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Protein content and volume of early porcine blastocysts
Animal Reproduction Science, 5 (1982/1983) 207--212
207
Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands
PROTEIN CONTENT AND VOLUME OF EARLY PORCINE BLASTOCYSTS 1
RAYMOND W. WRIGHT, Jr., JEAN GRAMMER, KENNETH BONDIOLI, F R A N K KUZAN and A L F R E D MENINO, Jr.
Department of Animal Science, Washington State University, Pullman, WA 99164 (U.S.A.) 1Scientific Paper No. 5848. College o f Agriculture Research Center, Washington State University, Pullman. Project 0313. (Accepted 21 July 1982)
ABSTRACT Wright, R.W., Jr., Grammer, J., Bondioli, K., Kuzan, F. and Menino, A., Jr., 1983. Protein content and volume o f early porcine blastocysts. Anita. Reprod. Sci., 5: 207--212. Mean protein and volume o f 222 blastocysts collected on 6 to 9 days o f pregnancy were measured. E m b r y o protein differed (P < 0.05) for each day o f development studied. Protein content o f embryos doubled between days 6 and 7 and days 7 and 8 (1.2 ± 0.04, 2.0 ± 0.14, and 3.7 ± 0.2 , g , respectively). A dramatic increase from 3.7 ± 0.2 to 56.0 ± 3.4 , g was observed between days 8 and 9. Blastocyst volume increased (P < 0.05) from 0 . 5 6 ± 0 . 0 3 x 10 -~ mms to 1.11 ± 0.04 x 10 -~ m m 3 between days 6 and 7, and then increased 10-fold on day 8 and five-fold on day 9. Blastocyst volume was not correlated with protein for days o f development and females studied. Approximately 20% o f all blastocysts within a single female contained less protein than the average protein content of all embryos from the same uterus. The results indicate that day 6 o f development marks the onset o f an exponential increase in e m b r y o protein. Also, blastocyst volume is n o t correlated with blastocyst protein, suggesting that e m b r y o viability is difficult t o estimate by size alone. Further, approximately 20% o f the blastocysts collected from a single female m a y exhibit reduced viability, based on reduced protein content, as early as day 6 of development.
INTRODUCTION
Since pigs have a large number of ovulations with a fertilization rate of more than 90%, the potential for reproductive efficiency in this species is great. However, full reproductive efficiency in this polytocous species is seldom achieved because of a high degree of embryonic loss before day 25 of gestation (Lerner et al., 1957; Perry and Rowiands, 1962). The mechanisms affecting this early embryonic loss are n o t well understood. Few studies have examined the protein content of early porcine embryos with the assumption that embryo protein is an indicator of embryo
0378-432018310D00--00001503.00 © 1983 Elsevier Scientific Publishing Company
208
growth and viability. Wright et al. (1980) measured porcine embryo protein content from fertilization through day 9 of development and found that protein values increased only slightly before day 5 and exponentially from days 6 through 9. Similarly, Anderson (1978) found porcine blastocyst protein to increase exponentially from days 9 through 18 of development. The first dramatic rise in embryo protein was observed between days 6 and 9 of development, a time coincident with blastocyst expansion and "hatching" of the blastocyst from the surrounding zona pellucida (Wright et al., 1980). The objective of this study was to examine the volume and protein content of embryos within and among pigs from day 6 through 9 of development. Volume and protein content of individual embryos were compared within pigs as an indication of overall embryo viability. MATERIALS AND METHODS Twenty gilts were hand-mated, and their reproductive tracts were collected at slaughter 6 to 9 days after the onset of estrus (day 0). The uteri were flushed with phosphate buffered saline (PBS). Embryos were identified and evaluated for stage of development under a dissecting microscope, and only embryos that appeared morphologically normal were used. Overall embryo diameter (OD) was measured at 100X magnification with an ocular micrometer, with each micrometer graduation representing 6.6 nanometers. Two measurements of OD were taken perpendicular to each other, and each was considered a specific data point and used to calculate an OD mean for each embryo. Embryo volume was calculated by the formula for an ellipsoid, v = 4/3 ~rabc, where a = c, with the mean OD (Lindner and Wright, 1978). Embryos were washed twice in PBS and transferred to 12 × 75 mm disposable culture tubes containing 2 ml of distilled water. Embryos were frozen and thawed three times and sonicated for two 5-s pulses to ensure cell disruption. Embryos were held at --20°C before protein analysis. Total protein was determined by the Bio-Rad microassay method, a dye binding technique described by Bradford (1976). This method has been slightly modified for the microassay system as described by Bio-Rad Laboratories (Bull. 1069, Bio-Rad Laboratories, Richmond, CA 94804). Between 0.8 and 19 ~g ~ or T globulin protein (bovine, fraction II) served as the protein standard. Protein standards were run in triplicate, and a standard curve was constructed for each assay. All measurements were made with a Beckman, D.U. Spectrophotometer (Beckman Instruments, Inc., Fullerton, CA 92634) at a wavelength of 595 nm, and a linear regression line was calculated to estimate embryo protein. Data were analyzed statistically by analysis of variance. Significant differences among means were determined by Duncan's (1955) new multiple range test. Simple correlation coefficients were computed for each animal.
209
RESULTS
Mean protein c o n t e n t and volume for blastocysts collected from a single female for day 6 through 9 of development are presented in Table I. Protein c o n t e n t of e m b r y o s was different (P <~ 0 . 0 5 ) o n each day of development studied, b u t n o t different for blastocysts collected the same day from different females. E m b r y o protein approximately doubled between days 6 and 7 and days 7 and 8 (1.2 +- 0.04 to 2.0 + 0.14 to 3.7 -+ 0.2 pg, respectively). A dramatic increase from 3.7 -+ 0.2 to 56.0 +- 3.4 pg was observed between days 8 and 9 o f development. Mean blastocyst volume increased (P < 0.05) from 0.56 -+ 0.03 X 10 -2 mm 3 to 1.11 -+ 0.04 X 10 -2 mm 3 between days 6 and 7 of development. This rise was followed by a 10-fold increase to 11.56 -+ 0.6 × 10 -2 mm 3 at day 8 and a five-fold increase to 55.96 +- 1.67 × 10 -2 mm 3 at day 9. Volume did n o t differ between blastocysts collected on the same days from different females (Table I). Blastocyst volume was n o t correlated with protein in embryos collected from a single female on days 6 through 9 of development (Table II). T w o or three e m b r y o s contained less protein than other blastocysts colTABLE I
Protein c o n t e n t
and volume of porcine blastocysts from days 6 through 9 of
Animal
Days of
No. of
Protein (.g)
V o l u m e ( x 1 0 -2 m m 3)
no.
development
embryos
Mean ± SEM
Mean ± SEM
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
6 6 6 6 6 7 7 7 7 7 8 8 8 8 8 9 9 9 9 9
10 11 11 10 12 10 11 12 13 10 11 12 12 11 12 11 10 12 11 12
1.31 1.17 1.08 1.16 1.13 1.92 2.09 2.03 2.25 1.85 3.61 3.87 3.84 3.59 3.62 55.51 55.23 55.83 61.42 52.39
0.77 0.64 0.52 0.34 0.52 1.16 1.07 1.09 1.08 1.18 5.45 9.74 12.22 14.39 15.40 45.22 56.64 59.83 57.52 59.91
aJ~lMeans
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.11 a 0.07 a 0.09 a 0.08 a 0.06 a 0.31 b 0.29 b 0.32 b 0.33 b 0.34 b 0.50 c 0.52 c 0.44 c 0.49 c 0.42 c 7.62 d 7.79 d 7.99 d 7.45 d 7.79 d
in the same column without common
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
development
0.08 a 0.07 a 0.07 a 0.03 a 0.04 a 0.14 b 0.10 b 0.10 b 0.66 b 0.08 b 0.89 c 0.80 c 0.41 c 0.02 c 0.98 c 3.55 d 3.31 d 3.67 d 4.59 d 2.04 d
superscripts are different (P < 0.05).
210 TABLE II Correlation coefficients of porcine blastocyst volume on protein Animal no.
Days o f development
Individual females (r)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
6 6 6 6 6 7 7 7 7 7 8 8 8 8 8 9 9 9 9
20
9
0.51 0.18 0.17 --0.13 --0.40 0.58 --0.07 --0.20 ---0.03 --0.10 0.14 --0.26 0.09 0.27 ---0.13 --0.13 -0.01 -0.28 -0.06 0.16
90.
A
z
0.25
0.03
0.02
-0.08
A
A
A A C B
3..
A
Different females (r)
A B
~
60
A
sB A
A
w F0 n 0 30 w
A A
A 8
ANIMAL
B
B
8 A
NUMBER
Fig. 1. Protein content o f blastocysts collected from a single female on day 9 of development. Letters A, B and C represent one, two and three embryos, respectively. lected from the same female on days 6 through 9 of development. This obs e r v a t i o n w a s m o s t e v i d e n t o n d a y 9, w h e n b l a s t o c y s t p r o t e i n w a s i n c r e a s i n g e x p o n e n t i a l l y , b u t w a s a l s o e v i d e n t o n d a y s 6, 7 a n d 8 o f d e v e l o p m e n t ( F i g . 1).
211 DISCUSSION
The protein content of mouse embryos (Brinster, 1967) and of rat embryos (Schiffner and Spielmann, 1976) increases at the blastocyst stage, but not as dramatically as observed for porcine blastocysts. This exponential increase in blastocyst protein is consistent with a large increase in blastocyst volume and is supported by the findings of Lindner and Wright (1978). Anderson (1978) found that the length of the pig conceptus was highly correlated with embryo protein between days 9 and 18 of development. In this study, embryo protein was not correlated with volume in any of the days examined. The apparent differences between the two studies might be explained by the fact that blastocysts become filamentous on day 11 but remain spherical until this time (Anderson, 1978). In addition, Anderson (1978) measured length of the conceptus rather than blastocyst volume (OD) which was measured in this study. The lack of correlation between blastocyst protein, an indicator of cell growth, and voIume point to the difficulty of attempting to evaluate quality simply by appearance (Dorst et al., 1975). The large increase in blastocyst protein and volume from days 6 and 9 of development suggests a period of rapid embryonic development. This development increases exponentially from day 9 to 18 of development (Anderson, 1978); however, the onset of exponential growth begins on day 6 (Wright et al., 1980). This period coincides with the time of blastocyst expansion and escape from the zona peUucida ("hatching"). Culture studies have shown that a high degree of embyro loss occurs in the pig during "hatching". This observation suggests that blastocyst expansion and "hatching" may be critical periods for embryo survival. Anderson (1978) found a 17% reduction in the potential number of embryos during the first 18 days of gestation, and this loss was unrelated to uterine capacity. This report supported the magnitude of earlier estimates of embryonic loss in pigs (Perry and Rowlands, 1962; Vincent et al., 1976). Reduced protein values observed in blastocysts in this study suggest that some embryos may be declining in viability as early as day 6 of development. The percentage of blastocysts with reduced protein corresponded well to the estimated embryo loss previously reported. ACKNOWLEDGEMENTS
This work was supportedin part by Pacific N o ~ h w e s t S w i n e P r o d u c e ~ Grants 6 3 1 3 a n d 4313. REFERENCES Anderson, L.L., 1978. Growth, protein content and distribution of early pig embryos. Anat. Rec., 190: 143--148. Bradford, M.H., 1976. A rapid and sensitive method for the quantitation of mic~gram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248.
212 Brinster, R.L., 1967. Protein content of the mouse embryo during the first five days of development. J. Repro& Fertil., 13: 413--420. Drost, M., Anderston, G.B., Cupps, P.T., Horton, M.B., Warner, P.U. and Wright, R.W., Jr., 1975. A field study on embyro transfer in cattle J. Am. Vet. Med. Assoc., 166: 1176--1182.
Duncan, M.B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1--18. Lerner, E.H., Mayer, D.T. and Lasley, I.F., 1957. Early embryonic mortality in strained cross gilts. Missouri Agric. Exp. Stn. Res. Bull., 629: 1--15. Lindner, G.M. and Wright, R.W., Jr., 1978. Morphological aspects of the development of swine embryos in vitro. J. Anita. Sci., 46: 711--716. Perry, J.S. and Rowtands, I.W., 1962. Early pregnancy in the pig, J. Reprod. Fertil., 4: 175--188. Schiffner, J. and Spielmann, H., 1976. Fluorometric assay of the protein content of mouse and rat embryos during preimplantation development. J. Reprod. Fertll., 47: 145--147. Vincent, F., Wintenberger-Torres, S., Paquignon, M. and Du Masnil Du Buisson, F., 1976. Developpemant embryonnaire chez la truie au 17~me jour de hi gestation. Relation avec hi taille des comes uterines. J. Rech. Porc. France, 1: 185--191. Wright, R.W., Jr., Crammer, J.C., Bondioli, K.R., Kuzan, F.B. and Menino, A.R., Jr., 1980. Total protein and volume of porcine embryos. In: Western Section Proceedings 1980, Hilo, I-Iawafi. J. Anim. Sci., 31 (Suppl. 1): 201--204.
207
Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands
PROTEIN CONTENT AND VOLUME OF EARLY PORCINE BLASTOCYSTS 1
RAYMOND W. WRIGHT, Jr., JEAN GRAMMER, KENNETH BONDIOLI, F R A N K KUZAN and A L F R E D MENINO, Jr.
Department of Animal Science, Washington State University, Pullman, WA 99164 (U.S.A.) 1Scientific Paper No. 5848. College o f Agriculture Research Center, Washington State University, Pullman. Project 0313. (Accepted 21 July 1982)
ABSTRACT Wright, R.W., Jr., Grammer, J., Bondioli, K., Kuzan, F. and Menino, A., Jr., 1983. Protein content and volume o f early porcine blastocysts. Anita. Reprod. Sci., 5: 207--212. Mean protein and volume o f 222 blastocysts collected on 6 to 9 days o f pregnancy were measured. E m b r y o protein differed (P < 0.05) for each day o f development studied. Protein content o f embryos doubled between days 6 and 7 and days 7 and 8 (1.2 ± 0.04, 2.0 ± 0.14, and 3.7 ± 0.2 , g , respectively). A dramatic increase from 3.7 ± 0.2 to 56.0 ± 3.4 , g was observed between days 8 and 9. Blastocyst volume increased (P < 0.05) from 0 . 5 6 ± 0 . 0 3 x 10 -~ mms to 1.11 ± 0.04 x 10 -~ m m 3 between days 6 and 7, and then increased 10-fold on day 8 and five-fold on day 9. Blastocyst volume was not correlated with protein for days o f development and females studied. Approximately 20% o f all blastocysts within a single female contained less protein than the average protein content of all embryos from the same uterus. The results indicate that day 6 o f development marks the onset o f an exponential increase in e m b r y o protein. Also, blastocyst volume is n o t correlated with blastocyst protein, suggesting that e m b r y o viability is difficult t o estimate by size alone. Further, approximately 20% o f the blastocysts collected from a single female m a y exhibit reduced viability, based on reduced protein content, as early as day 6 of development.
INTRODUCTION
Since pigs have a large number of ovulations with a fertilization rate of more than 90%, the potential for reproductive efficiency in this species is great. However, full reproductive efficiency in this polytocous species is seldom achieved because of a high degree of embryonic loss before day 25 of gestation (Lerner et al., 1957; Perry and Rowiands, 1962). The mechanisms affecting this early embryonic loss are n o t well understood. Few studies have examined the protein content of early porcine embryos with the assumption that embryo protein is an indicator of embryo
0378-432018310D00--00001503.00 © 1983 Elsevier Scientific Publishing Company
208
growth and viability. Wright et al. (1980) measured porcine embryo protein content from fertilization through day 9 of development and found that protein values increased only slightly before day 5 and exponentially from days 6 through 9. Similarly, Anderson (1978) found porcine blastocyst protein to increase exponentially from days 9 through 18 of development. The first dramatic rise in embryo protein was observed between days 6 and 9 of development, a time coincident with blastocyst expansion and "hatching" of the blastocyst from the surrounding zona pellucida (Wright et al., 1980). The objective of this study was to examine the volume and protein content of embryos within and among pigs from day 6 through 9 of development. Volume and protein content of individual embryos were compared within pigs as an indication of overall embryo viability. MATERIALS AND METHODS Twenty gilts were hand-mated, and their reproductive tracts were collected at slaughter 6 to 9 days after the onset of estrus (day 0). The uteri were flushed with phosphate buffered saline (PBS). Embryos were identified and evaluated for stage of development under a dissecting microscope, and only embryos that appeared morphologically normal were used. Overall embryo diameter (OD) was measured at 100X magnification with an ocular micrometer, with each micrometer graduation representing 6.6 nanometers. Two measurements of OD were taken perpendicular to each other, and each was considered a specific data point and used to calculate an OD mean for each embryo. Embryo volume was calculated by the formula for an ellipsoid, v = 4/3 ~rabc, where a = c, with the mean OD (Lindner and Wright, 1978). Embryos were washed twice in PBS and transferred to 12 × 75 mm disposable culture tubes containing 2 ml of distilled water. Embryos were frozen and thawed three times and sonicated for two 5-s pulses to ensure cell disruption. Embryos were held at --20°C before protein analysis. Total protein was determined by the Bio-Rad microassay method, a dye binding technique described by Bradford (1976). This method has been slightly modified for the microassay system as described by Bio-Rad Laboratories (Bull. 1069, Bio-Rad Laboratories, Richmond, CA 94804). Between 0.8 and 19 ~g ~ or T globulin protein (bovine, fraction II) served as the protein standard. Protein standards were run in triplicate, and a standard curve was constructed for each assay. All measurements were made with a Beckman, D.U. Spectrophotometer (Beckman Instruments, Inc., Fullerton, CA 92634) at a wavelength of 595 nm, and a linear regression line was calculated to estimate embryo protein. Data were analyzed statistically by analysis of variance. Significant differences among means were determined by Duncan's (1955) new multiple range test. Simple correlation coefficients were computed for each animal.
209
RESULTS
Mean protein c o n t e n t and volume for blastocysts collected from a single female for day 6 through 9 of development are presented in Table I. Protein c o n t e n t of e m b r y o s was different (P <~ 0 . 0 5 ) o n each day of development studied, b u t n o t different for blastocysts collected the same day from different females. E m b r y o protein approximately doubled between days 6 and 7 and days 7 and 8 (1.2 +- 0.04 to 2.0 + 0.14 to 3.7 -+ 0.2 pg, respectively). A dramatic increase from 3.7 -+ 0.2 to 56.0 +- 3.4 pg was observed between days 8 and 9 o f development. Mean blastocyst volume increased (P < 0.05) from 0.56 -+ 0.03 X 10 -2 mm 3 to 1.11 -+ 0.04 X 10 -2 mm 3 between days 6 and 7 of development. This rise was followed by a 10-fold increase to 11.56 -+ 0.6 × 10 -2 mm 3 at day 8 and a five-fold increase to 55.96 +- 1.67 × 10 -2 mm 3 at day 9. Volume did n o t differ between blastocysts collected on the same days from different females (Table I). Blastocyst volume was n o t correlated with protein in embryos collected from a single female on days 6 through 9 of development (Table II). T w o or three e m b r y o s contained less protein than other blastocysts colTABLE I
Protein c o n t e n t
and volume of porcine blastocysts from days 6 through 9 of
Animal
Days of
No. of
Protein (.g)
V o l u m e ( x 1 0 -2 m m 3)
no.
development
embryos
Mean ± SEM
Mean ± SEM
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
6 6 6 6 6 7 7 7 7 7 8 8 8 8 8 9 9 9 9 9
10 11 11 10 12 10 11 12 13 10 11 12 12 11 12 11 10 12 11 12
1.31 1.17 1.08 1.16 1.13 1.92 2.09 2.03 2.25 1.85 3.61 3.87 3.84 3.59 3.62 55.51 55.23 55.83 61.42 52.39
0.77 0.64 0.52 0.34 0.52 1.16 1.07 1.09 1.08 1.18 5.45 9.74 12.22 14.39 15.40 45.22 56.64 59.83 57.52 59.91
aJ~lMeans
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.11 a 0.07 a 0.09 a 0.08 a 0.06 a 0.31 b 0.29 b 0.32 b 0.33 b 0.34 b 0.50 c 0.52 c 0.44 c 0.49 c 0.42 c 7.62 d 7.79 d 7.99 d 7.45 d 7.79 d
in the same column without common
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
development
0.08 a 0.07 a 0.07 a 0.03 a 0.04 a 0.14 b 0.10 b 0.10 b 0.66 b 0.08 b 0.89 c 0.80 c 0.41 c 0.02 c 0.98 c 3.55 d 3.31 d 3.67 d 4.59 d 2.04 d
superscripts are different (P < 0.05).
210 TABLE II Correlation coefficients of porcine blastocyst volume on protein Animal no.
Days o f development
Individual females (r)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
6 6 6 6 6 7 7 7 7 7 8 8 8 8 8 9 9 9 9
20
9
0.51 0.18 0.17 --0.13 --0.40 0.58 --0.07 --0.20 ---0.03 --0.10 0.14 --0.26 0.09 0.27 ---0.13 --0.13 -0.01 -0.28 -0.06 0.16
90.
A
z
0.25
0.03
0.02
-0.08
A
A
A A C B
3..
A
Different females (r)
A B
~
60
A
sB A
A
w F0 n 0 30 w
A A
A 8
ANIMAL
B
B
8 A
NUMBER
Fig. 1. Protein content o f blastocysts collected from a single female on day 9 of development. Letters A, B and C represent one, two and three embryos, respectively. lected from the same female on days 6 through 9 of development. This obs e r v a t i o n w a s m o s t e v i d e n t o n d a y 9, w h e n b l a s t o c y s t p r o t e i n w a s i n c r e a s i n g e x p o n e n t i a l l y , b u t w a s a l s o e v i d e n t o n d a y s 6, 7 a n d 8 o f d e v e l o p m e n t ( F i g . 1).
211 DISCUSSION
The protein content of mouse embryos (Brinster, 1967) and of rat embryos (Schiffner and Spielmann, 1976) increases at the blastocyst stage, but not as dramatically as observed for porcine blastocysts. This exponential increase in blastocyst protein is consistent with a large increase in blastocyst volume and is supported by the findings of Lindner and Wright (1978). Anderson (1978) found that the length of the pig conceptus was highly correlated with embryo protein between days 9 and 18 of development. In this study, embryo protein was not correlated with volume in any of the days examined. The apparent differences between the two studies might be explained by the fact that blastocysts become filamentous on day 11 but remain spherical until this time (Anderson, 1978). In addition, Anderson (1978) measured length of the conceptus rather than blastocyst volume (OD) which was measured in this study. The lack of correlation between blastocyst protein, an indicator of cell growth, and voIume point to the difficulty of attempting to evaluate quality simply by appearance (Dorst et al., 1975). The large increase in blastocyst protein and volume from days 6 and 9 of development suggests a period of rapid embryonic development. This development increases exponentially from day 9 to 18 of development (Anderson, 1978); however, the onset of exponential growth begins on day 6 (Wright et al., 1980). This period coincides with the time of blastocyst expansion and escape from the zona peUucida ("hatching"). Culture studies have shown that a high degree of embyro loss occurs in the pig during "hatching". This observation suggests that blastocyst expansion and "hatching" may be critical periods for embryo survival. Anderson (1978) found a 17% reduction in the potential number of embryos during the first 18 days of gestation, and this loss was unrelated to uterine capacity. This report supported the magnitude of earlier estimates of embryonic loss in pigs (Perry and Rowlands, 1962; Vincent et al., 1976). Reduced protein values observed in blastocysts in this study suggest that some embryos may be declining in viability as early as day 6 of development. The percentage of blastocysts with reduced protein corresponded well to the estimated embryo loss previously reported. ACKNOWLEDGEMENTS
This work was supportedin part by Pacific N o ~ h w e s t S w i n e P r o d u c e ~ Grants 6 3 1 3 a n d 4313. REFERENCES Anderson, L.L., 1978. Growth, protein content and distribution of early pig embryos. Anat. Rec., 190: 143--148. Bradford, M.H., 1976. A rapid and sensitive method for the quantitation of mic~gram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248.
212 Brinster, R.L., 1967. Protein content of the mouse embryo during the first five days of development. J. Repro& Fertil., 13: 413--420. Drost, M., Anderston, G.B., Cupps, P.T., Horton, M.B., Warner, P.U. and Wright, R.W., Jr., 1975. A field study on embyro transfer in cattle J. Am. Vet. Med. Assoc., 166: 1176--1182.
Duncan, M.B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1--18. Lerner, E.H., Mayer, D.T. and Lasley, I.F., 1957. Early embryonic mortality in strained cross gilts. Missouri Agric. Exp. Stn. Res. Bull., 629: 1--15. Lindner, G.M. and Wright, R.W., Jr., 1978. Morphological aspects of the development of swine embryos in vitro. J. Anita. Sci., 46: 711--716. Perry, J.S. and Rowtands, I.W., 1962. Early pregnancy in the pig, J. Reprod. Fertil., 4: 175--188. Schiffner, J. and Spielmann, H., 1976. Fluorometric assay of the protein content of mouse and rat embryos during preimplantation development. J. Reprod. Fertll., 47: 145--147. Vincent, F., Wintenberger-Torres, S., Paquignon, M. and Du Masnil Du Buisson, F., 1976. Developpemant embryonnaire chez la truie au 17~me jour de hi gestation. Relation avec hi taille des comes uterines. J. Rech. Porc. France, 1: 185--191. Wright, R.W., Jr., Crammer, J.C., Bondioli, K.R., Kuzan, F.B. and Menino, A.R., Jr., 1980. Total protein and volume of porcine embryos. In: Western Section Proceedings 1980, Hilo, I-Iawafi. J. Anim. Sci., 31 (Suppl. 1): 201--204.