The effects of mycotoxins on swine reproduction

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 Thessaloníki Greece

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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.

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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 .

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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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