Effects of dietary kelp (Ascophylum nodosum) supplementation on survival rate and reproductive performance of mink challenged with Aleutian mink disease virus

Abstract: Infection with Aleutian mink disease virus (AMDV) has negative effects on reproductive performance and survival rate of American mink (Neovison vison). The objectives of this study were to assess the effects of kelp (Ascophylum nodosum) supplementation on survival, growth rate, and reproductive performance of mink challenged with AMDV. AMDV-free female black mink (n = 75) were intranasally inoculated with a local AMDV strain. Mink were fed a commercial pellet supplemented with 1.5% or 0.75% kelp or were kept as controls (received no kelp) for 451 d. Body weight and rectal temperature were recorded on days 0, 31, 56, 99, 155, 366, and 451 post inoculation (PI). Annual mortality rates were 13.6%, 20.0%, and 31.8% for mink fed 1.5%, 0.75%, or 0.0% kelp, respectively (P = 0.29). Mink which were fed 1.5% kelp had a significantly (P < 0.01) greater daily weight loss during breeding and post-breeding periods (days 155–366 PI), and outperformed (P < 0.01) the other groups in regard to litter sizes at birth and weaning. Differences among treatments were not significant for the number of females mated, or whelped of those exposed to males, kit survival from birth to weaning, or rectal temperature. It was concluded that 1.5% kelp supplementation had beneficial effects on survival rate of adult mink and litter size.


Introduction
Ascophyllum nodosum, a brown seaweed that grows in abundance along the shores of North Atlantic (Allen et al. 2001), has been fed to livestock for many decades (Evans and Critchley 2013). Reports on chemical composition and nutritional properties of various seaweed species have been extensively reviewed in recent years (O'Sullivan et al. 2010;Pangestuti and Kim 2011;Wijesekara et al. 2011;Evans and Critchley 2013;Ngo and Kim 2013;Collins et al. 2016;Pérez et al. 2016), concluding that seaweeds contain an array of natural bioactive compounds and trace minerals with antioxidant, antibacterial, antiviral, and anti-inflammatory properties. Although there are inconsistencies in the results of various experiments, the consensus of those reviews is that dietary use of seaweed and their extracts have positive effects on health status of humans and animals. Phaeophyta (brown seaweeds) are particularly rich sources of polysaccharides (alginate, fucoidan, and laminarin) and polyphenols (phenolic acids, flavonoids, stilbenes, and lignans) (O'Sullivan et al. 2010;Kim et al. 2014;Collins et al. 2016). Among Phaeophyta, A. nodosum, including Tasco®, the proprietary product marketed by Acadian Seaplant (http://www.acadianseaplants. com/), has been studied most extensively (reviewed by Evans and Critchley 2013;Luthuli et al. 2019).
In vivo and in vitro studies have shown that dietary use of seaweed extracts, particularly polysaccharides, increase the abundance of beneficial bacteria and reduce levels of pathogenic bacteria in the gut, modulate the immune system, and increase beneficial volatile fatty acids in livestock (O'Sullivan et al. 2010;Sweeney and O'Doherty 2016). Similarly, feeding whole seaweed caused favorable changes in the abundance of beneficial and pathogenic bacteria in the digestive tract of pigs (Dierick et al. 2009), layer hens (Kulshreshtha et al. 2014), and rats (Liu et al. 2015); increased the concentration of short-chain fatty acids in hens (Kulshreshtha et al. 2014) and rats (Liu et al. 2015); increased villi height and crypt depth of the small intestine in chickens (Kulshreshtha et al. 2014); and increased the depths of the colonic crypt, mucosa, externa muscularis, and colonic total wall in rats (Liu et al. 2015). On the contrary, Michiels et al. (2012) found no positive effect of A. nodosum supplementation on gut microbial profile, plasma oxidative status, or gut tissue morphology in piglets fed a balanced diet.
The effects of A. nodosum supplementation on mink infected with Aleutian mink disease virus (AMDV) has not been studied but could be of great interest to mink producers in countries where AMDV positive mink are not culled. AMDV causes Aleutian disease (AD), resulting in serious health problems for mink in most minkproducing countries. The AD is an immune-complexmediated syndrome characterized by persistent antiviral antibody production, hypergammaglobulinemia, plasmacytosis, and progressive renal disease (reviewed by Bloom et al. 1994) and negatively affects reproductive success and kit survival (Alexandersen 1986;Hansen and Lund 1988;Broll and Alexandersen 1996;Reichert and Kostro 2014;Andersson et al. 2017). There is no cure nor effective vaccine against AD (Aasted et al. 1998;Castelruiz et al. 2005;Liu et al. 2018) and the virus cannot be easily destroyed by heating or composting (Hussain et al. 2014). Testing of mink for antibodies against AMDV and removal of seropositive animals has not been effective in permanent virus eradication (Themudo et al. 2011;Farid et al. 2012).
Macrophages and cytokines, especially interleukin-6 (IL-6), have been implicated in the pathogenesis of AD (Bloom et al. 1994). Because pigments and sulfated polysaccharides in seaweeds have antiinflammatory activities by modulating macrophage functions and by production of anti-inflammatory cytokines, such as IL-6 (reviewed by Pangestuti and Kim 2011;Wijesekara et al. 2011;Sweeney and O'Doherty 2016), it seems logical to assume that seaweeds could alleviate the harmful effects of AMDV infection. In addition, animals of various species which were under stressful conditions, such as those challenged with bacteria or viruses, exposed to heat or experienced long-hall transportation showed stronger positive responses to seaweed supplementation than those which were not stressed and fed balanced diets, possibly because stress causes immune suppression, resulting in higher susceptibility to infection by pathogens (reviewed by Allen et al. 2001 andCritchley 2013), making AMDV infection an attractive case for testing the effects of seaweed supplementation to mink. The objectives of this study were to assess the effects of A. nodosum supplementation on survival, weight gain, and reproductive performance of mink inoculated with AMDV.

Statement of animal care
Mink were managed according to the industry standards (NFACC 2013) and sampling protocols were performed according to the standards of the Canadian Council for Animal Care (http://www.ccac.ca) after approval by the Institutional Animal Care and Use Committee. Prior to inoculation and weighing, animals were anesthetized by intramuscular injection of ketamine hydrochloride (Ketalean, Bimeda-MTC Animal Health Inc., Cambridge, ON, Canada) and xylazine hydrochloride (Rompun 2%, Bayer Animal Health, Mississauga, ON, Canada) at the dose of 10 and 2 mg kg −1 live weight, respectively.

Source of seaweed
Ascophylum nodosum (kelp) was hand harvested by Tidal Organics (http://tidalorganics.com/) from small skiffs using a cutter rake. It was harvested from June through October in south-western Nova Scotia's coastal region from Lunenburg to Pubnico, mechanically dried, and particle sizes reduced with a hammer mill to 14 mesh.

Animal management and experimental design
A total of 75 five-month-old female black American mink (Neovison vison) were purchased in September 2013 from an AMDV-free farm in Nova Scotia, Canada. Animals were transferred to the Aleutian Disease Research Center (ADRC), which is a building designed to minimize the chance of viral introduction or escape. Animals were individually kept in 61.0 cm × 30.5 cm × 20.3 cm wire-meshed cages with a wooden nest box containing aspen shavings. Cages were separated by a solid plastic sheet to prevent direct physical contact. Animals had free access to water via nipples connected to waterlines which were heated during the winter. Three days after arrival at ADRC, animals were anesthetized and intranasally inoculated with 60 μL of a 10% (w/v) passage 2 of a local strain of AMDV prepared from the spleens of mink harvested 10 d post inoculation (PI) and stored at −80°C, as previously described (Farid et al. 2015). All mink were infected by AMDV on day 56 PI (unpublished data).
The diet gradually changed from the wet feed used on the farm of origin to a commercial dry pellet (National Feeds Inc., Maria Stern, OH, USA) with kelp supplementation of 1.5% (T1.5), 0.75% (T0.75), or 0% (T0, control) of the feed. The pellet's nutritional composition changed based on the production cycles of the mink. To enhance the attachment of the kelp to the dry pellets, a combination of 96.0% pellet, 2.5% flour, and 1.5% kelp was thoroughly mixed to make the T1.5 diet. The composition of T0.75 diet was 96.75% pellet, 2.5% wheat flour, and 0.75% kelp, and no flour was added to the control diet. Animals had free access to the feed which was added to feeders daily. Some kelp particles settled out in feeders, which could not be measured. One row of cages was divided into two blocks of three sections of 12 or 13 cages each, and the three treatments were assigned at random within each block. On PI days 0 (before inoculation, 9 Sep. 2013), 31 (10 Oct.), 56 (5 Nov.), 99 (17 Dec.), 155 (11 Feb. 2014), 366 (9 Sep.), and 451 (4 Dec.), animals were sedated, and their body weights were recorded. Rectal temperature was measured by a digital thermometer on days 31, 56, 99, and 155 PI.

Breeding
Females which survived until breeding season (n = 62) were exposed to three different AMDV-free males up to six times between 2 Mar. and 20 Mar. 2014. Females whelped between 26 Apr. and 9 May, and the number of dead and live kits was recorded at birth and on day 7 postpartum. Kits were weaned on 28 June at 50-64 d of age, were fed the control diet (0% kelp) after weaning, and their body weights were recorded on 30 Oct. 2014, at an average age of 180 d.

Statistical analyses
Data were analyzed with SAS version 9.4 for Windows (SAS Institute Inc., Cary, NC, USA). Prior to analyses, data were checked for normality by the Shapiro-Wilk test implemented in the UNIVARIATE procedure. Visual inspection of the pattern of changes of body weights over time showed minor fluctuations during the early period after inoculation followed by sharp decreases in all treatments ( Fig. 1), suggesting that segmented regression would more accurately describe the pattern of changes of body weight over time. To determine the breakpoint, the nonparametric LOESS procedure of SAS was used, which fits a local regression function to the data and determines a graphical diagnostic of trends in the data. The ODS GRAPHICS statement of PROC LOESS was used to overlay the fit plot on the scatterplot of the data. Visual inspection of the fit plots and residual plots suggested that day 155 PI was the breaking point. Body weight from day 0 to 155 and 155 to 451 PI, and rectal temperature were analyzed using linear mixed models (PROC MIXED) which included the fixed effects of treatment, linear, and quadratic regression of sampling dates and their interactions with the treatment. The interactions between treatment and sampling dates were not significant and were deleted from the final models. The REPEATED statement was used to take the repeated observations on animals over time into account. The appropriate correlation structures were determined after fitting five models with different correlation structures. The models with the smallest Bayesian information criterion value were the autoregressive for body weights and heterogeneous autoregressive for rectal temperature, which were used in the final analyses. Restricted maximum likelihood estimation and type 3 tests of fixed effects were used with the Kenward-Roger correction. Post hoc comparisons among least-squares means were performed with the Tukey-Kramer adjustment. The effects of treatment on average daily gains from day 0 to 155 PI, 155 to 366, and 366 to 451, and 0 to 451 PI were separately analyzed by the generalized linear model procedure. The effects of maternal supplementation with kelp, sex of kits, and their interactions on body weight of the progenies at 180 d of age were tested by PROC GLM. Kaplan-Meier estimates of survival functions and comparison of survival curves among treatments were performed using the nonparametric LIFETEST procedure. Paired comparisons were carried out using the Šidák multiple-comparison adjustment. Number of kits born and weaned deviated from normality and treatment effects were tested by the nonparametric Kruskal-Wallis test. In cases where this test was significant at α < 0.05, pairwise comparison of treatment means was performed by the Mann-Whitney U test with Bonferroni correction. The likelihood ratio χ 2 test was used to compare treatments for the number of females mated and whelped.

Survival rate
Mortalities during the first 21 d PI were 3, 1, and 3 in T1.5, T0.75, and T0, respectively, which could not have been the result of AMDV infection and were excluded from the analysis of mortality data. To confirm that AD was not the cause of death, these animals were necropsied and none showed signs of AD in the liver, spleen, or kidneys. Four of the dead mink had suffered from fatty liver. Mortalities from 1 Oct. 2013 to 1 Oct. 2014 were 13.6%, 20.0%, and 31.8% in mink fed T1.5, T0.75, and T0 diets, respectively (Table 1), but the differences among treatments for cumulative mortality at the termination of the experiment were not significant (χ 2 (2 df) = 2.47, P = 0.29). There was no mortality from 1 Oct. 2014 until the termination of the experiment on 4 Dec. 2014. The means and standard errors of survival times measured by LIFETEST for uncensored data were 401.7 ± 12.3, 377.9 ± 19.7, and 351.8 ± 26.2 d PI for T1.5, T0.75, and T0, respectively, and the differences were not significant (χ 2 (2 df) = 2.50, P = 0.28).

Body weight and average daily gain
The MIXED model analysis revealed that treatment effect on body weight was not significant from days 0 to 155 but approached significance (P = 0.08) from day 155 to 451. Mink which were fed 1.5% kelp tended to have lower average body weight than the other two groups ( Table 2). Regressions of body weight on the first segment of sampling dates were quadratic and for the second period were linear. The regression equations of body weight on sampling dates, adjusted for the treatments, were (1196.29 ± 26.03) + (1.6135 ± 0.340) X − (0.00782 ± 0.00172) X 2 for days 0-155 and (1315.53 ± 38.50) -(0.6595 ± 0.07649) X for days 155-451, where X is Note: Row total and % exclude the mink which died during September (within the first 21 d after inoculation) and did not show signs of Aleutian disease. Note: Treatment means within a row followed by different lowercase letters (a, b) differ at the P < 0.10. Treatment means within a row followed by different lowercase letters (c, d) differ significantly at the P < 0.05. Day 155 was 11 Feb. 2014 (prebreeding) and day 366 was 9 Sep. (after breeding, pregnancy, and lactation periods).
number of days PI. Differences among treatments for average daily gain from days 0 to 155 PI, 366 to 451 PI, and during the entire period (days 0 to 451) were not significant, but mink on T1.5 diet lost almost twice as much weight as those on T0.75 and T0 diets from days 155 to 366 PI (P < 0.01) ( Table 2).

Reproductive performance
Mink which were fed 1.5% kelp outperformed the other two groups for the percentages of females mated of those exposed to males, whelped of those mated, and whelped of those exposed to males, but the differences were not significant (Table 3). Mink supplemented with 1.5% kelp significantly outperformed the other two groups for the total number of kits born (dead or alive), kits born alive, kits alive on day 7 postpartum, and kits weaned per females whelped, per females mated, and per females exposed to males (Table 4), but differences between mink on T0.75 and T0 were not significant for any of the measurements. Kelp supplementation did not have any effect on mortality of live-born kits to day 7 postpartum or until weaning (Table 4).

Rectal temperature
The MIXED model analysis showed that the effects of the treatment and treatment by sampling day interaction on rectal temperature were not significant, but changes in rectal temperature over time, although small, were significant (P < 0.01). Mean rectal temperature was the highest on day 31 PI and was significantly different from those on other sampling dates (Table 5). Rectal temperature decreased by almost 1°C to its lowest levels on days 56 and 99 PI, which were not statistically different, followed by a minor increase on day 155 PI.

Body weight of progeny at 180 d of age
The dam's diet had negligible effects on body weight of progeny at 180 d of age (P = 0.41), and male kits were significantly heavier than females at this age (Table 5). The interaction between maternal diets and sex of the kits was not significant.  Note: Treatment means within a row followed by different lowercase letters differ significantly at the P < 0.017 when tested by the Mann-Whitney U test. P values are in brackets.

Discussion
The two novel aspects of this study were the long duration of kelp supplementation to mink (451 d) and measuring the effects of kelp supplementation on reproductive performance of mink challenged with AMDV. To our knowledge, no trial with seaweed longer than 100 d has previously been conducted, and there are no studies on the effects of seaweed supplementation on reproductive performance of animals of any species challenged with any pathogen. Whole A. nodosum meal was tested in this study because it is abundantly available in Nova Scotia at a relatively low cost. Seaweed meals contain a large array of bioactive compounds and trace minerals with unknown effects on the digestive and immune systems of animals and could have different effects than any specific seaweed extract on enzymatic digestion (Chater et al. 2016) and animal performance. The downfall of feeding whole seaweed meal is the variation amongst different batches caused by the location and season of harvest and other climatic and processing conditions, which are reported for A. nodosum (Apostolidis et al. 2011;Kim et al. 2014;Tabassum et al. 2016) and other species of seaweed (Rioux et al. 2009).
The rather high annual mortality rate of the control group was likely because the virus isolate used in this study was moderately pathogenic (Farid and Hussain 2019). The observation that mink which were fed 1.5% and 0.75% kelp had 18.2% and 11.0% lower mortality and lived 49.9 and 23.9 d longer than the control group, although statistically nonsignificant because of the small number of observations, has important economic implications for mink farms infected with AMDV. These findings warrant further investigation on the long-term effects of kelp supplementation on survival rate of AMDV-infected adult mink.
Information on the effects of feeding seaweeds on survival rate of animals challenged with pathogens is scarce. In a previous experiment, addition of crude sulfated galactans derived from red seaweed (Cryptonemia crenulata) to the diet of mice inoculated vaginally with herpes simplex virus type 2 resulted in 30% mortality in a 20 d experimental period compared with 100% mortality in the control group (Talarico et al. 2004). Diet supplementation of Nile tilapia with A. nodosum reduced the incidence of the lesions caused by infection with the Aeromonas hydrophila bacterium and healing was faster than in the control animals (Oliveira et al. 2014). Based on published reports on different animal species (Gardiner et al. 2008;Pérez et al. 2016), it may be hypothesized that feeding seaweed improves health of AMDV-infected mink by improving gut health, reducing harmful pathogens in the gut, diminishing viral replication, and improving immune response to infection.
The LOESS procedure and segmented regression showed that changes in body weight over time and daily gain were considerably different before and after breeding. Infection with AMDV decreases appetite (Jensen et al. 2016), which was manifested as weight loss or slow rate of gain in this experiment. The significantly greater weight loss of the mink fed 1.5% kelp from day 155 (February) to 366 PI (September), which encompassed the pregnancy and lactation periods, was likely the result of significantly larger litter size of this group. Mink with large litters had high nutritional requirements, but the high mineral content (Rupérez, 2002;Rioux et al. 2009;Evans and Critchley 2013), or the taste of the kelp might have caused reduced feed consumption.
The results of studies on diet supplementation with seaweed or seaweed extracts on weight gain and feed intake of farm animals have been variable. Pigs that were exposed to porcine reproductive and respiratory syndrome virus supplemented with various amounts of Tasco® during a 5 wk nursery period showed significantly higher weight gain, higher feed intake, and better feed conversion than the controls (Allen et al. 2001). Note: Means within a column followed by different lowercase letters differ significantly at the P < 0.05. PI, post inoculation.
The effects of seaweed supplementation were not significant on body weight or feed intake of piglets in a 28 d trial (Michielset al. 2012), or of lambs after 74 d ), on body weight of layer hens after 30 d (Kulshreshtha et al. 2014), broilers after 21 d (Abudabos et al. 2013), goats after 84 d (Yates et al. 2010), or rats after 21 d (Liu et al. 2015). Supplementing finishing lamb diet with 1% or 2% of A. nodosum for 7, 14, or 28 d did not affect feed intake or weight gain (Bach et al. 2008). Supplementing the diet of rabbits with 1% green seaweed (Ulva lactuca) meal did not affect body weight in two experiments, but 2% supplementation significantly decreased body weight .
Seaweed extract supplementation significantly decreased average daily gain of growing-finishing pigs in a 61 d feeding trial with no effect on feed intake. The negative effect of seaweed on weight gain was attributed to the high levels of phenolic compounds, chelated metals, or alginates (Gardiner et al. 2008). In contrast, seaweed extracts did not have a significant effect on weight gain of weaned piglets after feeding for 28 d (Sweeney et al. 2012) or 40 d (O'Shea et al. 2014, whereas it significantly improved growth rate of piglets after weaning in 25 d (O'Doherty et al. 2010) and 32 d (Heim et al. 2014) feeding trials, respectively. It may be concluded from the current and previous studies that dietary supplementation of seaweed at the rate of 1.5% of dry matter intake has negligible effect on weight gain in growing-finishing animals, and more information is needed on the effect of seaweed supplementation on feed consumption and weight gain when animals' nutrient requirements are high.
Maternal supplementation with kelp did not have any effect on body weight of their progeny at 180 d of age (pelting time). Body weight was recorded at this age because maternal supplementation with a seaweed extract from day 83 of gestation and throughout the lactation period was shown to enhance the immune response of weaned pigs challenged with Escherichia coli (Heim et al. 2014). Dietary supplementation of pregnant sows with seaweed extracts had positive effects on health and growth rate of piglets before and after weaning, including increased serum immunoglobulin G concentrations, favorable changes in the gut's microflora, enhanced immune function, and improved villous architecture of small intestine (reviewed by Sweeney and O'Doherty 2016).
The negative effects of AMDV infection on reproductive success and kit survival have been widely reported (Padgett et al. 1967;Alexandersen 1986;Hansen and Lund 1988;Broll and Alexandersen 1996;Andersson et al. 2017), and the magnitude of the effect of infection is determined by the genotype of the mink and the strain of the virus (Hadlow et al. 1983). In a previous study, the incidence of females which did not whelp of those exposed to males was 11.8%, 12.4%, and 2.5% in two AMDV-infected and one AMDV-free farm in Poland, respectively, and the corresponding values for the number of kits born alive were 3.4, 3.1, and 5.8, and for the number of kits weaned were 2.2, 2.1, and 4.9 (Reichert and Kostro 2014), showing substantial negative effects of AMDV infection on reproduction. The measures of female fertility and litter size in the control group in the current study could be considered the estimates of reproduction of black mink in Nova Scotia infected with a moderately pathogenic local AMDV isolate. AMDV infection in the current study resulted in a low percentage of females whelping of those exposed to males in the control group (66.7%), which is lower than 31% and 14% barren females on two AMDV-infected farms in Sweden (Andersson et al. 2017), and 11.8% and 12.4% barren females on two AMDV-infected farms in Poland (Reichert and Kostro 2014).
The most noteworthy result of the current study was the observation that all the 13 measures of reproduction (female fertility, number of kits born, and weaned) were higher in mink fed 1.5% kelp than the controls and those fed 0.75% kelp, although only the measures of litter size were statistically significant. The small number of kits born alive per female whelped in the controls (2.67) was considerably smaller than the numbers of live-born kits per female mated and per female whelped (5.71 and 6.29, respectively) in wild-type AMDV-infected mink in Argentina (Martino and Villar 1990). The average number of kits born alive per female whelped in mink fed 1.5% kelp (6.71) was 4.04 kits (151%) greater than that in the control group. This estimate is in the upper range of the reported values for uninfected mink, namely 5.8 and 5.28 in two studies in Poland (Socha and Markiewicz 2002;Reichert and Kostro 2014), 5.68, 7.17, and 7.46 live kits 24 h postpartum on three Danish farms (Thirstrup et al 2014a), 6.75 and 5.0 kits within 48 h after birth in brown and black mink, respectively, in Denmark (Thirstrup et al. 2014b), 5.0 and 5.67 kits in two studies in Denmark (Malmkvist et al. 1997;Hansen et al. 2010), 5.77 in Nova Scotia (Karimi et al. 2018), and 5.6 in Sweden (Elofson et al. 1989). The number of kits weaned per female whelped for mink fed 1.5% kelp was greater than in the controls by 174.8% and was marginally smaller than 4.8 to 5.12 for uninfected mink in previous studies (Socha and Markiewicz 2002;Hansen et al. 2010;Reichert and Kostro 2014;Karimi et al. 2018). The reason for the low reproductive performance of mink supplemented with 0.75% kelp is not clear. The findings somewhat agree with the report that green seaweed (Ulva lactuca) supplementation at 2% of dry matter intake decreased conception rate but increased litter size in female rabbits, but supplementation at 1% level had no effect on these parameters . Contrary to the result of the current study, dietary supplementation of pregnant sows with brown seaweed extracts from day 83 of gestation had no effect on the number of liveborn piglets or piglet birth weight (Heim et al. 2014).
In the present study, pre-weaning mortality rates in mink fed the three diets were higher than 25.8% kit mortality from birth to weaning at 4 wk of age on two AMDVinfected farms in Argentina (Martino and Villar 1990). Pre-weaning mortality rates were also higher than those of uninfected mink, which ranged between 11.3% and 27.4% (Schneider and Hunter 1993;Malmkvist et al. 1997;Hansen et al. 2010;Farid et al. 2018;Karimi et al. 2018). The lack of any effect of 1.5% kelp on pre-weaning survival was probably partly the result of significantly greater litter size in this group which negated the possible positive effects of kelp supplementation. It is also possible that beneficial effects of kelp were not passed into milk, and the amount of solid feed consumed by kits before weaning was too low to influence kit health and survival. The small litter size, low female fertility, and low survival rate of kits in the control group were likely because the mink used in the current study have never been exposed to the virus and thus suffered more than those which had a history of exposure to AMDV.
The negligible effect of kelp supplementation on rectal temperature in the current study contradicts the results of previous studies that Tasco® slightly increased rectal temperature of piglets infected with the porcine reproductive and respiratory syndrome virus compared with the controls after a 5 wk suckling period (Allen et al. 2001) and significantly increased rectal temperature of goats exposed to high environmental temperatures (Yates et al. 2010). On the contrary, body temperature of lambs which were fed 2% A. nodosum extract were lower than that for controls when exposed to stress conditions (heat and transportation) (Archer et al. 2008).

Conclusions
The results of this long-term feeding experiment showed that 1.5% kelp supplementation improved litter size and tended to increase survival rate of adult mink. Feed supplementation with 1.5% kelp showed a significantly higher weight loss compared with the control group during the pregnancy and lactation periods when the nutrient requirements of female mink were high. Further research is needed to validate the positive effects of 1.5% kelp supplementation.