Physiological responses of rats fed loline and ergot alkaloids from endophyte-infected tall fescue.

Sixty-three male sprague Dawley rats were randomly assigned to dietary treatments containing 1) N-formylloline alkaloid (NFL), 2) N-acetylloline alkaloid (NAL), 3) NFL + NAL, 4) NFL + a mixture of ergot alkaloids, 5) NAL + a mixture of ergot alkaloids, 6) NFL + NAL + a mixture of ergot alkaloids, 7) a mixture of ergot alkaloids, 8) endophyte-free tall fescue seed (EFTF), and 9) endophyte-infected tall fescue (EITF) seed (negative control). All diets were prepared by mixing the prepared treatments and Laboratory Chow (1:1 ratio) and were fed at a maximum of 15 g per rat per day. All rats were killed at termination (d 18). Rats fed the EITF consumed less (P < .05) than those fed all other treatments. Feed intake for rats fed the NFL + ergot alkaloids was lower than for those fed NAL, NAL + ergot alkaloids, NFL + NAL + ergot alkaloids, ergot alkaloids, and EFTF treatments. Average daily weight gains (ADG) followed a trend similar to feed intake with some exceptions. Rats fed the NFL + NAL treatments had higher (P < .05) ADG than those fed all other treatments except the NFL + NAL + ergot alkaloid treatment. Rats fed the EITF had lower (P < .05) ADG than those fed all other treatments except those fed ergot alkaloids and NAL + ergot alkaloids. Epididymides, testes, hypothalamus, corpus striatum weights, prolactin and alkaline phosphatase content were not altered by dietary treatments. Results suggest that loline alkaloids may have a slight depressing effect on feed intake.

Onset of puberty in CD-1 mouse pups exposed prenatally through weaning to endophyte-infected tall fescue seed.

This study was undertaken to investigate the effects of feeding endophyte - infected (Acremonium coenophialum) tall fescue seed to CD-1 mouse dams (P(1)) during gestation and lactation, and on the subsequent growth and sexual maturity (onset of puberty) of their male and female offspring (F(1)). Forty-eight 21 d old pups (24 male and 24 female F(1) mice) were weaned from dams fed one of two diets containing 50% rodent chow (w/w) and 50% KY-31 tall fescue (Festuca arundinacea) seed. The seed in Diet 1 was noninfected, while the seed in Diet 2 was 80% endophyte-infected. At weaning (21 d), the F(1) pups were fed rodent chow, ad libitum throughout the remaining experimental period. At 24 d, they were paired with sexually mature non-treated virgin CD-1 mice (fed 100% rodent chow) for one parturition cycle. Male F(1) mice were sacrificed at 84 d to determine testicular development. The age at the birth of the first litter for Diet 2 F(1) male (76.8 +/- 2.2 d) and female (58.4 +/- 2.1 d) was significantly greater (P<0.05) than the age at parturition for Diet 1 male and female F(1) test mice (64.1 +/- 1.8 and 51.9 +/- 1.2 d, respectively). At parturition, the female F(1) mice showed no significant differences (P>0.05) in either mean parturition weight or number of F(2) pups born per litter. However, total F(2) litter wight (11.38 +/- 1.14 g) and mean weight per F(2) pup (1.40 +/- 0.04 g) for Diet 2 female F(1) mice litters were lower (P<0.05) when compared with Diet 1 females (14.53 +/- 0.57 g and 1.66 +/- 0.02 g, respectively). No significant differences were observed between the two male F(1) treatment groups, for total F(2) litter weight or the number of pups born per F(2) litter. Although Diet 2 F(1) males weighed significantly less (P<0.05) at weaning and at pairing, final body weights at sacrifice (84 d) were not different (P>0.05) from the Diet 1 males.

Tall fescue endophyte: effect on congenital development and pup growth in mice.

This study was designed to assess effects of a diet containing endophyte (Acremonium coenophialum)-infected tall fescue seed on litter growth and development in mice. Twenty pairs of mature CD-1 mice were allocated to dietary treatments containing 50% (wt/wt) rodent chow plus either 50% endophyte-infected tall fescue seed or seed that was not infected. At parturition, six pups of approximately equal BW and sex ratio were selected from each litter and fostered to each of the dams for suckling, resulting in four suckling groups. Pups born to dams fed the infected diet weighed less at birth and experienced developmental delays in BW gain and in eye opening (16.7 vs. 13.7 d) regardless of the foster dam's diet. Pups born of dams fed the diet that was not infected attained the greatest growth rates and weaning weights; those suckling dams fed the diet that was not infected demonstrated the largest gains. Pups born of and suckled by dams fed the infected diet demonstrated greatly reduced growth rates and weaning weights compared with the other groups. Results suggest that the congenital effect on pups born of dams fed an infected diet was significant in lowering the growth rate of the pups during suckling. The dam's ability to provide nourishment to the pups also was lowered when fed this diet. When these two factors were combined, the differences were greater than for either factor separately.

Effects of feeding endophyte-infected tall fescue seed on reproductive performance in male CD-1 mice by competitive breeding.

The objective of this study was to assess the effects of endophyte-infected tall fescue (KY-31) seed (80% infected, containing Acremonium coenophialum ) on the reproductive performance of male CD-1 mice measured by competitive breeding. Sixteen mature CD-1 male mice were randomly allocated to one of two dietary treatments containing 50% mouse chow and either 1) 50% noninfected tall fescue seed, restricted to the daily feed intake of dietary treatment 2, or 2) 50% infected tall fescue seed (w/w) fed ad libitum. Mice were preconditioned on their respective diets for 50 d prior to 96 h of cohabitation with 10 mature CD-1 females. Following removal of males, the females were carried to full term. Males were sacrificed, and testicular, epididymal weights, sperm motility and progressive sperm motility were assessed. The results showed no differences (P>0.05) in average daily weight gain (ADG) and body weight changes during the course of the experiment between the two dietary treatments. There were significant reductions (P<0.05) in total testes weight (0.238 vs 0.214 g), epididymal weight (0.087 vs 0.073 g), sperm motility (82.5 vs 58.7%), and progressive motility (3.7 vs 2.6; scale 0 to 4) between dietary treatments 1 and 2, respectively. Similarly, significant differences in pregnancy rates were 53.8 and 45.0%, and the average numbers of pups born per litter were 10.67 and 9.84, respectively. These data suggest that 50% endophyte-infected fescue seed in the diet of male CD-1 mice can affect their reproductive capacity by influencing the testicular-epididymal weights, and subsequently, the quality of spermatozoa and overall fecundity.

Effects of feeding endophyte infected tall fescue seed on reproductive performance of female CD-1 mice via competitive breeding.

This study was conducted to determine the effects of feeding endophyte-infected (Acremonium coenophialum ) tall fescue (Festuca arundinacea ) seed on the growth and reproductive performance of female CD-1 mice via competitive breeding. One hundred sixty female mice were randomly allocated to groups of ten and fed one of two diets. Diet 1 consisted of 50% mouse chow and 50% noninfected Ky-31 tall fescue seed (w/w). Diet 2 contained 50% chow and 50% tall fescue seed that was 80% infected with A. coenophialum . After 50 d of preconditioning on their respective diets, a single male was introduced into each group of 10 females and allowed to cohabitate for 96 h. The males were removed after the cohabitation period and the females continued through gestation on their respective diets. Body weight of dams and litter weights were recorded at parturition. There were no differences (P>0.05) in pregnancy rates between dietary Treatments 1 and 2 (50% vs 48.8%, respectively). However, the average number of pups born per litter (11.10 pups) and average total litter weight (17.21 g) was greater for those females consuming Diet 1 than for females consuming Diet 2 (9.33 pups per litter and 13.97 g total litter weight). The incidence of dead and cannibalized pups was more frequent with Diet 2 than Diet 1 (0.13 vs 0.0 dead; 0.21 vs 0.03 cannibalized, respectively). The obtained data suggest that although the pregnancy rate was similar between the two dietary treatments, the reproductive capacity (litter size and litter weight) of female CD-1 mice was affected by the consumption of endophyte infected fescue seed.

Fertilization rates and embryonic development in CD-1 mice fed fungal endophyte-infected tall fescue seed.

The objective of this study was to assess the effects of endophyte-infected tall fescue (KY-31) seed (80% infected, containing Acremonium coenophialum ) on the fertilization rates and embryonic development of CD-1 mice. Twenty-four pairs of mature CD-1 mice were randomly allocated to two dietary treatments containing 40% mouse chow and 1) 60% noninfected tall fescue seed or 2) 60% infected tall fescue seed (w/w). The mice were preconditioned on their respective diets for 60 d prior to 96 h of cohabitation between pairs of males and females. Following the removal of the males, equal numbers of females (n = 4) from each dietary treatment were sacrificed at 80 h, 6 and 10 d after vaginal plug establishment. The female reproductive tracts were excised and flushed to recover eggs and embryos or dissected to identify the number and status of the implanted fetuses. The number of eggs-embryos and/or fetuses per mouse present at the time of sacrifice were 9.8, 8.8 and 11.5 vs 8.2, 5.3 and 4.8 for the mice fed the noninfected and infected fescue seeds, respectively. Similarly, the conception rates (%) were 100, 100 and 100 vs 73, 50 and 50 for the two treatments. The mice sacrificed at 80 h and fed the noninfected fescue had 9.8 expanded blastocysts per animal, whereas those fed the infected fescue had only 4.0 expanded blastocysts. The results suggest that the infected fescue seed consumed by these mice had a significant effect on fertilization rates. More importantly, the infected fescue seed influenced negatively the development of these embryos during the embryonic-preimplantation period.

Reproductive responses in male rats fed extracts of fescue seed infected by the endophytic fungus Acremonium coenophialum.

This study was undertaken to determine whether a methanol extract of endophyte-infected tall fescue seed contained the factor(s) that were responsible for the decrease in male reproductive potential when rats were fed toxic tall fescue seed. Twenty-four Sprague-Dawley male rats (ca. 225 g.) were randomly allocated to one of three dietary treatments containing 50% (w/w) rodent chow and 50%: (1) noninfected tall fescue seed; (2) noninfected tall fescue seed treated with concentrated methanol effluent from serial methanol extraction of infected seed; and (3) the extracted seed residue of seed that was greater than 95% infected by Acremonium coenophialum. Parameters assessed were testicular weight, epidydimal weight, parenchymal weight, progressive sperm motility, sperm motility, sperm count and daily sperm production potential. Significant reduction (P less than 0.05) for feed intake, average daily gain, testicular weight, epidydimal weight and daily sperm production potential were observed for treatment 2. These data suggest that the serial methanol extract contains the factor(s) responsible for the lowered reproductive potential in male rats. The extract contained 15,578 micrograms/ml of pyrrolizidine alkaloids and only trace amounts of ergopeptides. These results suggest that N-formyl and N-acetyl loline may play an important role in the fescue toxicity noted in the exposed rats.

Lactation in mice fed endophyte-infected tall fescue seed.

This study was conducted to assess the effects of endophyte-infected Acremonium coenophialum tall fescue (KY-31) seed (80% infected) on lactation in CD-1 dams and suckling performance of pups as measured by pup survival and growth rates. Twenty-four pairs of mature CD-1 mice were randomly allocated to four dietary treatments: 1) 100% mouse chow ad libitum; 2) 40% endophyte-infected tall fescue seed and 60% mouse chow (w/w); 3) reduced intake (100% chow), adjusted daily to the intake level of Treatment 2; and 4) 60% infected tall fescue seed and 40% chow. The mice were preconditioned on their respective diets for 100 d prior to 96 h of cohabitation between pairs of males and females. At parturition the litters were removed, and each dam was given a litter of six pups of equal weight, size and sex ratio to suckle for 15 days. All pups given to all the dams were born to other mice that were not part of the study and had not been exposed to endophyte-containing diets. Dams and litter weights were measured daily for 15 consecutive days. The combined body weight measurements of litters from dams fed the tall fescue containing diets (Treatments 2 and 4) were significantly lower (2.07 +/- 0.41 g/d) than that of litters from dams fed the chow containing diets (Treatments 1 and 3) during the suckling trial (P<0.05). Similarly, nine of ten (90%) dams fed the chow containing diets maintained five or more pups (5.5 +/- 0.2) throughout the study as compared to five of nine (55.6%) dams fed the tall fescue containing diets that maintained less than five pups (4.5 +/- 0.2).

The effect of feeding tall fescue seed infected by Acremonium coenophialum on pregnancy and parturition in female rats.

1. Female Sprague-Dawley rats (Rattus norvegicus) were randomly assigned to various dietary treatments containing: (1) 100% Purina rodent chow, ad libitum; (2) same as 1, but restricted to daily intake of 7; (3) 50% rodent chow (w/w) and 50% endophyte-free tall fescue (Festuca arundinacea) seed; (4) same as 3, but restricted to intake of 5; (5) 50% rodent chow, 25% endophyte-free tall fescue seed and 25% endophyte-infected (Acremonium coenophialum) tall fescue seed; (6) 50% rodent chow, 12.5% endophyte-free and 37.5% endophyte-infected tall fescue seed; and (7) 50% rodent chow and 50% endophyte infected tall fescue seed. 2. Average daily feed intakes and average daily weight gains decreased with higher levels of endophyte infected seed. 3. Frequency of litter production was affected by all endophyte-infected containing diets. 4. Conception was reduced only in dietary treatment (7). 5. Litter weights, number of pups per litter and weight per pup were proportionally reduced as higher levels of infected seed were incorporated in the ingested diets.

Effects of feeding endophyte-infected tall fescue seed on the reproductive performance in male and female CD-1 mice by combination crosses.

The present study was undertaken to assess the effects of endophyte-infected Acremonium coenophialum tall fescue (KY 31) seed (80% infected) on the reproductive performance of male and female CD-1 mice by combination crosses. Forty-eight male and 48 female 30-d-old mice were randomly allocated to two diet treatments. Twenty-four males and 24 females were fed Diet 1, consisting of 50% non-infected fescue and 50% mouse chow; the remaining animals were fed Diet 2, containing 50% infected fescue. At the end of 50 d, the animals were paired in groups (n = 12) as follows: 1) Diet 1 males and females; 2) Diet 2 males and Diet 1 females; 3) Diet 1 males and Diet 2 females; and 4) Diet 2 males and females. The pairs were maintained on the diets of their female partners and were allowed to cohabitate for 96 h. After this period the males were removed and the females were carried to term. Pregnancy rates among the four treatment groups were 100, 83.3, 75.0 and 54.5%, respectively. Similarly, the average number of pups born per litter among the four groups were 11.5, 8.0, 9.7, and 7.5, respectively. Also, the weights (grams) of pups born per litter were 17.5, 12.6, 12.4, and 9.8, respectively. The results point out that the reproductive capacity of both male and female mice are affected (P < 0.05) by the ingestion of endophyte-infected fescue seeds. Such negative effects were more severe in the female than the male (P < 0.05).

Effect of feeding fungal endophyte (Acremonium coenophialum )-infected tall fescue seed on reproductive performance in CD-1 mice through continuous breeding.

This study was designed to assess the effect of endophyte-infected (Acremonium coenophialum ) tall fescue (KY-31) seed (80% infected) on reproductive performance in CD-1 mice by continuous breeding. Twenty-four pairs of 70-d-old CD-1 mice were randomly allocated to four diets: 1) mouse chow ad libitum; 2) 40% infected fescue seed and 60% chow (w/w); 3) reduced intake (100% chow) similar to the intake, adjusted daily, in Diet 2; and 4) 60% infected fescue seed and 40% chow. Males and females were randomly paired (six pairs/treatment) and placed on the above diets. The mice were fed the corresponding diets for 80 d, although the pairs were separated on Day 60 (prior to the birth of the 3rd litter) and the females were monitored for one additional gestation period (20 d). The pregnancy data (litters produced) among the four treatments were 100.0 (18), 77.8 (14), 100.0 (18) and 80.0% (12) respectively. Similarly, the average number of pups born per litter among the four treatments was 11.8, 9.3, 10.1, and 9.8. When the chow treatment (1 and 3) and the fescue treatments (2 and 4) were pooled and compared, the percent pregnancy was 100.0 (n = 36) and 78.8 (n = 26), and the pups born per litter (means +/- SEM) were 11.0 +/- 0.5 and 9.5 +/- 0.6, respectively. Also the intervals between the three litters born during the 60-d cohabitation period were 21.6 +/- 1.1 and 24.5 +/- 0.9 d for the chow and fescue treatments, respectively. The results point out that 40 and 60% infected fescue seed in the diet of mice does influence (P < 0.05) their reproductive capacity as measured by percent pregnancy and litter size.

The effect of feeding endophyte infected tall fescue seed on reproductive performance in female rats.

Female Sprague-Dawley rats (Rattus norvegicus) were randomly assigned to diets containing mixtures of rat chow and tall fescue (Festuca arundinacea) seed with 0, 5, 10, 20 and 40% infection levels of Acremonium coenophialum to assess the effect of the diets on the reproductive potential of rats. Rats fed 40% infected seed had decreased body weight, decreased mean percent body weight of uteri, failed to maintain normal estrous cycles and were unable to become pregnant. Animals fed a diet of 20% infected fescue seed had extended estrous cycles. There were no significant differences among the 0, 5 and 10% dietary treatments.

Effect of feeding tall fescue seed infected by endophytic fungus (Acremonium coenophialum ) on reproductive performance in male rats.

An experiment was conducted to determine the effect of feeding tall fescue seed infected by an endophytic fungus Acremonium coenophialum on the reproductive performance of male rats. Thirty 70-day-old rats were randomly allocated to four treatments: (I) fed 50% rat chow and 50% healthy fescue seed ad libitum (control; N=9; (II) same as I but restricted to the daily feed intake of III (N=7; (III) 50% fungal-infected fescue seed and 50% chow (N=7); and (IV) a mixture of 50% laboratory chow, 25% healthy fescue seed and 25% fungal-infected fescue seed (N=7). The rats were fed these diets for 42 days. During this time, body weights were taken weekly and feed intake was taken daily. At the end of the experiment, the rats were sacrificed and the testes and epididymides were excised and measured. Sperm parameters were assessed (concentration, percentage of motility and progressive motility) at the site of the cauda epididymis; the testes were homogenized and assessed for daily sperm production potential (DSP). Concentrations of spermatozoa (x 10(6)) among the various treatments were 645.6, 486.5, 387.4 and 457.1; motility and progressive motility measurements ranged from 48 to 50% and 2.3 to 2.4 (0-4), respectively. DSP values (per gram) and testicular weight were reduced (P<0.05) in Diet III. The data suggests that 50% fungal-infected fescue seed in the diet of rats does influence the DSP, testicular parechyma and epididymal weight in the rat.

Insect feeding deterrents in endophyte-infected tall fescue.

The presence of an endophytic fungus, Acremonium coenophialum, in tall fescue (Festuca arundinacea) deterred aphid feeding by Rhopalosiphum padi and Schizaphis graminum. Both species of aphid were unable to survive when confined to endophyte-infected tall fescue plants. Feeding deterrents and toxic factors to R. padi and Oncopeltus fasciatus, large milkweed bug, were primarily associated with a methanol extract obtained when endophyte-infected tall fescue seed was serially extracted with hexane, ethyl acetate, and methanol. The concentrations of pyrrolizidine alkaloids were determined to be 30 to 100 times greater in the methanol extract than in the hexane and ethyl acetate extracts.