Vitamin E: Role in Fertilization of Bovine Ova1,2

Selenium/Vitamin E: Role in Fertilization of Bovine Ova ~ ,2 E. C. SEGERSON, JR., F. A. M U R R A Y , A. L. MOXON, D. R. REDMAN3, and H. R. CONRAD 4 Department of Animal Science Ohio Agricultural Research and Development Center Wooster 44691 ABSTRACT

determined. The purpose was to evaluate the effect of a supplement of combined selenium and vitamin E upon fertility in ova collected from superovulated beef cattle maintained on either inadequate or adequate nutrition.

The effect of combined supplementation with selenium and Vitamin E upon fertilization of ova was evaluated in beef cattle maintained on either an adequate or inadequate nutrition. Fertilization of ova was 100% in those females receiving supplemental selenium/vitamin E and on an adequate plane of nutrition. Interaction between plane of nutrition and selenium/vitamin E was significant for percent fertility.

EXPERIMENTAL PROCEDURE Animals and Treatments

INTRODUCTION

During an experiment to develop improved methods for culture and transfer of ova, fertility of ova was impaired in a group of experiment station cows and in cows transferred from Greene County to Wayne County in Ohio. Percent fertilization was low in comparison to fertility data (35.6% vs. 67.5%) in our laboratory (12). An inadequate plane of nutrition relative to protein and energy has impaired fertility of bovine ova (9). Selenium and vitamin E have been implicated as necessary for proper functioning of various reproductive characteristics in the mammalian female (5, 10), but a possible role for these nutrients in the fertilization process of mammalian ova has not been reported. Field observations in ewes in New Zealand (7) indicated that selenium supplementation of different forage diets significantly improved lambing rate; however, any possible effect of selenium on fertilization of ova and/or embryo viability after fertilization was not

Received December 16, 1976. l Journal Article No. 157-76, Ohio Agricultural Research and Development Center, Wooster 44691. 2This work supported in part by a grant from E. J. Nutter, Xenia, OH. 3Department of Veterinary Science. 4Department of Dairy Science.

Thirty-six females of mixed beef breeding (from 2 to 3 yr of age and weighing between 300 to 450 kg) were allotted selectively by breed, number of prior superovulation treatments, and surgeries to one of four treatment groups within a 2 × 2 factorial experiment. The treatment groups consisted of: 1)supplemental selenium/vitamin E with an adequate plane of nutrition (SSe-APN); 2) no supplemental selenium/vitamin E with an adequate plane of nutrition (NSe-APN); 3) supplemental selenium/vitamin E with an inadequate plane of nutrition (SSe-IPN); 4) no supplemental selenium/vitamin E with an inadequate plane of nutrition (NSe-IPN). Analytical Methods

Quantitation of nutrients in the roughage and concentrate rations was generally by standard laboratory procedures. Percent crude protein was determined by the Kjeldahl method, and combustible energy was determined by an adiabatic oxygen bomb calorimeter. Calcium, phosphorus, and potassium analyses were with a direct reading emission spectrograph; carotene was determined by a standard chromatographic procedure (1) and selenium with the fluorometric method of Olson (11). Percent fiber and lignin were determined with the use of detergents (15). Digestible protein and metabolizable energy were computed from National Research Council (1976) values for the respective constituents of the ration. Initially selenium was measured in the plasma of the cattle to establish the status of selenium nutrition. Mean

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SEGERSON, JR., ET AL.

selenium in plasma was below .035 ppm in each animal, considered low (3).

TABLE 1. Daily feed intake for cattle on adequate or inadequate planes of nutrition (APN and 1PN). Plane of nutrition (daily intake) a

Diets and Injections

Cattle in adequate nutrition groups (APN) each was fed approximately 5.5 kg of orchardgrass hay daily supplemented with 2.3 kg of a concentrate ration (82.7% ground ear corn, 6% alfalfa meal, 10% soybean meal, .8% limestone, and .5% trace mineralized salt). The cows in IPN groups were fed approximately 5.5 kg of orchardgrass hay and trace mineralized salt in lick blocks. A 10-cc intramuscular injection of the selenium/vitamin E preparation MuSe (Burns, Biotec Lab., Oakland, CA), 50 mg of selenium as sodium selenite and 680 international units (equivalent to 500 rag) of vitamin E as alpha tocopherol acetate was administered initially to animals designated SSe in the groups described above. Subsequent injections of 25 mg of selenium and 340 international units of vitamin E were given at 30 and 45 days following the initial injection. The selenium preparation MuSe contains vitamin E for use as a vehicle for selenium; therefore, additional vitamin E was administered by injection. However, the vitamin E content of the rations (Table 1), as calculated from standardized tables (4), was adequate for normal reproductive function (6). Daily intake of protein, energy, carotene, and phosphorus was below maintenance requirements as suggested by the National Research Council for cattle in the IPN groups and above maintenance requirements in the A P N groups. Dietary daily feed intakes are in Table 1. The cattle were maintained on these diets for at least 45 days prior to superovulative treatment. Ovulation and Fertilization

Animals with an established estrus date after 45 days from the onset of the experiment received an injection of 2000 international units of pregnant mare serum gonadotropin (PMSG) on day 15 after estrus (estrus = day O) to induce a superovulatory response for maximizing the number of ova for examination of fertility. All females responding to the hormone were inseminated artificially with semen of proven fertility at approximately 10 and 24 h after onset of first estrus following PMSG Journal of Dairy Science Voi. 60, No. 6

Diet (units)

IPN

APN

Total protein (kg) Digestible protein (kg) Gross energy (Meal) Metabolizable energy (Mcal) Acid detergent fiber (kg) Acid detergent lignin (kg) Calcium (g) Phosphorus (g) Potassium (g) Provitamin A (carotene, rag) Selenium (mg) Tocopherols (rag)

.26 .14 23.82 9.76 2.46 .47 13.00 8.70

.62 .36 33.29 16.37 2.70 .50 27.95 19.74

47.96

68.89

24.30 .44 70.85

40.14 .53 111.25

aAll values on a dry matter basis.

treatment. At each breeding, semen was deposited into the uterine lumen. On day 3, ova were recovered by surgical methods. Initially animals were anesthetized with thiamylal sodium followed by closed circuit anesthesia with fluothane and oxygen. The ovaries and uterine horns were exposed through a mid-line incision, and a polyvinyl tube was inserted approximately 24 mm into each oviduct. The uterine horns then were infused with Dulbecco's Modified Eagle Medium (GIBCO, Grand Island, NY) with the fluid passing through the tubes in the oviducts and into sterile collection vessels. The recovered fluids were examined with microscopy to locate and count the ova and to determine whether fertilization had occurred. Uniform cleavage was the primary criterion for estimating fertilization although presence of two polar bodies and sperm within the zona pellucida additionally were used in questionable instances to evaluate fertility. Plasma and Tissue Selenium

Ten milliliters of jugular venous blood were obtained from all females at the time of surgery for subsequent evaluation of selenium in the plasma. The animals were slaughtered within 3 days after surgery to obtain liver samples for selenium analysis. Selenium in plasma and liver tissue was analyzed by a fluorometric procedure (11) in 2 ml plasma and appropriate

TECHNICAL NOTE s a m p l e weights up to 2 g o f f r o z e n liver tissue. Statistical Methods

D e t e r m i n a t i o n s f o r t h e main e f f e c t s o f plane o f n u t r i t i o n (PN), Se/E, and a possible PN X Se/E i n t e r a c t i o n u p o n p e r c e n t fertility and n u m b e r o f fertile ova r e c o v e r e d per g r o u p were f r o m least squares analysis o f variance (8) with d i f f e r e n c e s b e t w e e n t r e a t m e n t m e a n s for these and o t h e r variables a n a l y z e d b y t h e least significant d i f f e r e n c e t e s t (13). R e s p o n s e b y individual animals t o ova fertilization was anal y z e d a m o n g g r o u p s by Chi-square (X 2 ) analysis (13). RESULTS A N D DISCUSSION

Ova were recovered successfully f r o m 20 females. Mean values w i t h i n t r e a t m e n t s for s e l e n i u m o f plasma and liver, ovulation, and ova r e c o v e r y rate, n u m b e r o f fertile ova recovered, and fertilization r a t e (%) are in Table 2 R e c o v e r e d fertile ova w e r e generally at the f o u r t o eight-cell stage o f d e v e l o p m e n t . Ova classified as u n f e r t i l i z e d failed to m e e t any o f t h e criteria f o r evaluating fertilization. No sperma-

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t o z o a were w i t h i n and s u r r o u n d i n g t h e zona pellucida o f t h e unfertilized ova, suggesting t h a t failure o f f e r t i l i z a t i o n may have r e s u l t e d f r o m a lack in t r a n s p o r t o f s p e r m to t h e site o f fertilization. Analysis o f variance indicated t h a t t h e A P N diet increased fertility ( P < . 0 5 ) w h e r e a s t h e addition of supplemental selenium/vitamin E alone had no significant e f f e c t o n fertility. However, significant PN × Se/E i n t e r a c t i o n was d e m o n s t r a t e d f o r p e r c e n t fertility ( P < . 0 3 ) and n u m b e r o f fertile ova ( P < . 0 4 ) r e c o v e r e d per group. P e r c e n t fertility in t h o s e animals receiving s e l e n i u m / v i t a m i n E and t h e APN diet was greater ( P < . 0 1 ) t h a n p e r c e n t fertility in t h e remaining t r e a t m e n t groups. N u t r i t i o n a l imp r o v e m e n t was associated with o b t a i n i n g 100% fertilization in collected ova. This was t h e m a j o r objective o f t h e e x p e r i m e n t . The u n e x p e c t e d i n t e r a c t i o n w h i c h r e s u l t e d in a higher n u m b e r o f fertile ova in t h e NSe-IPN t r e a t e d c o w s t h a n in either t h e NSe-APN or SSe-IPN cows c o u l d be associated with t h e failure o f t h e r a n d o m i z a t i o n p r o c e d u r e t o d i s t r i b u t e the c o w s on previous fertility. This is logical f r o m c o m p a r i n g p r e v i o u s fertility o f t h e treat-

TABLE 2. Selenium of plasma and liver, ovulation and ova recovery rate, number of fertile ova recovered, and fertilization rates of ova recovered from superovulated cattle. Data are limited to those females in which at least one ovum was recovered at surgery, with the exception of the data on previous fertilization rate.

Parameters measured No. animals Plasma selenium (ppm) Liver selenium (ppm on wet basis) No. ova ovulated No. ova recovered No. fertile ova recovered Previous percent fertilizationg (fertilized ova/animal/group) Percent fertilization (fertilized ova/animal/group) Percent fertilization (fertilized ova/t0tal recovered)

SSe-APN

Treatment groupsa, b NSe-APN SSe-IPN

NSe-IPN

5

5

4

6

.067 c .328 c 5.8 e 2.6 2.6 e

.036 d .171 d 5.8 e 4.0 ,6 f

.076 c .481 c 7.3 ef 3.8 .8 f

.036 d .183 d 9.2 f 4.5 2.3 e

Pooled SE

.005 .059 1.3 1.3 .8

43.3 e

20.0 ef

0.0 f

47.5 e

16.7

100.0 c

40.0 d

19.3 d

40.7d

17.0

100.0 e

15.0 f

20.0 f

51.9 f

19.6

aSupplemental selenium/vitamin E (SSe) ; no supplemental selenium/vitamin E (NSe) ; adequate plane of nutrition (APN); inadequate plane of nutrition (IPN). bMeasurements are least square mean values ± pooled standard error of the mean (SE). C'dMeans on the same line with no superscript in common differ (P<.O1). e'fMeans on the same line with no superscript in common differ (P<.05). gThe mean values include previous ova fertility data obtained from any of the initial 36 animals before allotment to specific treatment groups for this experiment. Journal of Dairy Science Vol. 60, No. 6

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TABLE 3. Response by individual animals to ova fertilization (%) during various feeding regimes. Treatment groups SSe-APN NSe-APN SSe-IPN NSe-I PN

Observed n

100%

Expected

<100%

100% 2 2 1.6 2.4

5 5 4 6

5 2 0 1

0 3 4 5

20

8

12

< 100% 3 3 2.4 3.6

X2 7.5** 0 2.11 1.63 11.64

aSupplemental selenium/vitamin E (SSe); no supplemental selenium/vitamin E (NSe); adequate plane of nutrition (APN); inadequate plane of nutrition (IPN). **Significantly (P<.01) different from other treatment groups by Chi-square (;(2) analysis.

m e n t groups in Table 2. Since previous fertility of all animals within each group was n o t known, a second approach in Table 3 was to clarify further fertility differences a m o n g groups. Chi-square analysis for successful biological responses (100% fertilization) within animals was c o m p u t e d . T h e SSe-APN group was different (P<.01) f r o m o t h e r treated groups. Previous studies have d e m o n s t r a t e d the effectiveness of supplemental selenium injections (with and w i t h o u t vitamin E) in decreasing t h e incidence of retained placenta in the dairy c o w (10, 14), thus suggesting an i m p r o v e m e n t of uterine muscular f u n c t i o n . Since uterine contractions are of primary i m p o r t a n c e in mammalian sperm transport (2), one m a y t h e o r i z e that a uterus exhibiting p o o r muscular f u n c t i o n may be incapable of eliciting the necessary contractions for t h e transport o f sperm to the site of fertilization. A l t h o u g h fertility within t h e NSe-APN group was lower t h a n e x p e c t e d f r o m previous observations o f fertility in our l a b o r a t o r y (12), t h e e x p l a n a t i o n m a y be a t t r i b u t e d to the 4 m o prior to initiation of t h e e x p e r i m e n t in which the cattle were fed orchardgrass ration only. What enduring effect, if any, was imposed u p o n the fertilization process in females in this t r e a t m e n t group and those females in the IPN groups due to the previous diet is u n k n o w n . The data, however, indicate that o p t i m u m fertility (100%) occurred in those cattle receiving supplemental selenium/vitamin E and on a d e q u a t e nutrition. The absence o f any of these nutritional conditions resulted in l o w fertility. The differential effects o f selenium and vitamin Journal of Dairy Science Vol. 60, No. 6

E u p o n fertilization warrants f u r t h e r study, but most o f t h e i n f o r m a t i o n suggests that selenium was one interacting nutrient. ACKNOWLEDGMENT

F. M. Byers, W. E. Julien, A. P. Grifo, P. Bicksler, P. Cunningham, W. Yaussy, and R. M c C o r m i c k are acknowledged for their assistance. REFERENCES

1 Association of Official Agricultural Chemists (AOAC). 1960. Official methods of analysis. 9th ed. Ass. Offic. Agr. Chem., Washington, DC. 2 Blandau, R. J. 1969. The mammalian oviduct. University of Chicago Press, Chicago, 1L. 3 Conrad, H. R., W. E. Julien, and A. L. Moxon. 1976. Plasma selenium levels in supplemented and selenium deficient dairy cows. Distillers Feed Res. Coun. Proc. 31:49. 4 Dicks, M. W. 1965. Vitamin E content of foods and feeds for human and animal consumption. Wyoming Agr. Exp. Sta. Bull. 435. 5 Evans, H. M., and K. S. Bishop. 1922. On the existence of a hitherto unrecognized dietary factor essential for reproduction. Science 56:650. 6 Gullickson, T. W., S. L. Palmer, W. L. Boyd, J. W. Nelson, F. C. Olson, C. E. Calverley, and P. D. Boyer. 1949. Vitamin E in the nutrition of cattle. I. Effect of feeding vitamin E poor rations on reproduction, health, milk production, and growth. J. Dairy Sci. 32:495. 7 Hartley, W. J., and A. B. Grant. 1961. A review of selenium responsive diseases of New Zealand livestock. Fed. Proc. 20:679. 8 Harvey, W. R. 1960. Least squares analysis of data with unequal subclass numbers. USDA, ARS 20-8. 9 Hill, J. R., D. R. Lamond, D. M. Henricks, J. F. Dickey, and G. D. Niswender. 1970. The effects of undemutrition on ovarian function and fertility in beef heifers. Biol. Reprod. 2:78.

TECHNICAL NOTE 10 Julien, W. E., H. R. Conrad, J. E. Jones, and A. L. Moxon. 1976. Selenium and vitamin E and incidence of retained placenta in parturient dairy cows. J. Dairy Sci. 59:1954. 11 Olson, O. E. 1969. Fluorometric analysis of selenium in plants. J. Ass. Offic. Agr. Chem. 52:677. 12 Segerson, E. C., D. R. Redman, and F. A. Murray. 1976. Bovine embryo transplants. Ohio Rep. 61:57. 13 Snedecor, G. W., and W. G. Cochran. 1967.

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Statistical methods. The Iowa State University Press, Ames, Iowa. 14 Trinder, N., C. D. Woodhouse, and C. P. Rentan. 1969. The effect of vitamin E and selenium on the incidence of retained placentae in dairy cows. Vet. Rec. 85:550. 15 Van Soest, P. J. 1963. Use of detergents in the analysis o f fibrous feeds. II. A rapid method for the determination of fiber and lignin. J. Ass. Offic. Agr. Chem. 46:829.

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