Effect on abortion of feeding Korean pine needles to pregnant Korean native cows

Abstract

We studied the frequency and timing of abortion and the serum levels of 17β-estradiol and progesterone in Korean native cows fed pine needles during pregnancy. Fifteen pregnant cows were randomly assigned to groups of 5. The control group was fed a concentrate and rice straw, and the other 2 groups were fed, in addition, either 1.3 or 2.7 kg (dry weight) of Korean pine needles daily, starting at an average of 91 d of gestation and continuing until 245 d of gestation. The health status of the dams and the viability of the fetuses were ascertained by rectal palpation and ultrasound scanning during pregnancy. Simultaneously, blood samples were collected for analysis of serum 17β-estradiol and progesterone. Two abortions in mid-pregnancy (at 126 and 150 d of gestation) occurred in the group ingesting the higher daily amount of pine needles. Premature parturition occurred at 259 d of gestation in this group and at 241 and 252 d of gestation in the group ingesting the lower daily amount of pine needles. The serum 17β-estradiol concentration was significantly higher (P < 0.05) at 4 mo of gestation and the serum progesterone level significantly lower (P < 0.05) at 8 mo of gestation in the group ingesting more pine needles daily than in either of the other 2 groups. These results suggest that the ingestion of pine needles may play a role in abortion in Korean native cows by increasing the serum 17β-estradiol concentration and decreasing the serum progesterone concentration.

Introduction

The ingestion of Ponderosa pine needles by beef cattle in the western United States has induced premature parturition or abortion (1,2,3), resulting in large economic losses (4) due to retention of the placenta, death of premature calves, and lowered subsequent fertility of the dam (1). The mechanism of action by the ingested pine needles seems to involve constriction of the maternal cotyledon arterial bed (5), which results in a profound decrease in uterine blood flow, fetal stress, and parturition (6). Feeding pine needles induces a premature rise in the levels of cortisol and 17β-estradiol (associated with parturition), as well as an abrupt rise in the progesterone concentration after feeding and a sharp decrease before parturition (7).

The abortion response to ingested pine needles is variable in both field and controlled experimental conditions (1) and may be related to the stage of pregnancy (2). Premature parturition resulting from pine needle ingestion is very frequent when cows are in late pregnancy (2,7,8). Short and colleagues (2) reported that no abortion occurred when cattle were fed 5.4 kg daily for 21 d starting at day 116 of gestation, whereas the proportion of cows aborting increased significantly, and the interval to abortion decreased considerably, when cattle were fed the same amounts for 21 d starting at day 167, 215, or 254 of gestation. However, the variations in abortion response have not been clearly determined. The amount and duration of feeding, as well as the stage of pregnancy at the time of ingestion, may be related to the variations. Thus, continuous feeding of pine needles at different doses from early to late pregnancy may give accurate information on the frequency and timing of abortion. Our study was designed to yield such information and to determine changes in serum concentrations of 17β-estradiol and progesterone in pregnant Korean native cows.

Materials and methods

Animals

Fifteen pregnant Korean native cows (Bostaurus coreanae), weighing 380 to 530 kg, were used for the experiment at Kyeongbuk Livestock Research Institute, Korea. Pregnancy was confirmed by rectal palpation and ultrasound scanning (Sonoace 600; Medison Company, Seoul, Korea) with a 6.5-MHz transucer before feeding. Breeding dates were recorded and parturition dates calculated on the basis of a 285-d gestation period. All experiments were performed with the approval of the Animal Ethics Committee at the College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Korea.

Experimental treatment

The branches and twigs of Korean pine needles (Pinus koraiensis) remaining after forest tending, were collected in Gapyung County, Kyonggi-do, Korea. These needles are quite different from those of Ponderosa pine. There are clusters of 5 needles, and the needles contain different chemicals, such as terpineol and b-pinene.

The needles were ground in a crusher, then mixed with 20 mL of 98% formic acid, 20 mL of 99% propionic acid, 25 g of urea (46% nitrogen), and 25 g of calcium carbonate per 5 kg of needles. Water was added to the mixture to obtain a moisture content of 60 to 65%, and the mixture was fermented in plastic bags for 1 mo to improve the digestibility and softness of the needles, which contain nondigestible lignin, and to increase the content of beneficial organic acids. At the time of feeding, the fermented needles were mixed with the basal diet.

Fifteen cows were randomly assigned to 3 treatment groups of 5 animals each. The daily diet offered to each group, on a dry-weight basis, was as follows, per animal: a) control diet, 2.2 kg of concentrate and 4.9 kg of rice straw; b) pine-needle diet A, 2.2 kg of concentrate and 4.0 kg of rice straw, plus 1.3 kg of pine needles; and c) pine-needle diet B, 2.2 kg of concentrate and 3.0 kg of rice straw, plus 2.7 kg of pine needles. The diets were started at a mean of 91 [standard deviation (s) 12] d of gestation and continued until 245 (s 13) d of gestation.

Clinical diagnosis

Diet ingestion and abortion were checked for daily. In addition, the reproductive health of the dams and the viability of the fetuses were ascertained by rectal palpation and monitored by ultrasound every 2 mo (at 4, 6, and 8 mo of gestation), from 1 mo after the start of the experimental diet. At the same time, blood samples were collected from the jugular vein for analysis of serum 17β-estradiol and progesterone.

Hormone assays

Serum concentrations of 17β-estradiol and progesterone were determined by means of enzyme-linked immunosorbent assay (ELISA) (Immuno-Biological Laboratories, Hamburg, Germany). The sensitivities of the assays were 10 pg/mL and 0.05 ng/mL, respectively, and the inter- and intra-assay variation was less than 10% in both cases.

Statistical analysis

The data, calculated as means and s, were analyzed by analysis of variance (ANOVA), followed by Tukey's multiple-comparison test. A P-value less than 0.05 was considered statistically significant.

Results

Two mid-pregnancy abortions occurred, 39 and 40 d after the start of pine-needle diet B (at 126 and 150 d of gestation). Premature parturition occurred in 2 cows receiving pine-needle diet A at 154 d, and 1 cow receiving pine-needle diet B at 153 d, after the start of the experimental diets (at 241, 252, and 259 d of gestation, respectively). All 3 premature calves died immediately s or shortly after birth. The cows that did not abort after ingesting 1.3 or 2.7 kg of pine needles daily had live calves after normal gestation periods [292.7 (s = 8.2) and 289 (s = 4.2) d, respectively]; the mean gestation period in the control group was 285.8 (s = 6.9) d.

At 4 mo of gestation but not at 6 or 8 mo, the mean serum concentration of 17β-estradiol was significantly higher (P < 0.05) in the group receiving pine-needle diet B than in the groups receiving pine-needle diet A or the control diet (Figure 1). In addition, at 8 mo of gestation, but not at 4 or 6 mo, the serum progesterone concentration was significantly lower (P < 0.05) in the group receiving pine-needle diet B than in the groups receiving pine-needle diet A or the control diet (Figure 2).

Figure 1. Means and standard deviations (s) for the serum 17β-estradiol concentration, determined by enzyme-linked immunosorbent assay (ELISA), at 4, 6, and 8 mo of gestation in groups of 5 pregnant Korean native cows fed a control diet or a diet containing 1.3 (A) or 2.7 (B) kg of pine needles (dry weight) daily. ^a Significant difference (P < 0.05) compared with the means of the other 2 groups.

Figure 2. Means and s for the serum progesterone concentration, determined by ELISA, at 4, 6, and 8 mo of gestation in the same groups of cows. ^a Significant difference (P < 0.05) compared with the means of the other 2 groups.

Discussion

Feeding Korean pine needles from 3 to 8 mo of gestation resulted in mid-pregnancy abortions and premature parturition among pregnant Korean native cows. Only the higher daily intake of pine needles (2.7 kg) was associated with mid-pregnancy abortion, at 126 and 150 d of gestation (as well as premature parturition). Christenson and associates (5) found that ingestion of 2.7 kg of pine needles daily at 250 d of gestation resulted in premature parturition among 5 of 8 cows, at a mean of 260.2 d of gestation. However, Short and colleagues (2) found that feeding 5.4 kg daily for 21 d did not induce abortion when started at 116 d, whereas abortion occurred when feeding started at 157 d.

The difference between these studies may be related to the different species of cattle and perhaps of pine. In addition, the ensiling process may have affected the content of the active compounds. The duration of feeding pine needles also needs to be considered: the duration in our study was greater than the duration in the study of Short and colleagues (2). Thus, extended feeding of 2.7 kg of pine needles daily, for as long as 39 to 40 d, may induce mid-pregnancy abortion.

The mechanism appears to be a reduction in the blood flow to the uterus (5,6,9). Christenson and associates (5) have demonstrated vasoactive components in the plasma of cows fed pine needles. In addition, they demonstrated an 8.9% reduction in uterine arterial blood flow to the gravid horn of cows fed 2.7 kg of pine needles daily from days 250 to 258 of gestation (6). This mechanism would explain the 3 premature births in our study. The mechanism of the 2 mid- pregnancy abortions is not clear but could be the same as that of premature parturition. However, we cannot rule out other mechanisms, including embryotoxic effects (10,11). Further studies needed to be done to elucidate the mechanism of mid-pregnancy abortion.

The significantly higher mean serum 17β-estradiol level at 4 mo of gestation in the group fed 2.7 kg of pine needles daily than in the other 2 groups suggests that the 2 mid-pregnancy abortions might be related to an elevated 17β-estradiol concentration. The significantly lower mean serum progesterone level at 8 mo of gestation in the same group, compared with the other 2 groups, may be associated with premature parturition. Short and coworkers (7) demonstrated that the serum 17β-estradiol concentration increased after the start of pine-needle feeding, up to the level normally seen at parturition, whereas the progesterone level increased after the start of pine needle feeding but subsequently declined sharply. It is possible that the components of Korean pine needles include flavones, which have phytoestrogenic activity, as does the abietic acid in pine wood (12) and aglycone flavonoids in Kouglas (13). Unfortunately, the blood collections in our study were insufficient to establish definitively that the hormonal changes were associated with abortion or premature parturition.

In conclusion, the ingestion of 1.3 kg (dry weight) of Korean pine needles daily induced premature parturition, at 241 and 252 d of gestation, whereas ingestion of 2.7 kg daily induced mid-pregnancy abortion, at 126 and 150 d of gestation, as well as premature parturition at 259 d. An increase in the serum level of 17β-estradiol and a decrease in the serum level of progesterone may play an associated role.