THE EFFECTS OF MYCOTOXINS ON SWINE REPRODUCTION
Panagiotis Tassis Assistant Professor of Swine Medicine and Reproduction Clinic of Farm Animals, School of Veterinary Medicine, Aristotle University of ThessalonÃki Greece
1
The significance of reproductive performance for the outcome of swine production is undoubtable. Both sides, gilts/ sows and boars are the basis of proper and financially rational production in intensive swine farms globally. Genetic lines of hyperprolific sows, as well as high durability and increased performance boars are needed for increasing production demands and high-quality pork meat.
A variety of major issues, such as
Controlling the major viral or
reproductive management, selection of
bacterial swine pathogens affecting
gilts and boars, introduction of gilts to
the genital tract and reproductive
farm’s reproductive program,
performance, as well as proper
environmental conditions affecting
nutrition in terms of ingredient/
reproductive performance (e.g. heat
nutrient requirements and a proper
stress, etc.), along with maintenance of
feeding schedule during various
high health status and fulfillment of
stages of gestation and boar’s
specific nutritional requirements, should
life, are significant parts of proper
be taken care during the rearing of
reproduction management.
hyperprolific sows and boars.
As already suggested by various research efforts in the past 50 years, mycotoxins can also pose a significant threat to the health and reproductive e!ciency of swine.
2
Mycotoxins are secondary
The most important mycotoxin
significant for their devastating effects
metabolites of certain fungi
producing fungi belong to the genera
on pig production worldwide.
species that can be found in
Aspergillus, Penicillium, Fusarium, Alternaria and Claviceps3.
Other mycotoxins such as T-2
grains worldwide. They are produced before (fungi as plant
toxin, nivalenol or ergot alkaloids
pathogens) or after the harvest
Out of more than 500 mycotoxins, some
have been suggested as somewhat
of grains, or even during storage
are considered extremely significant
significant for swine, especially in
(fungi growing saprophytically).
for pig health and productivity.
particular geographical regions1,4.
In particular regions, the mycotoxin
Namely, aflatoxins (AF) B1, B2, G1 and
menace seems to be higher
G2, deoxynivalenol (DON), zearalenone
resulting in severe outbreaks
(ZEN), fumonisins (FB1, FB2, FB3) and
of intoxication (mycotoxicosis)
ochratoxin A (OTA) are considered
threatening humans and animals1. The significant impact of mycotoxins includes loss of human and animal life, increased health care and veterinary care costs, and reduced livestock production . 2
ZEN
Even though several mycotoxins have been suggested as capable of inducing or contributing to reproductive disorders (DON, ZEN, FB, T-2) in various species5, it is obvious that the most “interesting� in terms of reproductive performance is ZEN and its metabolic derivatives.
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In this article, we will try to synopsize effects of the most important mycotoxins ingested separately, on the two parts of reproduction, i.e. the female part (gilts, sows and the offspring) and the male part which includes the boars. Predominantly direct toxic effects of AF, OTA, DON, T-2, FB and ZEN are described, since presentation of indirect effects (e.g. increased susceptibility to infections due to impaired immunity that could affect reproduction, or effects of reduced protein synthesis or feed refusal on gestation and litter weight) is practically more or less extremely extensive. In vivo and
in vitro effects along with aspects related to the hormonal alteration of reproduction according to relative literature are also described.
Reproductive effects of Aflatoxins in swine Aflatoxins (AFs) are produced
The most common AFs are
mainly by Aspergillus flavus, A.
AFB1, AFB2, AFG1 and AFG2.
parasiticus and A. nomius and are detected usually in maize, peanuts and cottonseed. AFB1 is considered as an active hepatocarcinogen and is the most significant in terms of toxicity in swine6. The liver is considered the primary target organ of AFB1.
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AFs, apart from being hepatotoxic, they have mutagenic, and possibly teratogenic effects in animals. They are categorized as Class 1 human carcinogens by the International Agency for Research on Cancer (IARC). AFs decrease the absorption of nutrients and reduce weight gain in pigs, while chronic exposure to low doses results in jaundice (pale-yellowish appearance of the liver) with hemorrhaging sites in the liver and variable levels of fibrosis and cirrhosis, diffused centrilobular necrosis and fat degeneration7. Few reports have connected AFs with
Sows are capable of normal gestation and reproduction when fed AF at levels between 500 and 700 ppb, whereas their piglets show growth retardation due to AF excretion in milk8,9.
reproduction in swine. Abortion is not
Reduced piglet birth weight has been reported after
expected at AF toxicosis cases7.
ingestion of 800 ppb AFB1 in feed of sows during the second half of gestation and the suckling period10.
In vitro detrimental effects on oocyte maturation through epigenetic modifications (increasing DNA methylation levels), induction of oxidative stress, excessive autophagy and apoptosis, have been presented after oocyte exposure to 50 ÎźM AFB111.
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It has been further suggested that AFB1 can impair porcine early embryonic development (blastocyst formation was impaired with treatment of 1 nM AFB1) through oxidative stress (excessive reactive oxygen species), induce DNA damage, disrupt DNA damage repair process, while also induces apoptosis and consequently autophagy12. Possible adverse effects on boar reproductive efficiency have been suggested13, where lower sperm concentration, lower survival of spermatozoa, and a larger proportion of abnormal spermatozoa were found simultaneously with great levels of AFB1 in seminal plasma.
Reproductive effects of Ochratoxin A in swine Ochratoxin A (OTA) is produced by several
Aspergillus and Penicillium species, such as Penicillium verrucosum A. ochraceus, A. westerdijkiae, A. steynii, A. carbonarius and A. niger14. Its primary target organs are the kidneys (nephrotoxicity), however it can induce several toxic effects such as teratogenic, embryotoxic, genotoxic, neurotoxic, immunosuppressive and carcinogenic14,15. OTA has been classified by the IARC as a possible human carcinogen (group 2B). In a previous study with boars (250 kg weight), which were given 0.08 Îźg/kg OTA orally for 6 weeks, reduction in sperm viability, initial forward motility, and motility were observed after 24 hours storage16.
Additionally, OTA is able to negatively
Moreover, it was suggested that OTA (fed to boars at
affect porcine oocyte maturation
high concentrations) might have the potential to affect
in vitro (reduced rate of porcine oocyte polar body extrusion) at levels of exposure exceeding 5 ÎźM18.
sperm production and boar semen quality by inducing a reduction of initial motility and longevity of spermatozoa17.
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Reproductive effects of Fumonisin B in swine Fumonisin Bs (FBs) are mycotoxins produced mainly by Fusarium verticillioides and
F. proliferatum, commonly in maize. The predominant FB 1corresponds to 70% of FBs and is classified as a potential human carcinogen (Class 2B) by IARC19. Its main toxic mechanism is based on the disruption of sphingolipid biosynthesis, with inhibition of ceramide synthase that results in the accumulation of sphinganine and sphingosine. Acute intoxication of swine with high levels of FB (>100 ppm) is characterized by pulmonary edema7. Nevertheless, FBs have been well implicated in the impairment of immune response [e.g. modification of Th1/ Th2 (T-helper 1/T-helper 2) cytokine balance], as well as in significant effects in the gastrointestinal tract.
FB1 has been associated with the induction of intestinal barrier integrity alterations and its function/permeability [e.g. decreased transepithelial electrical resistance (TEER), reduced expression of occludin and E-cadherin in ileum], as well as with the modulation of digestive and absorptive processes (e.g. reduced aminopeptidase activity in jejunum) and reduction of intestinal defense during pathogen exposure (increased colonization and shedding of Escherichia coli)7,20.
In regard to reproduction, FBs have been also linked with delay in sexual maturity and reproductive functionality alterations21. Specifically, they have been suggested as responsible for the reduction of testicular and epididymal sperm reserves and daily sperm production in boars22,23. Additionally, detrimental effects on semen quality and motility after 6 months of exposure to FBs have been suggested23.
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Furthermore in vitro, FB1 produced inhibitory effects on granulosa cell proliferation24. FB1 was found to influence the steroidogenic capacity of porcine granulosa cells (stimulation of progesterone production but no effect on estradiol production), as well as to inhibit their proliferation, thus it could compromise the normal follicle growth and oocyte survival in swine5,24,25.
In vivo, FB induced abortions 1–4 days after acute spontaneous toxicosis which was probably consequence of fetal anoxia due to severe pulmonary edema in the dam26, 27. Concentrations of 100 ppm FB1 fed to sows in the last 30 days of gestation did not induce pulmonary edema, abortions, or fetal abnormalities7.
Reproductive effects of Trichothecenes in swine Effects of Deoxynivalenol on swine reproduction
All trichothecenes are known to
DON toxicosis has been
affect reproductive performance
associated with gastrointestinal
in pigs. Deoxynivalenol (DON)
signs such as abdominal
belongs to the trichothecene
discomfort, diarrhea,
family of mycotoxins and it has
vomiting, anorexia and
been proved able to significantly
reduced weight gain.
inhibit protein synthesis.
DON heavily affects
Particular acetylated and
the intestinal barrier
modified forms of the parent
integrity and function,
toxin, such as 3-acetyl-DON
while can also modulate
(3-Ac-DON), 15-acetyl-DON
immune response7,20.
(15-Ac-DON) and DON-3glucoside [DON3G, main plant metabolite of DON], occur simultaneously in grains25.
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Evidence of oocyte maturation and
DON has been associated
increased proliferation, but greater
embryo development impairment
with particular reproductive
concentration (3.4mM) have
along with the reduction of feed
effects, mainly through in vitro
the opposite effect], inhibition
intake are the main reasons behind
studies on porcine oocytes.
of progesterone and estradiol
the DON-induced detrimental reproductive effects in pigs.
In vivo, ingestion of DON contaminated feed by pregnant gilts, may result in reduced weight and body length of piglets28. A significant passage of DON through the placenta from exposed sows to fetuses that could potentially affect fetal function has been demonstrated29,30. Furthermore, several in vivo negative effects on fertility have been associated with consumption of combined DON and ZEN contaminated feed . 31
In vitro effects of DON include disturbance of porcine oocytes maturation through the induction of abnormalities of the meiotic spindles and by altering oocyte cytoplasmic maturation32,33,34. In addition to the DON-induced impairment of oocyte maturation, findings of autophagy/ apoptosis and epigenetic modifications in porcine oocytes have been presented35.
production [induced by FSH plus Insulin-like growth factor I (IGF-I)] and CYP19A1 and CYP11A1 mRNA abundance5,36. Exposure of ovarian explants to 10 ÎźM DON affected the process of follicular maturation with a decrease of the reserve pool of follicles, resulting in a significant decrease in the number of normal follicles, as well as an increase of pyknotic oocytes number in all stages of
Moreover, DON has been
follicular development37, whereas
associated with dose-dependent
treatment with 1 ÎźM DON
effects on porcine granulosa
decreased the rate of polar body
cell proliferation [biphasic
extrusion in porcine oocytes18.
effect: lower concentration (0.034 mM) of DON results in
Taken together, it seems that DON can have a direct ovarian effect that could impact reproductive performance in swine.
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Effects of T-2 toxin on swine reproduction Besides DON, which is the main representative of the trichothecenes group, T-2 toxin and its deacetylated form HT-2 toxin (type A trichotecenes) can be considered as quite significant for swine, according to recent studies. They are produced in crops (e.g. wheat, maize, barley) by various Fusarium species such as Fusarium sporotrichioides, F. poae and F. langsethiae, either in the field or during storage. Pigs are very susceptible animals towards their effects. HT-2 toxin is a natural contaminant in cereals but is also the main metabolite of T-2 toxin, thus T-2 toxin effects can be partially attributed to HT-2 toxin. T-2 toxin inhibits protein, RNA and DNA synthesis, inducing apoptosis and necrosis in particular cell types and has a detrimental effect on cell membrane integrity due to increased lipid peroxidation25.
In acute T-2 toxicosis cases, serous-haemorrhagic necroticulcerative inflammation of the digestive tract, vomiting, diarrhea, leukopenia (leukocyte apoptosis), hemorrhage, shock and death, oral/dermal irritation and immunosuppression can be observed. However, in chronic cases of mildly contaminated grains ingestion, growth retardation, weight gain suppression and feed refusal along with greater pro-inflammatory gene expression, are observed in pigs38.
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Quite similarly to DON, the effects of type A trichothecenes on reproduction are mainly
It has been also suggested that the treatment of porcine oocytes with 50 nM HT-2 toxin and
demonstrated through in vitro studies,
greater concentrations significantly decreased the
since in vivo clinical reproductive disorders
rate of polar body extrusion18.
in pigs, that could be directly attributed to their effects have not been presented. According to a study39, T-2 toxin may be able to alter the growth of the granulosa cell layer as well as affecting steroidogenesis. T-2 toxin had potent inhibitory effects on IGF-I and FSH-induced steroid production in cultured porcine granulosa cells, since dosages of 1, 3, 30
Failure of oocyte maturation after HT-2 toxin treatment has also been suggested, since the toxin inhibited porcine oocyte polar body extrusion and cumulus cell expansion, while also disrupted meiotic spindle morphology and disturbed actin distribution40. Oxidative stress, apoptosis and autophagy in the treated oocytes were also among the findings of the previously mentioned study.
and 300 ng/mL inhibited estradiol production, but progesterone production was inhibited with a dose of 30 and 300 ng/mL. An inhibitory effect on cell number was observed at 3 ng T-2 toxin/mL.
Taken together, these studies confirm the potential of T-2 toxin and its metabolites to impair reproductive function in pigs.
Reproductive effects of Zearalenone in swine Zearalenone (ZEN) is a phenolic resorcylic acid lactone mycotoxin produced by several Fusarium species, especially F. graminearum and may undergo modification in plants, fungi and animals (prehepatic, hepatic and extrahepatic) by phase I and phase II metabolism. Major metabolites of ZEN include α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol, zearalanone (phase I), whereas conjugated forms with glucose, sulfate and glucuronic acid are the outcome of phase II25,41.
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Pigs are very sensitive to ZEN, since the parent toxin is metabolized mainly to Îą-zearalenol in that species, which shows greater estrogenic potency than ZEN. ZEN toxicosis has also been associated with increased oxidative stress, reduction of nutrient digestibility and growth retardation. Toxic effects of ZEN on other tissues and systems outside the reproductive tract, such as liver and immune system have already been demonstrated42,43,44.
ZEN su!ciently resembles 17β-oestradiol that allows it to bind to estrogen receptors in various organs and induce estrogenic effects. Its effects depend on the dose, as well as on the time of administration in relation to estrous cycle5. It is considered the most important mycotoxin affecting swine reproduction and prepubertal gilts seem to be a very sensitive age group to the effects of the toxin. For more than 40 years, researchers have demonstrated the significant reproductive disorders that can be caused after ZEN ingestion in gilts and sows in
vivo (e.g. pseudopregnancy, diminished fertility, hyperestrogenism syndrome, reduced litter size). Placental passage of ZEN from sows to fetuses Moreover, in vitro studies have presented its
has been confirmed, as ZEN and its metabolites
negative effects on oocyte maturation and
have been detected in the bile of newborn piglets
porcine granulosa cells proliferation7,41.
from sows ingesting contaminated feed45.
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As regard to the effects of ZEN on
Multiple impairment of semen quality and
Additionally, ZEN and
boars’ reproductive function and
kinetics, including decrease of sperm
Îą-zearalenol can reduce the
quality of semen, results of in vivo
viability and progressive motility46-48 can
ability of boar spermatozoa
studies have suggested reduced
be the outcome of boar semen in vitro
to bind to the zona
serum testosterone levels, testis
exposure to ZEN.
pellucida46 and affect sperm
weights and spermatogenesis, as
chromatin integrity48,49.
well as feminization and suppressed libido in young boars5. Further
in vitro studies on boar semen have suggested ZEN toxicity.
Hyperestrogenic effects in newborn piglets in Greek swine farms.
Due to the significance and extent of ZEN-toxicosis outcome on farm reproductive health and performance, a summary of the major reproductive effects of ZEN and its basic metabolites in swine is presented in Table 1.
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Table 1. Summary of major ZEN, α- and β-zearalenol effects in gilts, sows and boars according to in vivo and in vitro studies5,7,25,40,45-50,52-62. IN VIVO EFFECTS Mycotoxin ZEN
Gilts
Sows
1–5 ppm: Edema and hyperemia of the uterus/ vulva. Could result in vaginal and rectal prolapse. Neonatal gilts: swelling of the vulva and mammary glands and edematous infiltration of the perineal region, ventral abdomen, and umbilicus. Edematic regions could also have exudative crusted inflammation. Possibly necrosis of the teats.
Boars
Hyperestrogenism syndrome: edema of the vulva and mammary gland, vulvo-vaginitis, enlargement of the uterus, retained corpora lutea, ovarian cysts, nymphomania or anoestrus, ovarian atrophy. Increase of weaning-to-estrus interval. 3–10 ppm can induce anestrus. Linear relation of anestrus length and ZEN concentration in feed.
Particular studies suggest presence of detrimental effects on puberty attainment, while others demonstrated absence.
Infertility, pseudopregnancy, reduced litter size. lower conception rate, increased numbers of repeat breeders, increased numbers of stillbirths.
Pseudopregnancy and prolonged estrous cycle in cyclic gilts prior to mating.
Birth of piglets with hyperestrogenism syndrome and splay-leg.
Preputial enlargement, possibly general loss of vigor and reproductive compromise. Young boars: reduced libido and decreased testicular size. At 9 ppm lower total and gel free volumes of semen with lower total motile sperm. Reduced serum testosterone levels, testis weights and spermatogenesis, as well as feminization in young boars. Mature boars: unaffected by concentrations < 200 ppm ZEN.
IN VITRO EFFECTS Mycotoxin ZEN
Oocytes/uterus/ovary (gilts and sows) Impairment of oocyte maturation. Reduction of polar body extrusion rates. Oocyte dies in the Graafian follicles. Signs of oestrus could be present but there is no ovulation. Suppressed pig oocyte progression through meiosis by inducing the malformation of meiotic spindles aneuploid embryos Interference with the initial chromatin status and maturation competence of oocytes. Reduction of healthy follicles quantity could result in premature oocyte depletion in adulthood.
Granulosa cells and steroid production (gilts and sows) Porcine granulosa cells: High concentrations (30–120 mM): apoptosis and impairment of porcine granulosa cells proliferation. Induction of disorders of the mitochondrial transmembrane potential and increase of the reactive oxygen species levels. Apoptosis or necrosis through the caspase-3/caspase-9 dependent mitochondrial pathway may induce atresia in porcine follicles. Increased expression of genes related to DNA damage and repair.
Hormonal balance (gilts and sows) Inhibition of FSH release and secretion (similarly to 17-β estradiol) depresses maturation of ovarian follicles during the preovulatory stage. May exhibit a luteotrophic property Prolongs corpus luteum life span to equal or longer than normal gestation period. Contradictory results on effects to LH. Absence of effect, or in prepubertal gilts at 3.2 ppm ZEN, decreased LH levels in serum.
Boars Decrease sperm viability and progressive motility. Significant detrimental effects on major kinetics parameters of boar semen (e.g. static, rapid motile spermatozoa, etc.). Reduces the ability of boar spermatozoa to bind to the zona pellucida. Affects sperm chromatin integrity.
Prepubertal gilts: > 2 ppm ZEN Increase of prolactin levels in serum.
Accelerated development of the ovaries (follicles) in post-weaning piglets promotes the autocrine action or expression of the ghrelin gene in piglet ovary. May affect oviduct tubal function (alterations in gene expression of tubal epithelial cells). Ovarian atrophy and changes in the endometrium (proliferation of uterine glands). Proliferation (hyperplasia) of the epithelial cell layer of the uterine and vaginal mucosa thickening and irregularity of the epithelium. Squamous metaplasia in uterus and hyperplasia of the endometrial glands. Increased genital organs size in gilts, along with hyperplasia of submucosal smooth muscles in the corpus uteri. Increased weight of the uteri and thickness of the myometrium and endometrium (greater growth hormone receptor expression in the uteri).
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Table 1. Summary of major ZEN, α- and β-zearalenol effects in gilts, sows and boars according to in vivo and in vitro studies5,7,25,40,45-50,52-62.
IN VITRO EFFECTS Mycotoxin
Oocytes/uterus/ovary (gilts and sows)
Granulosa cells and steroid production (gilts and sows)
α-zearalenol
Reduced rate of oocyte maturation (7.5 μΜ).
Primarily increased progesterone (P4) production induced by FSH and insulin-like growth factor-I (IGF1). Biphasic dose–response with 0.094 mM increasing and 9.4 mM inhibiting FSH plus IGF-I-induced estradiol production or absence of effects at high ZEN concentration.
Hormonal balance (gilts and sows) No effects on LH.
Boars Reduced viability and motility of boar semen. Reduces the ability of boar spermatozoa to bind to the zona pellucida. Modified DNA integrity and structural stability at pM levels.
Increased progesterone and decreased estradiol production by porcine follicles = indicator of follicular atresia. Decreased abundance of CYP19A1 and CYP11A1 mRNA induced by FSH plus IGF-I. β-zearalenol
Reduced rate of oocyte maturation (30μΜ).
Increases the curvilinear velocity (VCL parameter). Modified DNA integrity and structural stability at nM levels
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CONCLUSIONS
Diagnosing mycotoxin-induced
disorders, must be considered when
as the inclusion of proper
reproductive disorders in sows or
establishing differential diagnosis of
agents that would reduce
boars is definitely not an easy
reproductive inefficiency cases.
the level of mycotoxins available for absorption in
task. As presented in this review,
Feed analysis is of colossal
the extent of effects on the
importance along with evidence
reproductive system of swine is
of mycotoxins/metabolites
vast and includes a great variety
circulating in blood or detected
Proper management of sows
of direct and indirect mechanisms
in tissues and excreta.
(e.g. proper estrus detection,
Furthermore, the interactions of the abovementioned toxins in vivo and their final observed effects on sow and boar’s reproductive efficiency have not been fully elucidated yet and need further clarification.
of utmost importance51.
heat stress countermeasures)
of toxicity at the cellular and genomic levels.
the gastrointestinal tract are
Unfortunately, antidotes against
and regular semen viability
mycotoxins do not exist, thus, control
and kinetics analysis,
of such cases would need removal of
along with prevention of
contaminated feed or mixing with
infectious agents (e.g.
clear feed (usually at 1:10 rate), as
proper vaccination schedule
well as inclusion of agents that could
of the breeding stock), would
either adsorb or biotransform the
significantly assist on rapid
mycotoxins to non-toxic metabolites.
detection of abnormalities
Evidence so far suggests that
Moreover, clinical support should
ZEN and α-zearalenol are the
be given to splay-leg newborns
most important mycotoxins for
with signs of hyperestrogenism
swine reproduction and from a
that cannot receive the appropriate
clinical viewpoint they are probably
amount of colostrum/milk.
that could be associated with mycotoxicosis, thus proper treatment and prevention efforts would start timely.
the first to investigate in cases of reduced fertility on farm. However, such cases usually include concomitant DON ingestion via feed due to the “characteristically observed” combined mycotoxins contamination of pig feed.
In the majority of cases, removal of mycotoxins would lead to the improvement of fertility and reproductive parameters in due time through defense and repair mechanisms at cellular level50. However, time would be needed in order to achieve previous
From the diagnostic
reproductive rates again on farm.
standpoint, onset of reproductive signs right
The principle is that prevention of
after feed alterations
mycotoxicosis should be the basic tool.
on farm, as well as the absence of any significant impact of infectious agents or environmental or managerial factors that could induce reproductive
Regular feed screening (enrichment materials and other fiber sources can contain significant amounts of mycotoxins, thus they should be also included in the analysis), as well
Nevertheless, further intensive research efforts are needed on the field of reproductive failure due to mycotoxins ingestion, especially in terms of explaining underlying mechanisms associated with observed detrimental effects.
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