A S S O C I A T I O N BETWEEN E N D O M E T R I A L P H O S P H A T A S E A C T I V I T Y A N D F E R T I L I T Y IN BEEF C A T T L E 1 B. V. Able 2 , R. H. Dutt, F. A. Thrift and N. W. Bradley
University o f Kentucky, Lexington 40506
Reproductive performance and endometrial enzyme activity of 56 beef females, consisting of 48 cows of various ages that failed to conceive during a normal breeding season (will be referred to as subfertile) and eight nonparous yearling heifers, were studied. All females were checked twice daily for estrous, bred naturally at the second estrous during the experiment and slaughtered 3 days following breeding. Tissue samples were taken and analyzed for endometrial protein, alkaline and acid phosphatase. Ova were collected and examined. Similar tissues were collected from cows not showing estrus at the end of the 60-day test period. All females exhibiting estrus had ovulated. However, seven of the subfertile cows and one yearling heifer failed to exhibit estrous during the test period. Ova were recovered from 70.7% of the subfertile cows and from 85.7% of the yearling heifers that had ovulated, and ovum fertilization rates in the two groups were 65.5 and 50.5%, respectively. Mean endometrial acid phosphatase levels were 2.07 and 1.99 units per 10 mg of tissue per hour in the subfertile cows and yearling heifers, respectively. Mean endometrial alkaline phosphatase level of yearling heifers was significantly less than that of subfertile cows (15.2 vs 20.6 units per 2.5 mg tissue per hour). Significant negative correlations between endometrial protein and both alkaline and acid phosphatase ( - . 3 3 and - . 4 6 , respectively) were found in the subfertile cows. No significant correlations were found among these traits in the yearling heifers. Significant negative correlations between both alkaline ( - . 3 6 ) and acid pbosphatase ( - . 4 6 )
and endometrial protein occurred in cycling cows. There were no significant correlations among these traits in the non-cycling females. Cows with unfertilized ova had a significant negative correlation ( - . 5 6 ) between endometrial alkaline phosphatase and endometrial protein. In cows with fertilized ova, alkaline and acid phosphatase were both negatively related ( - . 6 0 and - . 4 2 , respectively) to endometrial protein. (Key Words: Endometrium, Fertility, Phosphatase, Beef Cattle, Reproduction.)
INTRODUCTION
All factors which affect fertilization of mammalian ova are not fully understood. Ovarian hormones are responsible for uterine development and muscular activity (Asdell, 1955); they also influence enzyme systems present in the uterus (Murdoch and White, 1969). Ovarian hormones stimulate blood flow, cause cellular changes and stimulate the enzyme and coenzyme systems responsible for energy production (Hafez, 1964). The endometrium metabolizes carbohydrates, lipids, a n d proteins required for the nutrition of the implanting blastocyst and the production of energy for the rapid proliferation of the uterine tissues (Hafez, 1964). Alkaline and acid phosphatase are the most frequently studied enzymes with respect to enzymatic systems of the uterus. It has been suggested that alkaline phosphatase plays an important role in placental transfer mechanisms (Bradfield, 1950; Borghese, 1957), may be associated with transfer of solutes across membranes (Moog, 1946, 1959; Manning et al., 1966), and is involved in hydrolytic functions 1Published with approval of the Director of the (Fuhrmann, 1963). Acid phosphatase has been Kentucky Agricultural Experiment Station as Journal found in high concentrations at the time of Article 73-5-102. fertilization and implantation of the ovum 2present address: Department of Animal Science (Fuhrmann, 1963). DeDuve (1959) and Lobel and Industry, Kansas State University, Manhattan 66506. et al. (1961) implicate acid phosphatase in 40O JOURNAL OF ANIMAL SCIENCE, Vol. 42, No. 2, 1976
Downloaded from https://academic.oup.com/jas/article-abstract/42/2/400/4699483 by 04860000 user on 12 January 2019
SUMMARY
ENDOMETRIAL PHOSPHATASE AND FERTILITY
~lts. The experiment reported herein was conducted to measure amounts of endometrial phosphatase activity and to determine whether these levels are related to fertilization in beef COWS.
MATERIALS AND METHODS
Fifty-six bovine females consisting of 46 Hereford, seven Shorthorn, and three Angus, were used in this experiment. Forty-eight of the females ranging in age from two to 15 years had been culled from the University herds because they were found to be open after the breeding season. Hereafter these will be referred to as subfertile cows. The remaining eight females were yearling Hereford heifers included for comparison purposes. At the start of the study, rectal palpation revealed all females had normal reproductive tracts. Females were checked twice daily for estrus during a 60-day experimental period with an aproned bull to establish whether they were cycling. Upon second estrus within the 60-day period the females were bred naturally to fertility tested males and slaughtered 3 days later. Females not showing estrus were slaughtered at the end of the 60-day experimental period. The complete reproductive tract of each female was removed and immediately taken to the laboratory for examination of any anatomical abnormalities. A 25-mm cross section of the uterine horn (taken at the internal junction of the uterine horns) was excised and frozen at - 4 0 C for later determination of enzyme activity and protein content. Ovulation was determined by the presence of newly-formed corpora lutea. The Fallopian tube corresponding to the side of ovulation was dissected from the tract. The contents of the excised tube were flushed with approximately 5 ml .85% saline into a watch glass. Ova were located by scanning the flushing
under a dissecting microscope. Once located they were stained and transferred to a hanging drop slide and examined at 4 3 0 x . Cleavage or the presence of sperm in the zona pellucida were considered to be evidence of fertilization. The frozen uterine sections were thawed and pinned to a dissecting pan where the endometrium was removed by careful dissection with a razor blade. A 750-rag sample of the endometrium was placed in 15 ml of cold (5 C) .85% saline and homogenized in a Virtis " 4 5 " homogenizer for exactly 1 minute. The blender cup was kept submerged in ice water during the homogenizing process. Immediately after homogenization, the samples were placed in a refrigerated centrifuge for 1 min to lightly pack the cellular debris. The resulting endometrial homogenate was quickly refrozen at - 7 9 C in dry ice and alcohol and stored at - 4 0 C until analyzed for enzyme activity. Phosphatase activity of the endometrial homogenates was determined by the method described by Sigma Chemical Company (1963). Total protein of the endometrium was estimated by turbidity measurements of trichloroacetic acid precipitates of endometrial homogenates as outlined by Stadtman e t al. (1951). Levels of enzyme activity were calculated and will be reported as Sigma Units per 10 mg of tissue per hour for acid phosphatase and per 2.5 mg tissue per hour for alkaline phosphatase. Differences in the means of the various group comparisons were tested for significance by use of Cochran's approximation for testing group comparisons, as described by Snedecor and Cochran (1956). The procedure for analyzing the correlation between the various traits and the significance of these correlations were also taken from Snedecor and Cochran (1956). RESULTS A N D DISCUSSION
Average length of the estrous cycle for the subfertile cows and yearling heifers, when those females that did not exhibit estrus and those having only one estrus were excluded, was 21 and 22 days, respectively. Normal estrous cycle length for beef cows is reported to be 21 days (Wagnon et al., 1967), while 20 days is considered normal for yearling heifers (Snapp and Newmann, 1960). Estrus was not detected during the 60-day experimental period in one yearling heifer and in seven of the subfertile cows. Eleven subfertile cows (23%) and two yearling heifers (25%) expressed estrus only
Downloaded from https://academic.oup.com/jas/article-abstract/42/2/400/4699483 by 04860000 user on 12 January 2019
autolytic or regressive processes as a result of the presence of this enzyme at uterine sites where these processes are occurring. Bredeck and Mayer (1955) found acid phosphatase content of the rat uterus was significantly positively correlated with number of implantation sites and number of viable embryos. Goode et al. (1965) found a significant negative correlation between the level of endometrial alkaline phosphatase and number of viable embryos at the 25th day of gestation in
401
ABLE ET AL.
402
TABLE 1. OVULATION RATE, PERCENT OF OVA RECOVERED AND MORPHOLOGY OF OVA FROM SUBFERTILE COWS AND YEARLING HEIFERS Subfertile cows
Yearling heifers
Number
Percent
Number
Percent
Number of females Females showing estrus Females ovulating Ova recovered Ova fertilized Abnormal ova Ova unaccounted for
48 41 41 29 19 7 12
8514 85.4 70.7 a 65.5 a 24.1 a 29.3 a
8 7 7 6 3 2 1
8715 87.5 85.7 a 50.0 a 33.3 a 14.3 a
aBased on the~total number of ovulations in respective groups.
once d u r i n g t h e 60-day t e s t period. All cows a n d yearling heifers e x h i b i t i n g estrus d u r i n g t h e t e s t o v u l a t e d as e v i d e n c e d b y t h e p r e s e n c e o f n e w l y - f o r m e d c o r p o r a l u t e a at t i m e of slaughter. However, o f 41 cows t h a t ovulated, ova were n o t r e c o v e r e d f r o m 12 (29.3%). Also, n o o v u m was f o u n d in t h e t r a c t of o n e y e a r l i n g h e i f e r (14.3%) w h i c h o v u l a t e d ( t a b l e 1). G r a d e n e t al. ( 1 9 6 8 ) r e p o r t e d ova were n o t r e c o v e r e d f r o m 17.3% o f a g r o u p o f dairy cows, even t h o u g h n o a b n o r m a l i t i e s were a p p a r e n t t o a c c o u n t for t h e i r absence. T h e f i m b r i a was a d h e r e d t o t h e ovary in f o u r of t h e 12 cows while n o a d h e s i o n s were f o u n d in t h e yearling heifers. T h e significant ( P < . 0 1 ) difference b e t w e e n cows a n d yearling heifers (29.3 vs 14.3%) in n o n r e c o v e r e d ova m a y a c c o u n t f o r p a r t o f t h e failure o f t h e cows to c o n c e i v e
d u r i n g t h e b r e e d i n g season. Fertilized ova were r e c o v e r e d f r o m 19 o f 4 8 (39.4%) subfertile cows a n d t h r e e o f eight (37.5%) yearling heifers. However, o f t h e 29 ova r e c o v e r e d in t h e s u b f e r t i l e c o w g r o u p 6 5 . 5 % (19 of 29) were fertilized. Yearling heifers h a d t h r e e of six (50%) r e c o v e r e d ova classified as fertilized. G r a d e n e t al. ( 1 9 6 8 ) f o u n d 55.8% of t h e ova r e c o v e r e d f r o m r e p e a t b r e e d e r dairy cows a n d heifers t o b e fertilized. Acid p h 0 s p h a t a s e activity of t h e e n d o m e t r i u m f r o m individual cows r a n g e d f r o m .92 t o 4.25 Sigma Units p e r 10 m g tissue p e r h o u r . T h e m e a n s for e n d o m e t r i a l acid p h o s p h a t a s e activity for t h e s u b f e r t i l e cows a n d yearling heifers were 2.07 a n d 1.99 units, respectively ( t a b l e 2). T h e d i f f e r e n c e s b e t w e e n t h e m e a n e n z y m e activity o f t h e s e g r o u p s was n o t signifi-
TABLE 2. ALKALINE AND ACID PHOSPHATASE ACTIVITY IN ENDOMETRIAL TISSUE OF SUBFERTILE COWS AND YEARLING HEIFERS WHEN GROUPED ACCORDING TO ESTRUS ACTIVITY AND FERTILITY STATUS Phosphatase activity Akaline a
Acid b
Female group
Mean
SD
Mean
SD
Subfertile cows Yearling heifers
19.9" 15.2
8;3 5.0
2;-8 1.99
;81 1.12
Cycling females Non-cycling females
19.36 18.6
7.0 7.5
2.09 1.70
.72 .39
Fertilized ova Unfertilized ova
20.1 20.3
6.1 9.3
2.12 1.91
.64 .70
asigma Units per 2.5 mg tissue per hour. bsigma Units per 10 mg tissue per hour. * Significant (P
University o f Kentucky, Lexington 40506
Reproductive performance and endometrial enzyme activity of 56 beef females, consisting of 48 cows of various ages that failed to conceive during a normal breeding season (will be referred to as subfertile) and eight nonparous yearling heifers, were studied. All females were checked twice daily for estrous, bred naturally at the second estrous during the experiment and slaughtered 3 days following breeding. Tissue samples were taken and analyzed for endometrial protein, alkaline and acid phosphatase. Ova were collected and examined. Similar tissues were collected from cows not showing estrus at the end of the 60-day test period. All females exhibiting estrus had ovulated. However, seven of the subfertile cows and one yearling heifer failed to exhibit estrous during the test period. Ova were recovered from 70.7% of the subfertile cows and from 85.7% of the yearling heifers that had ovulated, and ovum fertilization rates in the two groups were 65.5 and 50.5%, respectively. Mean endometrial acid phosphatase levels were 2.07 and 1.99 units per 10 mg of tissue per hour in the subfertile cows and yearling heifers, respectively. Mean endometrial alkaline phosphatase level of yearling heifers was significantly less than that of subfertile cows (15.2 vs 20.6 units per 2.5 mg tissue per hour). Significant negative correlations between endometrial protein and both alkaline and acid phosphatase ( - . 3 3 and - . 4 6 , respectively) were found in the subfertile cows. No significant correlations were found among these traits in the yearling heifers. Significant negative correlations between both alkaline ( - . 3 6 ) and acid pbosphatase ( - . 4 6 )
and endometrial protein occurred in cycling cows. There were no significant correlations among these traits in the non-cycling females. Cows with unfertilized ova had a significant negative correlation ( - . 5 6 ) between endometrial alkaline phosphatase and endometrial protein. In cows with fertilized ova, alkaline and acid phosphatase were both negatively related ( - . 6 0 and - . 4 2 , respectively) to endometrial protein. (Key Words: Endometrium, Fertility, Phosphatase, Beef Cattle, Reproduction.)
INTRODUCTION
All factors which affect fertilization of mammalian ova are not fully understood. Ovarian hormones are responsible for uterine development and muscular activity (Asdell, 1955); they also influence enzyme systems present in the uterus (Murdoch and White, 1969). Ovarian hormones stimulate blood flow, cause cellular changes and stimulate the enzyme and coenzyme systems responsible for energy production (Hafez, 1964). The endometrium metabolizes carbohydrates, lipids, a n d proteins required for the nutrition of the implanting blastocyst and the production of energy for the rapid proliferation of the uterine tissues (Hafez, 1964). Alkaline and acid phosphatase are the most frequently studied enzymes with respect to enzymatic systems of the uterus. It has been suggested that alkaline phosphatase plays an important role in placental transfer mechanisms (Bradfield, 1950; Borghese, 1957), may be associated with transfer of solutes across membranes (Moog, 1946, 1959; Manning et al., 1966), and is involved in hydrolytic functions 1Published with approval of the Director of the (Fuhrmann, 1963). Acid phosphatase has been Kentucky Agricultural Experiment Station as Journal found in high concentrations at the time of Article 73-5-102. fertilization and implantation of the ovum 2present address: Department of Animal Science (Fuhrmann, 1963). DeDuve (1959) and Lobel and Industry, Kansas State University, Manhattan 66506. et al. (1961) implicate acid phosphatase in 40O JOURNAL OF ANIMAL SCIENCE, Vol. 42, No. 2, 1976
Downloaded from https://academic.oup.com/jas/article-abstract/42/2/400/4699483 by 04860000 user on 12 January 2019
SUMMARY
ENDOMETRIAL PHOSPHATASE AND FERTILITY
~lts. The experiment reported herein was conducted to measure amounts of endometrial phosphatase activity and to determine whether these levels are related to fertilization in beef COWS.
MATERIALS AND METHODS
Fifty-six bovine females consisting of 46 Hereford, seven Shorthorn, and three Angus, were used in this experiment. Forty-eight of the females ranging in age from two to 15 years had been culled from the University herds because they were found to be open after the breeding season. Hereafter these will be referred to as subfertile cows. The remaining eight females were yearling Hereford heifers included for comparison purposes. At the start of the study, rectal palpation revealed all females had normal reproductive tracts. Females were checked twice daily for estrus during a 60-day experimental period with an aproned bull to establish whether they were cycling. Upon second estrus within the 60-day period the females were bred naturally to fertility tested males and slaughtered 3 days later. Females not showing estrus were slaughtered at the end of the 60-day experimental period. The complete reproductive tract of each female was removed and immediately taken to the laboratory for examination of any anatomical abnormalities. A 25-mm cross section of the uterine horn (taken at the internal junction of the uterine horns) was excised and frozen at - 4 0 C for later determination of enzyme activity and protein content. Ovulation was determined by the presence of newly-formed corpora lutea. The Fallopian tube corresponding to the side of ovulation was dissected from the tract. The contents of the excised tube were flushed with approximately 5 ml .85% saline into a watch glass. Ova were located by scanning the flushing
under a dissecting microscope. Once located they were stained and transferred to a hanging drop slide and examined at 4 3 0 x . Cleavage or the presence of sperm in the zona pellucida were considered to be evidence of fertilization. The frozen uterine sections were thawed and pinned to a dissecting pan where the endometrium was removed by careful dissection with a razor blade. A 750-rag sample of the endometrium was placed in 15 ml of cold (5 C) .85% saline and homogenized in a Virtis " 4 5 " homogenizer for exactly 1 minute. The blender cup was kept submerged in ice water during the homogenizing process. Immediately after homogenization, the samples were placed in a refrigerated centrifuge for 1 min to lightly pack the cellular debris. The resulting endometrial homogenate was quickly refrozen at - 7 9 C in dry ice and alcohol and stored at - 4 0 C until analyzed for enzyme activity. Phosphatase activity of the endometrial homogenates was determined by the method described by Sigma Chemical Company (1963). Total protein of the endometrium was estimated by turbidity measurements of trichloroacetic acid precipitates of endometrial homogenates as outlined by Stadtman e t al. (1951). Levels of enzyme activity were calculated and will be reported as Sigma Units per 10 mg of tissue per hour for acid phosphatase and per 2.5 mg tissue per hour for alkaline phosphatase. Differences in the means of the various group comparisons were tested for significance by use of Cochran's approximation for testing group comparisons, as described by Snedecor and Cochran (1956). The procedure for analyzing the correlation between the various traits and the significance of these correlations were also taken from Snedecor and Cochran (1956). RESULTS A N D DISCUSSION
Average length of the estrous cycle for the subfertile cows and yearling heifers, when those females that did not exhibit estrus and those having only one estrus were excluded, was 21 and 22 days, respectively. Normal estrous cycle length for beef cows is reported to be 21 days (Wagnon et al., 1967), while 20 days is considered normal for yearling heifers (Snapp and Newmann, 1960). Estrus was not detected during the 60-day experimental period in one yearling heifer and in seven of the subfertile cows. Eleven subfertile cows (23%) and two yearling heifers (25%) expressed estrus only
Downloaded from https://academic.oup.com/jas/article-abstract/42/2/400/4699483 by 04860000 user on 12 January 2019
autolytic or regressive processes as a result of the presence of this enzyme at uterine sites where these processes are occurring. Bredeck and Mayer (1955) found acid phosphatase content of the rat uterus was significantly positively correlated with number of implantation sites and number of viable embryos. Goode et al. (1965) found a significant negative correlation between the level of endometrial alkaline phosphatase and number of viable embryos at the 25th day of gestation in
401
ABLE ET AL.
402
TABLE 1. OVULATION RATE, PERCENT OF OVA RECOVERED AND MORPHOLOGY OF OVA FROM SUBFERTILE COWS AND YEARLING HEIFERS Subfertile cows
Yearling heifers
Number
Percent
Number
Percent
Number of females Females showing estrus Females ovulating Ova recovered Ova fertilized Abnormal ova Ova unaccounted for
48 41 41 29 19 7 12
8514 85.4 70.7 a 65.5 a 24.1 a 29.3 a
8 7 7 6 3 2 1
8715 87.5 85.7 a 50.0 a 33.3 a 14.3 a
aBased on the~total number of ovulations in respective groups.
once d u r i n g t h e 60-day t e s t period. All cows a n d yearling heifers e x h i b i t i n g estrus d u r i n g t h e t e s t o v u l a t e d as e v i d e n c e d b y t h e p r e s e n c e o f n e w l y - f o r m e d c o r p o r a l u t e a at t i m e of slaughter. However, o f 41 cows t h a t ovulated, ova were n o t r e c o v e r e d f r o m 12 (29.3%). Also, n o o v u m was f o u n d in t h e t r a c t of o n e y e a r l i n g h e i f e r (14.3%) w h i c h o v u l a t e d ( t a b l e 1). G r a d e n e t al. ( 1 9 6 8 ) r e p o r t e d ova were n o t r e c o v e r e d f r o m 17.3% o f a g r o u p o f dairy cows, even t h o u g h n o a b n o r m a l i t i e s were a p p a r e n t t o a c c o u n t for t h e i r absence. T h e f i m b r i a was a d h e r e d t o t h e ovary in f o u r of t h e 12 cows while n o a d h e s i o n s were f o u n d in t h e yearling heifers. T h e significant ( P < . 0 1 ) difference b e t w e e n cows a n d yearling heifers (29.3 vs 14.3%) in n o n r e c o v e r e d ova m a y a c c o u n t f o r p a r t o f t h e failure o f t h e cows to c o n c e i v e
d u r i n g t h e b r e e d i n g season. Fertilized ova were r e c o v e r e d f r o m 19 o f 4 8 (39.4%) subfertile cows a n d t h r e e o f eight (37.5%) yearling heifers. However, o f t h e 29 ova r e c o v e r e d in t h e s u b f e r t i l e c o w g r o u p 6 5 . 5 % (19 of 29) were fertilized. Yearling heifers h a d t h r e e of six (50%) r e c o v e r e d ova classified as fertilized. G r a d e n e t al. ( 1 9 6 8 ) f o u n d 55.8% of t h e ova r e c o v e r e d f r o m r e p e a t b r e e d e r dairy cows a n d heifers t o b e fertilized. Acid p h 0 s p h a t a s e activity of t h e e n d o m e t r i u m f r o m individual cows r a n g e d f r o m .92 t o 4.25 Sigma Units p e r 10 m g tissue p e r h o u r . T h e m e a n s for e n d o m e t r i a l acid p h o s p h a t a s e activity for t h e s u b f e r t i l e cows a n d yearling heifers were 2.07 a n d 1.99 units, respectively ( t a b l e 2). T h e d i f f e r e n c e s b e t w e e n t h e m e a n e n z y m e activity o f t h e s e g r o u p s was n o t signifi-
TABLE 2. ALKALINE AND ACID PHOSPHATASE ACTIVITY IN ENDOMETRIAL TISSUE OF SUBFERTILE COWS AND YEARLING HEIFERS WHEN GROUPED ACCORDING TO ESTRUS ACTIVITY AND FERTILITY STATUS Phosphatase activity Akaline a
Acid b
Female group
Mean
SD
Mean
SD
Subfertile cows Yearling heifers
19.9" 15.2
8;3 5.0
2;-8 1.99
;81 1.12
Cycling females Non-cycling females
19.36 18.6
7.0 7.5
2.09 1.70
.72 .39
Fertilized ova Unfertilized ova
20.1 20.3
6.1 9.3
2.12 1.91
.64 .70
asigma Units per 2.5 mg tissue per hour. bsigma Units per 10 mg tissue per hour. * Significant (P
