain includes factors like shelter quality, environmental
temperatures and humidity, outdoor access, environmental enrichments, handling prac-
tices, and management practices. The third domain, health, includes all aspects of ensuring
good health, elimination of pain, and genetic inputs. Behavior is the fourth domain. It is
recognized that not all species-typical behaviors are desirable—fighting for example—but
behaviors like grooming and moving around are considered important. The nutrition,
environment, health, and behavior domains contribute to the fifth (mental) domain. In each
of the five domains, applicable negative and positive factors affecting welfare are compared
to ultimately define the animal’s welfare state. The desired goal is to have the positives
greatly outweigh the negatives and to eliminate any significantly negative experience or
condition altogether.
Interpretations of animal welfare often have an ethical basis, which also changes over
time. For some people, animals might be the biological equivalent of a tree (indirect theory).
Because the animal would have no moral standing, it could be treated in any manner [
40
,
41
].
More commonly, people grant some moral consideration to animals but not so much that
they would have full, equal status to humans (direct but unequal theory) [
40
,
41
]. The third
ethical view (moral equality theory) gives animals and humans equal standing and moral
status [40,41].
In consideration of the use of horses for industrial production of natural products and
therapeutics, the first question to ask is whether the horse should be used at all? Are there
non-animal alternatives available or could they be created? If it is necessary to use the
horse, then it is critical they be managed in such a way that outcome-based assessments
ensure the animals are receiving the best welfare treatment.
4. Husbandry and Care Considerations to Improve Horse Well-Being
4.1. Procurement of Horses
Horses bred, purchased, or acquired for the purpose of being used for producing
bio-substances, including blood, should be in good mental and physical health. The overall
fitness for the intended use should be verified by a thorough veterinary examination of
each individual horse. Soundness and good physical health are important because of the
environmental and physical stresses the mares will undergo.
Horses should not exhibit any signs of stress or problem behavior and should be used
to handling and trained in a way that obtaining bio-substances, i.e., regular withdrawal
of blood, does not create unnecessary stress. The previous history of the horse and the
records from the veterinary examination should be known and recorded by the owner.
4.2. Identification of Horses
Horses should be individually identified. Hot iron branding is used in several facilities
to individually identify horses. However, hot branding is very painful [
42
] and other
methods of individual identification systems should be used, whenever possible. Freeze
branding is an alternative to iron branding, as it is less painful [
43
]. However, freeze
branding is more time consuming and requires more equipment than iron branding. In
addition, there are several safety issues to be considered when using freeze branding,
and therefore, adequate training of personnel is required. Radiofrequency identification
(RFID) microchip placement is also a reliable means of identifying individual horses and is
considered humane [44].
4.3. Transportation of Horses
Horses are typically transported long distances when first procured, when moving
from pastures to holding areas for blood collection, and when leaving the operation for
slaughter or rehoming. Prior to considering transportation, horses must be assessed to
ascertain fitness for transport [
45
]. Unfit horses and mares in the last 10% of their pregnancy
must not be transported, except for veterinary care. Stallions and mares with nursing
Animals 2021,11, 1466 5 of 11
foals must be separated from other animals during transportation. During transport,
the horse’s welfare should be a priority, including observing all applicable national and
regional regulations addressing the needs of these animals. Under all circumstances, it is
recommended to keep transport to a minimum duration and ensure sufficient ventilation
and adequate temperature are maintained.
Adequate rest periods should be part of the plan for transport, including unloading
with rest in a stable facility and the provision of food and water at regular intervals (every
4 h as a minimum is recommended). Transport should never exceed 8 h per 24 h [46–51].
4.4. Feeding
Horses should be fed a wholesome diet of a sufficient nutritional quality and quantity
to maintain them in good condition. The physiological demand for production and lactation
requires attention to avoid malnutrition.
Feed should always contain enough grass or roughage to ensure sufficient fiber intake
and chewing time of the horse throughout the day and night. Prolonged time without
access to grass or roughage should be avoided (ideally, no more than 3–4 h). Free access to
clean water should be always ensured [52–55].
4.5. Housing, Social Contact, and Exercise
The accommodation should be constructed for horses to ensure safety and according
to the specific needs of the individual type of horse. At a minimum, horses must be
able to lie down, rest in a natural position, turn around, get up unimpeded, and stand
in a natural position. The accommodation must also have adequate regulation of noise,
light, temperature, and ventilation [
45
]. Horses should be protected against adverse
weather conditions, as well as insects and possible predators. Sufficient shelter of a size
to accommodate all horses on the pasture should be available for all weather conditions.
Fences and the ground should be well maintained [56–61].
When maintained in barns, the space should fit the size of each horse. At all times,
horses should be able to lie down easily and rest in a natural position, turn around and
get up unimpeded. For this reason, tie stalls are discouraged. Passageways and lying
areas should have non-slippery surfaces. Lying areas should be provided with an adequate
amount of good-quality bedding to ensure a dry and comfortable resting area.
Social contact with other horses as well as the horse’s innate need for movement and
foraging should be observed, as much as possible. Horses should be given daily access to
paddocks or pasture for 4–8 h and where possible together with other horses [55,62–67].
Turn out on spacious pasture provides the best incentive for horses to move naturally
in a steady slow pace while grazing. If the hours of grazing are limited, care should
be taken to give the horses daily adequate exercise according to their individual needs.
Horses should not be confined in stalls or paddocks without sufficient time and room
for movement.
4.6. Handling and Training
Horses used to produce blood and urine products are subject to environments and
procedures that can be potentially frightening. For this reason, it is important that each
animal undergoes early and appropriate habituation to people as well as training to
minimize future distress during handling, moving to and from collection sites, application
of apparatus, and collection procedures [
68
,
69
]. Habituation training should begin as soon
as possible after purchased mares arrive at the collection facilities and proceed slowly over
several days at a pace appropriate to the learning rate of the individual mare [
68
,
70
–
72
].
This training begins with quiet interactions that allow each animal time to investigate new
areas and learn to be comfortable with people around and touching them. Desensitization,
a technique of introducing potential triggers very slowly, and chaining of simple-toward-
complex tasks using positive reinforcement are the two most useful types of training for the
various situations these horses will face [
72
,
73
]. It is critical that progress be slow enough to
Animals 2021,11, 1466 6 of 11
not result in an unwanted behavior and that this is done prior to the actual use of the horse
in production. Careful introductions to people, places, and happenings will gradually
build the animal’s confidence and make long-term handling easier and less dangerous.
4.7. Veterinary Attention and Hoof Care
All horses should be observed diligently. Pregnant mares near the end of pregnancy
and until two weeks after foaling, horses currently used for production, ill or injured
horses, or horses in less than normal body condition should be observed as often as the
condition requires and at least once daily. Any horse appearing ill or injured should be
given appropriate care without delay and body condition scores should be monitored [
74
].
If the horse does not respond to such care, or if pain is suspected, veterinary advice should
be obtained.
Facilities for temporary separation of ill or injured horses should always be available.
All medication and treatment of horses should always be done according to standards of
best practice and in a way so that the overall welfare of the horse is never compromised.
Records should be kept of veterinary interventions and treatments as well as mortality or
euthanasia logs.
For routine health care, a semi-annual veterinary examination is recommended. This
should include monitoring dental and endoparasite status as well as overall soundness
and health. Vaccination against tetanus and other enzootic diseases present in the given
locale is always recommended.
Trimming of the hooves at regular intervals by a trained professional is also recom-
mended to maintain a good and healthy condition [75].
4.8. Specific Husbandry Issues Related to eCG Production
Equine chorionic gonadotropin (eCG) is produced from around day 38–40 of gestation,
with peak production between day 55 to 70 of gestation. Production of equine chorionic
gonadotropin continues until about day 110 (range 100 to 140 days) of gestation, when
blood collection is discontinued. At this point, some farms allow mares to carry the foal to
term and then sell the foals, whereas on other farms the pregnancy is terminated so that
the mare can be rebred either through natural mating or artificial insemination for a second
blood collection period that year. Abortion is accomplished by injection of abortifacients,
such as prostaglandins, or by manually forcing open the cervix and rupturing the fetal
membranes [1].
Abortion of mares is not needed to produce eCG, and when it is done, its only purpose
is to increase the amount of eCG produced per mare and year. Abortion is likely to be
distressing and possibly painful for the animals. Thus, based on humane care principles
and on ethical grounds, abortions should only be conducted on the advice of a veterinarian
and then solely for therapeutic reasons.
4.9. Euthanasia
The horse, as a sentient being, deserves to be cared for in a responsible way from
birth to death. Euthanasia of horses should always be performed when a horse is suffering
and is not responding to treatment or when a horse has a chronic or incurable condition
that causes pain or distress. Under no circumstances should a horse be abandoned or left
to suffer.
A method of euthanasia or killing for slaughter is only considered acceptable when
guaranteed loss of consciousness occurs before cardiac or respiratory arrest. This can be
achieved through chemical suppression of the brain or mechanical disruption (e.g., captive
bolt, gunshot). When captive bolt or gunshot are used, bleeding should follow soon after.
Euthanasia or killing for slaughter should be performed by a veterinarian or a trained
professional with appropriate skills [76].
Animals 2021,11, 1466 7 of 11
5. Recommendations for Collection of Blood
5.1. Collection Procedure
General guidelines for blood collection in horses can be found in the World Health
Organization (WHO) guidelines [
16
]. In general, the room or area in which blood will
be collected should be sanitized and all tubes, needles, and collection bags prepared.
Ideally, animals should be weighed before blood collection to increase accuracy of volume
withdrawal estimates. A new, sterile needle should be used for each venipuncture if a
venipuncture attempt fails or if the needle becomes dislodged during blood collection.
No more than two attempts should be made on each side of the neck. The venipuncture
site, generally the external jugular vein in a horse, should be aseptically prepared for
blood collection, including clipping, cleaning, and wiping with disinfectant. To minimize
stress, animals should be habituated to handling and to the collection area, and at least
two compatible horses should be present in the collection area at the same time. A topical
anesthetic cream may be used to desensitize the skin during venipuncture. Horses should
be monitored carefully during the collection period and for the following hour and over
the subsequent 24 h. Blood collection should be terminated in any animal evincing signs of
anxiety, stress, or distress during collection, such as sweating, defecating, etc. The entire
blood collection procedure for a given horse should not last more than approximately 30
min. It is critical that adequate hemostasis be applied following blood collection. Potential
adverse effects of bleeding include hematoma formation from inadequate hemostasis, pain
at the collection site usually due to poor bleeding technique or inexperience, or an infection
at the blood collection site due to poor technique or dirty equipment.
5.2. Considerations for Maximum Volume of Blood That Can Be Obtained
On average, the circulating blood volume of most breeds of adult horses is approxi-
mately 75 mL/kg, with hot-blooded horses (e.g., Thoroughbreds) having up to 100 mL/kg
and draft breeds having less (65 mL/kg) [
77
]. For a 500 kg healthy, normoweight non-
pregnant horse, the total blood volume is approximately 37.5 L. The circulating blood
volume in pregnant, obese, or geriatric animals may be as much as 15% lower than that
of nonpregnant, healthy weight animals of the same breed and should be factored into
the calculation for total volume removed. No specific guidelines have been developed
for blood removal from horses; however, collection of blood is a common research animal
procedure, and safe multi-species research animal blood collection guidelines have been
developed and are widely used [
78
]. The recommended multi-species blood volume limits
for single and repeat sampling are provided in Table 1[
78
]. These suggest that for a single
blood draw, a maximum of 10% of the circulating blood volume can be removed without
the need to provide supplemental replacement fluids. The minimum volume of blood
necessary for production needs should be collected. If >10% blood volume is required, the
collected volume can be replaced by 3–fold volume of isotonic fluids (e.g., saline, dextrose,
lactated ringer’s solution). It is also important to consider accidental blood loss during
sampling, e.g., blood loss outside the designated tube or bag, blood loss from wounds or
injuries, etc.
Table 1.
Recommended blood volume limits and recovery periods (reprinted with permission and
modified from [78]).
Single Sampling * Repeat Sampling
% Circulating Blood Volume
Removed in A
Single Sampling
Approximate
Recovery Period
% Circulating Blood Volume
Removed in 24 h
Approximate
Recovery Period
7.5% 1 week 7.5% 1 week
10% 2 weeks 10–15% 2 weeks
15% * 4 weeks 20% 4 weeks
* For single sampling it is not recommended to remove >15% of the blood volume due to risk of
hypovolemic shock.
Animals 2021,11, 1466 8 of 11
Although one study has demonstrated that larger volumes can be collected from
horses without death (up to 25% of circulating blood volume), animals were reported
to have significant signs of distress during the blood collection, including tachypnea,
tachycardia, neck sweating, urination, and defecation, and heart and respiratory rates
remained elevated for several hours after collections were complete [
79
]. In this study,
although many blood components, such as albumin, returned to pre-bleed levels within a
few days of the bleed, total protein levels and, in particular, globulin levels required up
to 31 days to recover to near pre-bleed levels [
79
]. This suggests that routine collection of
larger blood volumes than indicated in Table 1may impair immunity of horses, which also
could impact their overall health and well-being.
A potentially interesting alternative to bleeding is plasmapheresis, i.e., the separation
of plasma from blood cells so that only plasma is extracted from the animal and all blood
cells are immediately returned into the animal’s body. Although plasmapheresis may be
advantageous in terms of animal welfare, care should be taken not to extract an excessive
amount of plasma and additional fluids should be administered to help replace those lost
during plasmapheresis. Hygiene and extraction practices would have to be improved to
ensure that blood cells remained sterile following initial collection [80].
6. Conclusions
While horses have been used in the manufacturing process of biologics for many years,
their welfare has only recently surfaced as a concern. Until such time as non-animal origins
can be found for certain products, use of the horse is likely to continue. The value of these
horses, as well as their sentience, makes it important that producers ensure the best welfare
using programs such as the one described in this paper.
Author Contributions:
All authors contributed to conceptualization of the paper; X.M.V. developed
the outline; and all authors contributed to writing and editing of the MS. All authors have read and
agreed to the published version of the manuscript.
Funding: Publication of this paper was provided by Charles River.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest:
The authors declare no conflict of interest. Charles River had no role in the
writing of the manuscript.
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