Influence of uterine flushings from superovulated cows on in vitro bovine morulae development

THERIOGENOLOGY

INFLUENCE OF UTERINE FLUSHINGS FROM SUPEROVULATED COWS ON IN VITRO BOVINE MORULAE DEVELOPMENT 13 R.J. Toole,lF.C. Gwazdauskas,' W.D. Whittier2 and W.E. Vinson 1 1

Department of Dairy Science andLCollege of Veterinary Medicine Virginia Polytechnic Institute & State University Blacksburg, VA 24061

Received for publication: October 5, 1987 Accepted: July 2.9,1988

ABSTRACT

To evaluate early embryo development, 248 good to excellent bovine morulae were cultured in Ham's F-IO medium, supplemented with 10% steer serum, uterine flushings from Days 6, 10 or 15 following estrus (0.01, 0.1, 1.0 and 10% protein; 64 mg protein/ml), and 1.0% uterine flushings and 10% steer serum. Final development scores for embryos in steer serum were significantly higher (range across experiments was: 4.06 to 4.37) than for embryos cultured in uterine flushings alone (-0.23 to 0.52). Treatment means were not different (P >0.05) when 10% steer serum was added to 1.0% uterine flushings. A higher percentage of embryos in 10% steer serum (92%) than in 13% steer serum plus 1.0% uterine flushing from Day 6 (33%), Day 10 (45%) and Day 15 (50%) developed to hatched blastocysts. Embryos cultured in 1.0% Day 6 uterine flushings plus 10% steer serum required more time to attain the early blastocyst and blastocyst stages, while embryos in 1.0% Day 15 uterine flushings and 10% steer serum developed at the same rate as controls to the expanded blastocyst stage, but hatched sooner (72.8 vs 96.5 11). These results suggest substance(s) in uterine secretions can have inhibitory and stimulatory influences on early bovine embryo development. Key words:

bovine, embryo, uterine secretions

INTRODUCTION The integrative action of ovarian steroids causes cyclic changes in numerous uterine responses including blood flow (l), uterine microstructure (2), and uterine protein profiles (3-5). Such changes result in the establishment of an intrauterine environment capable of

Acknowledgment The authors wish to express appreciation to the Upjohn Co. for supplying Lutalyse. ;iCorrespondenceand reprint requests.

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sustaining the bovine embryo. Early analysis of uterine secretions collected from cows found no difference in the total amount of protein between samples from open and pregnant heifers (6). Although total protein did not differ, a few specific proteins were present in lesser amounts and one particular protein was absent in a large number of the nonpregnant heifers. Laster (7) described a protein with a molecular weight between 50,000 and 60,000 that could be detected in the uterus of cows 15 d after mating only when blastocysts were present. Very few embryo culture experiments have been conducted using uterine secretions as a media supplement. If the production of uterine proteins is necessary for embryo development in vivo, it follows that the addition of uterine proteins to in vitro culture systems should maintain or enhance embryo development. This has been observed in the rabbit, where the addition of unfractionated uterine proteins from Day 5 pregnant rabbits was found to stimulate the growth of early embryos (8 to 16-cell, morulae, and Day 4 blastocysts) in culture (8). Also, El-Bana and Daniel (9) reported increased uptake of amino acids with addition of uterine flushings to rabbit embryo culture. Similar work in the bovine using uterine secretions from Day 12 uterine flushings as a supplement to Day 10 embryos in culture found increased survival rates and greater increases in diameter than embryos cultured in bovine serum albumin (BSA) control media (10). The objectives of this study were to evaluate early bovine embryo development after culture with lo%, l%, 0.1% and 0.01% concentrated uterine proteins obtained from cows flushed on Days 6, 10 or 15 following superovulation, and combining steer serum with 1% uterine proteins obtained from Days 6, 10 or 15.

MATERIALS AND METHODS A total of 98 superovulations were conducted on 49 Holstein and Jersey cows (14 used 2X; 6 used 3X; 5 used 4X; 2 used 5X). Thirty-six were dry cows maintained together as a group in an open lot without housing, while 13 were lactating cows (11 early lactation; 1 mid lactation; 1 late lactation). All cows with normal corpora lutea were superovulated with follicle-stimulating hormone (FSH-P; Burns Biotech, Omaha, NE) using various dosage regimens containing 28 to 50 mg (n = 4, 28 mg; n q 8, 30 mg; n q 19, 32 mg; n q 44, 35 mg; n = 19, 40 mg; n = 4, 50 mg) between Day 9 to 13 of the estrous cycle. A total of six to eight intramuscular (i.m.) injections were administered twice daily at 12-h intervals. Prostaglandin Fzo (PG; Lutalyse, Upjohn Co., Kalamazoo, MI) was administered to induce luteal regression at 3.5 Days after initiation of FSH-P injection. Cows observed in estrus were inseminated 3 times (at first observation of estrus and 12 and 24 h later). cows not observed in estrus were inseminated at 72 and 84 h post PG injection. Nonsurgical flushing procedures were modifications of Elsden et al. (11). Cows were flushed 6 d after the first insemination with 500 ml of Dulbeccos phosphate-buffered saline and embryos were morphologically evaluated using the procedure of Reynard and Heyman (12). Embryos were placed into one of five categories: excellent, good, fair, poor, or unfertilized ova. Only excellent and good embryos were utilized in the experimental treatments.

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‘Two hundred and forty-eight bovine morulae were randomly assigned to the experiment. In Experiment I, 10 embryos were cultured in Ham’s F-10 supplemented with 10% (v/v) steer serum (64 mg protein/ml), 10 in 10% Day 6 uterine flushings, 10 in 10% Day 10 uterine flushings and 10 in lo’% Day 15 uterine flushings from previously superovulated cows. In each #of Experiments II, III and IV, 40 embryos were used as in Experiment I except that the uterine flushings were at 1% (6.4 mg/ml) 0.1% (0.64 mg/ml) and 0.01% (0.064 rag/ml, v/v), respectively. In Experiment V, 25 embryos were cultured in 10% steer serum, 21 in 1% uterine flushings from Day 6 plus 10% steer serum, 22 in 1% uterine flushings from Day 10 plus 10% steer serum and 20 in 1% uterine flushings from Day 15 plus 10% steer serum. .?ay 6 uterine flushings were obtained from fluid recovered from nonsurgical embryo flushes. All cows were previously superovulated with 50 mg of FSH-P (13). Flushings from seven different cows were then pooled and concentrated using a 50 ml Amicon stir cell at 4OC under nitro::en pressure not exceeding 2069 mmHg. After concentration of the flushings, the protein concentration was adjusted with distilled water to 64 rag/ml. The osmolarity was 305 m0sm. The Day 6 uterine flushings were then heat-treated (30 min at 56O C), sterilized (0.45~ millipore filter), placed in 35-ul aliquots and frozen (-70°C) prior to their use. :3ay 10 uterine flushings were obtained nonsurgically by flushing previously super-ovulated cows 4 d after they had been flushed for embryos with 50 ml of a 1.5% (0.33 M) sterile sodium chloride solution. The fluid was recovered by a gentle massaging motion into a sterile 50-ml Erlenmeyer flask which was then placed on ice. Following collection, samples with red blood cell concentrations of more than 3.0 x lOs./ml were discarded and remaining samples were centrifuged at 12,602 x g to remove cellular and mucal debris. The samples were sterilized by filtering through a millipore filter (0.45 u) and then placed into 50-ml polypropylene storage bottles and frozen at -7OoC until concentrated. Samples from 14 different cows were pooled and concentrated via a 50-011 Amicon stir cell as described previously. The protein concentration was then determined and the sample was diluted Kith distilled water to a concentration of 64 rug/ml. Small aliyuots (35 ul) were heat-treated (30 min a-t 56oC) and frozen at -7OOC until used. Osmolarity was 297 m0sm. Day 15 uterine flushings were obtained nonsurgically by flushing previously superovulated cows 9 d after they had been flushed for embryos. The flushing procedures and laboratory preparations were the same as described for Day 10 uterine flushings. Samples from eight different cows were pooled, concentrated, and the protein concentration was adjusted to 64 mg/ml. Osmolarity was 292 m0sm. ‘Ihe culture procedures (14) were similar to those used by Wright et al. (115). Following morphological evaluation, the embryos were placed in a watch glass containing Ham’s F-10 and then placed in culture. First 10 ul of a protein supplement was added to a 150-pl culture dish. Next 10 ul of Ham’s F-10 containing a randomly selected embryo was added to the culture well and mixed with the protein supplement. Finally, 80 ul of Ham’s F-10 was added and stirred to make up a total culture volume of 100 ul/embryo.

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Embryos were cultured individually in lOO-ul microdrops under a drop of previously equilibrated paraffin oil (5% COz, 5% 02, 90% Nz). Embryos were cultured in an incubator at 37OC with an atmosphere of 5% 02 t 5% co2, and 90% N2, bubbled through distilled water. Cultured embryos were examined microscopically at 60x every 12 h and development was recorded. Treatment differences in final embryo development were evaluated using the system of Wright et al. (15). A numerical value of 0 to 5 was assigned to each embryo representing the most advanced stage of development reached in culture. A score of 0 corresponded to an embryo showing no development or one that remained a morula before degenerating; a score of 5 represented development to the hatched blastocyst stage. Mean developmental scores for each treatment were calculated and the data analyzed by analysis of variance. The dependent variable was the final developmental score; the independent variables in the model were treatment, initial yuaiity, and cowflush, which was the combination of the particular cow and her flush date effect. Mean developmental times to each stage were determined for all treatments. The dependent variables used were times to the early blastocyst, blastocyst, expanded blastocyst, hatching blastocyst, hatched blastocyst stages, and the time to embryo degeneration. Independent variables were treatment, initial embryo quality, and cowflush. Means were compared by LSD. RESULTS Individual analyses of variance for final development score revealed significant treatment effects (PcO.01) in Experiments I through IV . Also, there was a significant cow flush effect of final score in Experiment IV. Final development scores for embryos in Experiments I through IV are in Table 1. Of the embryos cultured in 0.01% uterine flushings 40, 30 and 30% of embryos advanced to the early blastocyst stage when cultured in Day 6, 10 or 15 uterine flushings, respectively. Time to reach the hatched blastocyst stage of development was 108.0 + 7.2 h (n q 6), 108.0 ? 6.2 h (n = 8), 108.0 2 6.2 h (n = 6), and 96.0 + 6.2 h (n = 6) for embryos cultured in steer serum in Experiments I to IV, respectively. Only one embryo in 10% Day 10 uterine flushings developed to the early blastocyst stage. One embryo in each of the Day 10 and Day 15 1% uterine flushings advanced to the early blastocyst stage, whereas, one cultured embryo in each treatment at 0.1% advanced this far. Experiment V utilized a combination of 10% steer serum and 1% uterine flushings from either Day 6, 10, or 15. This experiment was conducted to determine whether these uterine flushings contained inhibitory substances or if components in the uterine secretions might act synergistically with steer serum to promote early embryo development. Final mean development scores are presented in Table 2. Analysis of variance showed a cowflush effect on the final score. Embryos in steer serum had a final mean developmental score of 4.19.

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Table 1. Mean final morphological development scores (ASEM) for embryos cultured in Ham's F-10 supplemented with steer serum (SS), 10% l%, 0.1% and 0.01% uterine secretions from cows on Days 6, 10 and 15 after superovulation

Treatment ~__

NO.

E?cperimentI ss

4.16 2 0.18a

10

-0.18 f 0.20b

10

- Day 10

-0.10 f 0.18b

10

- Day 15

-0.20 + 0.19b

10

10% - Day 6

miment ss

II 4.06 ? 0.2f1~

10

0.32 -+0.29b

10

- Day 10

0.52 f 0.29"

10

- Day 15

0.46 ? 0.28b

10

4.37 k 0.22=

10

0.25 + 0.21b

10

- Day 10

0.20 2 0.19b

10

- Day 15

0.23 f 0.22b

10

4.26 2 0.18a

10

-0.09 -+0.18b

10

- Day 10

-0.21 + 0.19b

10

- Day 15

-0.23 + 0.19"

10

1% - Day 6

miment ss

III

0.1% - Day 6

miment ss

IV

0.01% - Day 6

a,b

Least squares means with different superscripts within experiment differ at P
Developmental scores for embryos cultured in Day 6, 10, or 15 uterine flushings were 2.57, 3.28, and 3.34, respectively. Final developmental stages for all embryos in Experiment V are in Table 3. Twenty-three of 25 embryos cultured in steer serum developed to the hatched blastocyst stage, which was greater (PCO.01) than with embryos cultured in 1% Day 6, 10 or 15 uterine flushings combined with 10% steer serum.

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Table 2.

Final mean developmental scores for embryos cultured in 10% steer serum (SS) or 1% Day 6, 10, and 15 uterine proteins combined with 10% SS

Least squares mean Score

Treatment

SEM

SS

4.19

0.30

Day 6 t SS

2.57

0.34

Day 10 t SS

3.28

0.33

Day 15 t SS

3.34

0.34

Final morphological development of bovine embryos after culture in 10% steer serum (SS), or 1% Day 6, 10, and 15 uterine proteins combined with 10% SS --__ Treatment ss Day 6 t SS Day 10 t SS Day 15 t SS Table 3.

Stage of development

A-&)

Morula

25

(100)

21

(100)

22

(100)

20

(100)

Early blastocyst

24

(96)

19

(90)

20

(91)

17

(85)

Blastocyst

24

(96)

17

(81)

20

(91)

17

(85)

Expanded blastocyst

24

(96)a

12

(57)b

15

(68)b

15

(75)a

Hatching blastocyst

23

(92)"

9

(43)b

12

(55)b

10

(50)b

Hatched blastocyst

23

(92)a

7

(33)b

10

(45)b

10

(50)b

no

no

no

a,b Numbers in a row with different superscripts differ at PCO.01.

Mean times to early blastocyst stage of development were 28.6 h for steer serum, 28.7 h for 1% Day 10, and 30.0 h for 1% Day 15 uterine flushings combined with 10% steer serum (Table 4). Mean time to the early blastocyst stage for embryos cultured in 1% Day 6 uterine flushings plus 10% steer serum was longer (35.6 h) and different (PCO.05) from the other treatments. Mean time to the blastocyst stage for embryos cultured in 1% Day 6 uterine flushings plus 10% steer serum also was longer (PCD.05) than the other treatments,

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Table 4. Least squares means (?SEM) for developmental time to each stage of development for embryos cultured in 10% steer serum (SS) or 1% Day 6, 10, and 15 uterine proteins combined with 10% SS

Treatiaent

ss

Day 6 + SS

Stage of development

Day 10 t SS

Day 15 + SS

Time (hours)

Early blastocyst

28.6 + 2.5"

35.6 2 3.0b

28.7 + 3.0a

30.0 f 3.28

Blastocyst

42.9 2 2.5a

50.0 2 3.1"

44.3 + 3.0a

44.7 2 3.28

Expanded blastocyst

59.4 _+2.5

58.8 t 3.7

63.0 + 3.4

57.3 + 3.4

Hatching blastocyst

77.6 f 3.3c

72.3 2 5.W

82.2 z!z 5.2c

61.7 2 5.6d

Hatched blastocyst

96.5 f 3.8"

90.7 zt7.4~

96.1 + 6.3c

72.8 + 6.5d

--*,b numbers in a row with different superscripts differ at PcO.05. cld numbers in a row with different superscripts differ at P
IEmbryoscultured in 1% Day 15 uterine flushings plus 10% steer serum had a mean time to the hatching blastocyst stage of 61.7 h, which was significantly less (PcO.05) than embryos cultured in 10% steer serum or 1% Day 10 uterine flushings combined with 10% steer serum, which had mean -timesto hatching of 77.6 and 82.2 h, respectively. Also, mean time to the hatched blastocyst stage for embryos cultured in 1% Day 15 uterine flushings combined with 10% steer serum was significantly reduced (72.8 h) compared to 96.5, 90.7 and 96.1 h for embryos cultured in steer serum and Day 6, or Day 10 uterine flushings plus 10% steer serum, respectively. DISCUSSION :!nthis study, different sources of uterine proteins were tested for their ability to allow early bovine embryo development. Steer serum was chosen as the control since studies by Allen et al. (16) and Canfield et al. (13) report successful bovine embryo development in culture media supplemented with steer serum. Reasons for the success of steer serum in supporting early embryo development are not well understood. Brinster (17) suggested that sera in general may provide an exogenous nitrogen source which is required by the embryo to support the marked increase in protein synthesis that occurs between the morula and early blastocyst stages. Saito et al. (18) suggested that mouse embryos also require some protein component in the large molecular weight fractj.onof serum for hatching and attachment. Saito et al. (19) reported that a medium containing at least 10% serum was necessary for culturing mouse embryos. Sera concentration requirements for the culture of bovine embryos have not been well established, but bovine embryo development has been obtained with sera concentrations ranging from 5 to 50% (1.6,20).

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Our results show that steer serum supports early bovine embryo development. Seventy-five percent of embryos cultured in steer serum developed to the hatched blastocyst stage. These results are higher than those reported by Canfield et al. (13), who found that bovine morulae had a 37.9% hatching rate in steer serum, but they agree with results obtained by Allen et al. (16), who reported a 67.0% hatching rate for embryos cultured in 10% steer serum. Time to hatching for all embryos cultured in steer serum averaged between 96 and 108 h, which is similar to the time of hatching in vivo (21). Uterine proteins collected from previously superovulated cows on Day 6, 10, or 15 after insemination were unable to support early embryo development when added to Ham's F-10 medium as the only supplemental protein source. There is a possibility that the uterine flushings may contain inhibitory substances. This hypothesis is supported by the fact that as uterine flushings were diluted from 10% to 0.1X, times to degeneration increased from about 49 h to 63 h. Development occurred in all treatments where 1% uterine proteins were combined with 10% steer serum. However, embryos cultured in 1% Day 6 uterine flushings combined with 10% steer serum had delayed times to the early blastocyst and blastocyst stages. This suggests that Day 6 uterine flushings may contain some substance(s) that accounts for this retardation of development in the early stages. Embryos cultured in 1% Day 6 uterine flushings plus 10% steer serum and which survived to the blastocyst stage exhibited times to the further developmental stages which were similar or less than that of the controls. These results suggest that the embryo may be more susceptible to certain inhibitory substances in the period from morula to blastocyst. Embryos cultured in 1% Day 15 uterine flushings combined with 10% steer serum had developmental stage times which were similar to that of controls up to the expanded blastocyst stage. However, the time to hatching was significantly reduced for embryos in 1% Day 15 uterine flushings plus 10% steer serum, with a mean time of 72.8 h compared to 96.5 h for control embryos. This suggests that Day 15 uterine flushings may contain a factor(s) which may shorten the time of embryo hatching in vitro. However, only 10 of 20 embryos reached the hatched blastocyst stage in the 1% Day 15 uterine flushings plus 10% steer serum treatment compared with 23 of 25 hatched blastocysts in steer serum alone. A possible factor contributing to the poor embryo development in uterine flushings collected from Days 6, 10 and 15 is the source of these flushings. Newcomb et al. (22) reported that the high percentage of abnormal embryos recovered from superovulated donors may result from an unfavorable uterine environment due to grossly distorted levels of circulating steroids in these animals. It is well known that estrogens are abnormally high at estrus in superovulated cows, and they are also known to be high after ovulation in a large number of sterile cows (23). Greve et al. (241 \~ , suggested that the process of superovulation has a profound effect on a dairy cow's normal endocrine balance, with resultant abnormal embryo development.

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Estrogens and progesterone are known to influence the production and concentrations of certain uterine flushings (5). Anderson et al. (4) reported that acidic proteins were unique to collected uterine flushings associated with progesterone treatments. Also, a basic protein was observed in 11 of 18 cattle receiving treatments including progesterone, but it was not found in any of the cows receiving only an estrogen treatment. Since superovulated cows have high concentrations of estrogens and progesterone, they may have a uterine environment that differs from normal cows and may thus be detrimental to early embryo development. Another possibility contributing to the poor development in uterine flushings is that the synchrony requirements between the embryo and the uterine flushing may not have been met. It is known that the composition of the uterine environment in early pregnancy is constantly changing (25,27). Furthermore, embryo transfer experiments have found the rleedfor stringent synchrony requirements between the donor and recipients. Deviations of more than 2 d caused dramatic decreases in pregnancy rates, and generally best results were obtained when exact synchrony occurred (28,29). Culturing Day 6 morulae in uterine flushings from Day 10 or Day 15 flushings may mean that we were placing the embrgos in an environment for which they were not prepared. The protein environment of the uterus may be a major contributor for the noed for synchrony of stages of the estrous cycle and embryo development. Additionally, the poor development observed in the uterine flushings treatments may indicate that certain inhibitory substances as well as the proteins may have been concentrated. Schumacher (30) reported the presence of large numbers of immunoglobulins and lysozymes in human uterine flushings. Upon concentration, these substances may brcone detrimental to early embryo development. He reported that IgG and IgA antibodies present in uterine flushings were capable of inhibiting in vivo and in vitro fertilization in the rabbit. Also, IgA type antibodies reacted with rabbit blastocyst cells and caused subsequent degeneration of the blastocyst. The presence of immunoglobulins in uterine flushings obtained from prev:!ouslysuperovulated cows has been documented in Day 10 and Day 18 flushings (31). Thus, it is possible that the procedures employed to concentrate the uterine flushings may have concentrated immunoglobulins that became toxic to the early bovine embryo. Data from our laboratory suggests that the addition of IgM and IgG to bovine embryo culture media interferes with the normal development process (32). REFERENCES 1. Ford, S.P., Chenault, J.R. and Echterncamp, S.E. Uterine blood flow of cows during the estrous cycle and early pregnancy: effect of the conceptus on the uterine blood supply. J. Reprod. Fertil. x:53-62 (1979). 2. Priedkalns, J. Female reproductive system. b: Delman, H.D. (ed.). 'Textbookof Veterinary Histology. Lee and Febiger, Philadelphia, 1976, pp. 319-330.

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3. Roberts, G.P. and Parker, J.M. Fractionation and comparison of proteins from bovine uterine fluid and bovine allantoic fluid. Biochem. Biophys. Acta =:69-76 (1976). 4. Anderson, G.W., Gwazdauskas, F.C., Whittier, W. D. and Vinson, W.E. Uterine protein profiles in ovariectomized dairy cows administered progesterone and estradiol-178. Anim. Reprod. Sci. u:161-172. (1986). 5. Bartol, F.F., Thatcher, W.W., Lewis, G.S., Bliss, E.L., Drost, M. and Baser, F.W. Effect of estradiol-17B on PGF and total protein content in bovine uterine flushings and peripheral plasma concentrations of 13, 14-dihydro-15-Keto-PGF2a. Theriogenology x:345-348 (1981). 6. Laster, D.B. Uterine proteins and pregnancy in cattle. Sci. =:216 (1974).

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7. Laster, D.B. A pregnancy specific protein in the bovine uterus. Biol. Reprod. =:682-689 (1977). 8. Maurer, R.R. and Beier, H.M. Uterine protein and development b vitro of rabbit preimplantation embryos. J. Reprod. Fertil. 48:33-41 (1976). 9. El-Bana, A.A. and Daniel, J.C. The effect of protein fractions from rabbit uterine fluids on embryo growth and uptake of nucleic acids and protein precursors. Fert. and Steril. 23:105-114 (1972). 10. Laster, D.B., Maurer, R.R. and Chenault, J.R. Uterine flushings on in vitro growth of bovine embryos. J. Anim. Sci. 47 (Suppl.l):373 -~ (1978). 11. Elsden, R.P., Hasler, J.F. and Seidel, G.E., Jr. Nonsurgical recovery of bovine eggs. Theriogenology $:523-532 (1976). 12. Reynard, J.P. and Heyman, Y. Variable development of superovulated bovine embryos between Day 6 and Day 12. Ann. Biol. Anim. Biochem. Biophys. B:1589-1598 (1979). 13. Canfield, R.W., Toole, R.J., Gwazdauskas, F.C., Vinson, W.E. and Whittier, W.D. In vitro development of bovine morulae in bovine serum albumin, normal steer serum and uterine flushings. Theriogenology =:561-568 (1986). 14. Rajamahendran, R., Canseco, R.S., Gwazdauskas, F.C. and Vinson, W.E. Observations on in vitro development of bovine morulae in Ham's F-10 and Dulbecco's phosphate buffered saline supplemented with normal steer serum. Theriogenology %:369-374 (1985). 15. Wright, R.W. Jr., Anderson, G.B., Cupps, P.T. and Drost, M. Successful culture -in vitro of bovine embryos to the blastocyst stage. Biol. Reprod. 4:157-162 (1976).

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Ann.

24. Greve, T., Callesen, H. and Hyttel, P. Plasma progesterone profiles and embryo quality in superovulated dairy cows. Theriogeneology ;a:23 (1984). 25. Lamothe, P. and Guay, P. Electrolytes of bovine intra-uterine secretions during infertility sine materia. Can. J. Comp. Med. :H:167-171 (1970). 26. Schultz, R.H., Fahning, M.L. and Graham, E.F. A chemical study of uterine fluids and blood serum of normal cows during the estrous cycle. J. Reprod, Fertil. a:355-367 (1971). 27. Bartol, F.F., Thatcher, W.F., Bazer, F.W., Kimball, F.A., Chenault, J.R., Wilcox, C.J. and Roberts, R.M. Effects of the estrous cycle and early pregnancy on bovine uterine, luteal, and follicular response. Biol. Reprod. z:756-776 (1981) 28. Newcomb, R. and Rowson, L.E.A. Conception rate after uterine i;ransferof cow eggs, in relation to synchronization of oestrus. Reprod. Fertil. B:517-524 (1975).

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29. Rowson, L.E.A., Lawson, R.A.S., Moore, R.M. and Baker, A.A. Egg transfer in the cow: synchronization requirements. J. Reprod. Fertil. a:427-431 (1972). 30. Schumacher, G.F.B. Humoral immune factors in the female reproductive tract and their changes during the cycle. &: Dhinsda, D.H. and Schumacher, G.F.B. (eds.). Immunological Aspects of Infertility and Fertility Regulation. Elsevier/North Holland, Inc., New York, 1980, pp. 93-142. 31. Gimenez, T., Fisher, S. and Henricks, D.M. Immunosuppressive activity associated with early pregnancy in the bovine. Biol. Reprod. a(Suppl.1): 178 abstr. (1984). 32. Canseco, R.S., Gwazdauskas, F.C., Rajamahendran, R. and Vinson, V.E. Effect of immunoglobulins on early bovine embryo development in vitro. J. Dairy Sci. 69(Suppl. 1): 237 abstr. (1986).

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