Effects of standing estrus and supplemental estradiol on changes in uterine ph during a fixed-time artificial insemination protocol 1

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1 Published December 5, 2014 Effects of standing estrus and supplemental estradiol on changes in uterine ph during a fixed-time artificial insemination protocol 1 G. A. Perry 2 and B. L. Perry Department of Animal and Range Sciences, South Dakota State University, Brookings ABSTRACT: Cows that exhibit estrus within 24 h of fixed-time AI have elevated concentrations of estradiol and greater pregnancy rates compared with cows not in estrus. Our objective was to determine whether estradiol, estrus, or both had an effect on uterine ph during a fixed-time AI protocol. Beef cows were treated with the CO-Synch protocol (100 µg of GnRH on d 9; 25 mg of PGF 2α on d 2; and 100 µg of GnRH on d 0). One-half of the cows received an injection of estradiol cypionate (ECP; 1 mg) 12 h after PGF 2α. Cows detected in standing estrus within 24 h of the second GnRH injection were considered to be in standing estrus. Uterine ph was determined in all animals 12, 24, and 48 h after the PGF 2α injection. For Exp. 1, ph was also determined 72 and 96 h after the PGF 2α injection; in Exp. 2, ph was also determined at 54, 60, 66, 72, 78, 84, 90, and 96 h after the PGF 2α injection or until ovulation. A treatment time interaction (P < 0.01) influenced concentrations of estradiol. All cows had similar (P > 0.15) concentrations of estradiol at the time of ECP administration, but after ECP treatment all cows treated with ECP and control cows that exhibited estrus had greater (P < 0.01) concentrations of estradiol compared with nontreated cows that did not exhibit estrus. In all animals, estradiol diminished 48 h after the PGF 2α (time of the second GnRH injection), but ECP-treated cows, regardless of estrus, had elevated (P < 0.02) concentrations of estradiol compared with control cows. There was a treatment time interaction (P < 0.001) on uterine ph. All cows had similar uterine ph (P > 0.19) 24 h after the PGF 2α injection. Control cows that did not exhibit estrus had a greater uterine ph compared with control cows that exhibited estrus (P < 0.01) and ECP cows that exhibited estrus (P = 0.05) 48 h after the PGF 2α injection (7.0 ± 0.1 vs. 6.7 ± 0.1 and 6.8 ± 0.1, respectively). Estradiol cypionate-treated cows not exhibiting estrus were intermediate (6.8 ± 0.1; P > 0.05). All cows had similar uterine ph 72 h after the PGF 2α injection through ovulation (P > 0.06). In summary, uterine ph was similar among all animals that exhibited estrus, regardless of treatment with ECP. Key words: estradiol, fixed-time artificial insemination, uterine ph 2008 American Society of Animal Science. All rights reserved. J. Anim. Sci : doi: /jas INTRODUCTION 1 This research was supported by the South Dakota Agricultural Experiment Station and approved for publication as Journal Series No by the director, South Dakota Agricultural Experiment Station, South Dakota State University. Mention of a proprietary product does not constitute a guarantee or warranty of the product by South Dakota Agricultural Experiment Station or the authors and does not imply its approval to the exclusion of other products that may also be suitable. We express our thanks to T. Glaus, South Dakota State University, Brookings, SD; C. Moret, South Dakota State University, Brookings, SD; and J. Nelson, South Dakota State University, Brookings, SD, for technical support and to IVX Animal Health, St. Joseph, MO, for the donation of ProstaMate and Ova- Cyst. 2 Corresponding author: George.Perry@sdstate.edu Received May 15, Accepted July 3, Efficient transportation of sperm through the female reproductive tract requires that the female be in estrus or under the influence of estrogen (Hawk, 1983). Furthermore, animals that exhibit standing estrus within 24 h of fixed-time AI had increased concentrations of estradiol and greater pregnancy success compared with animals that did not exhibit estrus (Perry et al., 2005, 2007). Estrogen may influence fertilization through both sperm transport and fertilization efficiency by altering the uterine environment around the time of fertilization. Sperm have minimal metabolic function after final maturation (Hammerstedt, 1993). Thus, sperm become completely dependent on their environment. Motility of bull sperm increased as ph increased (Goltz et al., 1988). Decreasing ph completely suppressed sperm motility, and this suppression was reversible for up to 24 h by increasing ph (Jones and Bavister, 2000). Therefore, decreasing ph increased the viable lifespan of sperm. In cattle, uterine ph was decreased on the day of estrus compared with the midluteal phase (Elrod and 2928

2 Effects of estrus and estradiol on uterine ph 2929 Butler, 1993), and this decrease occurred around the initiation of estrus (Perry and Perry, 2008). With the interval from initiation of estrus (or second GnRH injection) to ovulation being approximately 30 h (Pursley et al., 1995; Vasconcelos et al., 1999), a decrease in uterine ph at initiation of standing estrus may initially decrease sperm motility and therefore increase sperm longevity. Consequently, a decrease in uterine ph at estrus may play a vital role in sperm transport and the sustained viability of sperm until the time of ovulation. This might also allow time for selection of sperm for fertilization. Accordingly, the objectives of these studies were to determine the influence of estradiol, estrus, or both on uterine ph during a fixed-time AI protocol. MATERIALS AND METHODS This research was conducted in accordance with procedures approved by the Institutional Animal Care and Use Committee at South Dakota State University. Exp. 1 Experimental Design. Nonpregnant, nonlactating, multiparous Angus-crossed beef cows (n = 20) at the South Dakota State University Beef Breeding Unit were synchronized with the CO-Synch protocol. Cows were injected with GnRH (100 µg as 2 ml of OvaCyst i.m.; IVX Animal Health, St. Joseph, MO) on d 9, PGF 2α (25 mg as 5 ml of Prostamate i.m., IVX Animal Health) on d 2, and GnRH (OvaCyst; 100 µg i.m.) on d 0. On d 2 an injection of 1 mg of estradiol cypionate [ECP (Sigma-Aldrich, St. Louis, MO)] dissolved in 1 ml of sesame seed oil was administered to one-half (n = 10) of the animals (i.m.) 12 h after the administration of PGF 2α. Control animals received a 1-mL injection (i.m.) of sesame seed oil. Transrectal Ultrasonography and Uterine ph. Ovaries of all cows were examined by transrectal ultrasonography to characterize follicular development (d 0) and to determine ovulation (d 2) by using an Aloka 500V ultrasound instrument with a 7.5-MHz transrectal linear probe (Aloka, Wallingford, CT). All follicles >8 mm in diameter were recorded, and ovulation was defined as the disappearance of a dominant follicle that was previously recorded. One cow failed to ovulate after the second injection of GnRH and was removed from the analysis of serum concentration of estradiol and uterine ph. Uterine ph was determined in all animals 12, 24, 48, 72, and 96 h after the PGF 2α injection. Uterine ph was determined by inserting a sterile plastic infusion pipette through the cervix and into the uterus as described previously (Perry and Perry, 2008). Estrous Detection, Blood Collection, and RIA. Beginning on d 1, standing estrus was detected by visual observation and with the aid of Estrus Alert Patches (Western Point Inc., Apple Valley, MN). Blood samples were collected via venipuncture of the tail vein into 10-mL Vacutainer tubes (Fisher Scientific, Pittsburgh, PA) at the time uterine ph was measured to determine circulating concentrations of estradiol. Blood samples were allowed to clot at room temperature for 1 h before being placed in a 4 C refrigerator for 24 h. Samples were centrifuged at 1,200 g for 30 min, and serum was collected and stored at 20 C until RIA were performed. Circulating concentrations of estradiol-17β were analyzed in all serum samples by RIA using the methodology described by Perry and Perry (2008). Inter- and intraassay CV were 19 and 7.8%, respectively, and assay sensitivity was 0.4 pg/ml. Exp. 2 Experimental Design. Postpartum (20 to 33 d) multiparous Angus-crossed beef cows (n = 40) at the South Dakota State University Beef Breeding Unit were synchronized with the CO-Synch protocol as described previously in Exp. 1. Cows were housed as a single group, and calves were allowed to suckle without restriction between sampling periods. As described in Exp. 1, one-half (n = 20) of the cows received 1 mg of ECP (i.m.) dissolved in 1 ml of sesame seed oil 12 h after the administration of PGF 2α. Control animals received 1 ml of sesame seed oil (i.m.). Transrectal Ultrasonography and Uterine ph. Ovaries of all cows were examined by transrectal ultrasonography to record follicular development and time of ovulation by using an Aloka 500V ultrasound instrument with a 7.5-MHz transrectal linear probe. Ovulatory follicle size was determined on d 2 and 0 by averaging follicular diameter at the widest point and at a right angle to the first measurement by using the internal calipers on the Aloka 500V instrument. Beginning 72 h after the PGF 2α injection, ovaries were examined every 6 h until ovulation had occurred. Ovulation was defined as the disappearance of a previously recorded dominant follicle. Uterine ph was determined in all cows 12, 24, 36, and 48 h after the PGF 2α injection. Beginning 48 h after the PGF 2α injection (time of the second GnRH injection), uterine ph was determined every 6 h until ovulation had occurred. Uterine ph was determined as described previously in Exp. 1. Cows were removed from the analysis of serum concentration of estradiol and uterine ph if they failed to ovulate after the second injection of GnRH (n = 2 ECP-treated and n = 3 control cows). Estrus Detection, Blood Collection, and RIA. All cows were individually fitted with estrousdetection transmitters and monitored for behavioral estrus continuously with the HeatWatch Estrous Detection System (DDx Inc., Denver, CO) from d 2 until ovulation occurred. Cows were considered to be in standing estrus when 3 mounts of 2 s or longer in duration were recorded within a 4-h period. Blood samples

3 2930 Perry and Perry Figure 1. Influence of standing estrus and treatment with estradiol cypionate (ECP) on circulating concentrations of estradiol (n = 7, ECP Estrus; n = 3, ECP No Estrus; n = 4, Control Estrus; and n = 5, Control No Estrus) during the CO-Synch protocol (treatment P = 0.002, time P < 0.001, and treatment time interaction P = 0.001; Exp. 1). Arrows indicate the time of the ECP injection and the second GnRH injection. were collected each time uterine ph was measured to determine circulating concentrations of estradiol. Blood samples were collected via venipuncture of the tail vein into 10-mL Vacutainer tubes (Fisher Scientific), and serum was collected as described previously. Circulating concentrations of estradiol-17β were analyzed in all serum samples by RIA as described previously. Interand intraassay CV were 13 and 5.9%, respectively, and assay sensitivity was 0.4 pg/ml. Statistical Analysis. Effect of treatment with ECP and standing estrus on uterine ph and circulating concentrations of estradiol-17β were determined by ANOVA for repeated measures (PROC MIXED, SAS Inst. Inc., Cary, NC; Littell et al., 1998). All covariance structures were modeled in the initial analysis. The indicated best-fit covariance structure was used for the final analysis. The model included the independent variables of treatment, time, and treatment time. The effects of treatment on uterine ph and circulating concentrations of estradiol-17β were analyzed by using animal within treatment as the error term, and the effects of day and treatment day on uterine ph and estradiol-17β were analyzed by using the residual as the error term. Differences in ovulatory follicle size between treatments at the time of the second GnRH injection were analyzed by ANOVA in SAS by PROC GLM. When the F-statistic was significant (P < 0.05), mean separation was performed by using least significant differences (means ± SEM; Snedecor and Cochran, 1989). Exp. 1 RESULTS There was a treatment (P = 0.002), time (P < ), and treatment time interaction (P = 0.001) on circulating concentrations of estradiol. Control animals that did not exhibit standing estrus maintained basal concentrations throughout the entire experiment. Twelve hours after administration of PGF 2α (time of the ECP injection), all cows had similar (P > 0.15) circulating concentrations of estradiol. At the second GnRH injection, the time insemination would normally occur, control animals that exhibited standing estrus and animals treated with ECP, regardless of standing estrus, had greater concentrations of estradiol compared with control animals that did not exhibit standing estrus (Figure 1). In all animals, concentrations of estradiol diminished after the second GnRH injection, but ECP-treated cows, regardless of standing estrus, had elevated (P < 0.02) circulating concentrations of estradiol compared with control cows, regardless of standing estrus (Figure 1). There was a treatment (P = 0.01) and time (P < 0.01) effect on uterine ph, whereas the treatment time interaction tended (P = 0.065) to affect only uterine ph. Control cows that did not exhibit standing estrus (n = 5) maintained a uterine ph of 6.8 to 7.2 throughout the entire experiment (Figure 2). All cows had a similar uterine ph (P > 0.19) 12 h after the PGF 2α injection.

4 Effects of estrus and estradiol on uterine ph 2931 Figure 2. Influence of standing estrus and treatment with estradiol cypionate (ECP) on uterine ph (n = 7, ECP Estrus; n = 3, ECP No Estrus; n = 4, Control Estrus; and n = 5, Control No Estrus) during the CO-Synch protocol (treatment P = 0.01, time P < 0.01, and treatment time interaction P = 0.065; Exp. 1). Arrows indicate time of the ECP injection and the second GnRH injection. At the time of the second GnRH injection, the time insemination would normally occur, control animals that exhibited standing estrus (n = 4; P < 0.01) and ECPtreated animals that exhibited standing estrus (n = 7; P = 0.05) had lesser uterine ph values compared with control animals that did not exhibit standing estrus (Figure 2). Cows treated with ECP that did not exhibit standing estrus (n = 3) were intermediate (6.8 ± 0.1), but tended (P = 0.065) to be less than control animals not exhibiting estrus. At the end of the experiment (96 h after the PGF 2α injection), uterine ph in all animals were similar to the uterine ph observed at the beginning of the experiment (Figure 2). Exp. 2 All cows treated with ECP exhibited standing estrus. Cows exhibiting estrus, regardless of treatment, had similar (P > 0.16) but elevated (P < 0.01) estradiol concentrations from the time of PGF 2α through the second GnRH injection compared with cows not detected in standing estrus (Figure 3). After the second GnRH injection, circulating concentrations of estradiol decreased in all animals, but concentrations of estradiol remained elevated in ECP-treated cows (P < 0.05) compared with control cows throughout the sampling period. There was no difference (P = 0.43) in ovulatory follicle size 48 h after the PGF 2α injection, the time of the second GnRH injection, between cows treated with ECP and control cows (14.8 ± 0.32 mm and 14.2 ± 0.48 mm, respectively). There was an effect of estrus on ovulatory follicle size. Cows that exhibited estrus had larger (P = 0.007) follicles (14.9 ± 0.31 mm) compared with cows that did not exhibit estrus (13.2 ± 0.49 mm). However, there was no difference (P = 0.43) between ECP and control cows that exhibited estrus (14.8 ± 0.3 mm and 15.3 ± 0.68 mm, respectively). Treatment (P = 0.07), time (P < 0.001), and treatment time (P < 0.001) had an affect on uterine ph. Control cows that did not exhibit standing estrus (n = 8), similar to the first experiment, maintained a uterine ph between 6.9 and 7.2 throughout the entire experiment (Figure 4). In this experiment, all animals treated with ECP exhibited standing estrus within 24 h of the second injection of GnRH. Forty-eight hours after the PGF 2α injection, the time of the second GnRH injection, animals in standing estrus (control and ECPtreated cows) had a significantly less (P = 0.004) uterine ph compared with control animals not in standing estrus (Figure 4). Uterine ph in all animals at the end of the experiment was between 6.9 and 7.2, analogous to the uterine ph observed at the beginning of the experiment. When uterine ph was standardized to the onset of estrus as determined by the HeatWatch system, there was a treatment time interaction (P = 0.019) on uterine ph. Uterine ph decreased and was lowest at the initiation of standing estrus and increased after the initiation of estrus among cows that exhibited es-

5 2932 Perry and Perry Figure 3. Influence of standing estrus and treatment with estradiol cypionate (ECP) on circulating concentrations of estradiol (n = 18, ECP Estrus; n = 8, Control Estrus; and n = 9, Control No Estrus) during the CO-Synch protocol (treatment P < 0.001, time P < 0.001, and treatment time interaction P = 0.005; Exp. 2). Arrows indicate the time of the ECP injection and the second GnRH injection. trus. Uterine ph did not change among cows that did not exhibit estrus (Figure 5). Uterine ph had returned to between 6.8 and 7.0 by 12 h after the initiation of standing estrus. DISCUSSION In the present study, cows that were detected in standing estrus had ovulatory follicles and elevated concen- Figure 4. Influence of standing estrus and treatment with estradiol cypionate (ECP) on uterine ph (n = 18, ECP Estrus; n = 8, Control Estrus; and n = 9, Control No Estrus) during the CO-Synch protocol (treatment P = 0.07, time P < 0.001, and treatment time interaction P < 0.001; Exp. 2). Arrows indicate time of the ECP injection and the second GnRH injection.

6 Effects of estrus and estradiol on uterine ph 2933 Figure 5. Influence of standing estrus and treatment with ECP on uterine ph (n = 18, ECP Estrus; n = 8, Control Estrus; and n = 9, Control No Estrus) standardized to the initiation of standing estrus (treatment P = 0.09, time P < 0.001, and treatment time interaction P = 0.019; Exp. 2). Time 0 is the time of the initiation of standing estrus (ECP and Control Estrus) or time of the second GnRH injection (Control No Estrus). trations of estradiol similar to cows treated with ECP. In cattle, serum concentrations of estradiol begin to increase around the time of follicular selection (Kulick et al., 1999; Ginther et al., 2000). Expression of mrna for P450 side-chain cleavage, P450 17α-hydroxylase, P450 aromatase, 3β-hydroxysteroid dehydrogenase, and steroidogenic acute regulatory protein (StAR) in thecal or granulosal cells or both also increased during selection of the future dominate follicle (Xu et al., 1995; Bao et al., 1997a,b, 1998), and during this time, the follicles produced increasing amounts of estradiol (Martin et al., 1991). However, the effects of administering exogenous estradiol on fertility are not clear. When estradiol was administered at follicular wave emergence, fertility was reduced, but when estradiol was administered after follicular selection, when a dominant follicle was present on the ovary, it had no influence on fertility (Lane et al., 2001). These different effects on fertility may be related to the influence of estradiol on follicular development, timing of estradiol administration, and type of estradiol used. Estradiol administered during follicular wave emergence reduced the maximum diameter of the ovulatory follicle, but estradiol administered when a dominant follicle was present did not affect maximum ovulatory follicle size (Evans et al., 2003). When estradiol was administered prebreeding in sheep bred by natural mating or AI after detection in standing estrus, there was no effect on pregnancy success (Inskeep et al., 1979). Furthermore, administering estradiol before the GnRH injection and AI following a fixed-time insemination protocol improved fertility (Ahmadzadeh et al., 2003; Souza et al., 2007). Greater serum concentrations of estradiol and increased pregnancy rates were observed in cows and heifers exhibiting standing estrus within 24 h of fixedtime AI compared with animals not exhibiting standing estrus (Perry et al., 2005, 2007). The initiation of estrus has been tied to the peak in circulating concentrations of estradiol (Allrich, 1994). When an injection of estrogen corresponding to initiation of estrus was omitted in ovariectomized ewes, embryo survival after embryo transfer was decreased (Miller and Moore, 1976). In the present study, animals that exhibited standing estrus within 24 h of the second GnRH injection had greater concentrations of estradiol. Furthermore, an injection of 1 mg of ECP elevated the circulating concentration of estradiol to a concentration similar to that of animals that exhibited estrus spontaneously. In a recent review by Santos et al. (2004), fertilization failure in lactating beef cows ranged from 0 to 25% and in lactating dairy cows from 12 to 45%. Allison and Robinson (1972) and Bedford (1972) reported that sperm transport only occurred in ovariectomized females under the influence of estrogen. Noyes et al. (1959) arrived at similar conclusions in ovariectomized rabbits treated with a small dose of estradiol; sperm transport efficiency was

7 2934 increased, but at a larger dosages the effects were detrimental. Therefore, efficient transportation of sperm through the female reproductive tract requires that the female be in estrus or under the influence of estrogen (Hawk, 1983). Estrogen may influence fertilization through both sperm transport and fertilization efficiency by altering the uterine environment around the time of fertilization. In the present study, cows with elevated concentrations of estradiol (spontaneous estrus or ECP treatment) had decreased uterine ph at the time of the second GnRH injection compared with cows that did not receive ECP or did not exhibit estrus. In cattle, uterine ph was decreased on the day of estrus compared with d 7 of the estrous cycle (Elrod and Butler, 1993), and this decrease occurred around the initiation of standing estrus (Perry and Perry, 2008). Considerable evidence supports the notion that ph regulates sperm motility and longevity in invertebrates (Schackmann et al., 1981; Christen et al., 1982; Johnson et al., 1983; Lee et al., 1983). In vertebrate species, Wong et al. (1981) reported that sperm motility was dependent on sodium ion concentrations and concluded that an increase in ph leads to the initiation of motility. Furthermore, ph influenced the motility of sperm collected from the caudal epididymis (Acott and Carr, 1984), and Goltz et al. (1988) reported the motility of bull sperm increased as ph increased. Jones and Bavister (2000) demonstrated that decreasing ph by 0.5 ph units (from 6.9 to 6.4) completely suppressed sperm motility. This suppression was reversible for up to 24 h by increasing ph. Therefore, by decreasing ph, the lifespan of sperm was increased; motility returned when ph was increased. With the interval from the initiation of estrus (or second GnRH injection) to ovulation being around 30 h (Pursley et al., 1995; Vasconcelos et al., 1999), a decrease in uterine ph at the initiation of standing estrus may initially decrease sperm motility and therefore increase sperm longevity. Consequently, the decrease in uterine ph at estrus may play a vital role in sperm transport and the sustained viability of sperm until the time of ovulation, and induced ovulation of follicles in the absence of elevated concentrations of estradiol may result in decreased sperm survival until the time of ovulation. In summary, treatment of cows with 1 mg of ECP increased concentrations of estradiol to levels similar to cows that spontaneously exhibited estrus. Furthermore, cows that exhibited estrus or were treated with ECP experienced a decrease in uterine ph at the time of the second GnRH injection. Decreased ph has previously been reported to increase sperm longevity. Therefore, with the interval from the second GnRH injection to ovulation being around 30 h, a decrease in uterine ph at time of the second GnRH injection among cows that exhibit estrus within 24 h of fixed-time AI may initially decrease sperm motility and therefore increase sperm longevity and pregnancy success. Perry and Perry LITERATURE CITED Acott, T. S., and D. W. Carr Inhibition of bovine spermatozoa by caudal epididymal fluid: II. Interaction of ph and a quiescence factor. Biol. Reprod. 30: Ahmadzadeh, A., D. G. Falk, R. Manzo, C. B. Sellars, and J. C. Dalton Effect of incorporation of a low dose of estradiol cypionate (ECP) into a timed artificial insemination protocol on estrous behavior and conception rates in beef cattle. J. Anim. Sci. 81(Suppl. 1):180. (Abstr.) Allison, A. J., and T. J. Robinson The recovery of spermatozoa from the reproductive tract of the spayed ewe treated with progesterone and oestrogen. J. Reprod. Fertil. 31: Allrich, R. D Endocrine and neural control of estrus in dairy cows. J. Dairy Sci. 77: Bao, B., M. D. Calder, S. Xie, M. F. Smith, B. E. Salfen, R. S. Youngquist, and H. A. Garverick Expression of steroidogenic acute regulatory protein messenger ribonucleic acid is limited to theca of healthy bovine follicles collected during recruitment, selection, and dominance of follicles of the first follicular wave. Biol. Reprod. 59: Bao, B., H. A. Garverick, G. W. Smith, M. F. Smith, B. E. Salfen, and R. S. Youngquist. 1997a. Changes in messenger ribonucleic acid encoding luteinizing hormone receptor, cytochrome P450- side chain cleavage, and aromatase are associated with recruitment and selection of bovine ovarian follicles. Biol. Reprod. 56: Bao, B., H. A. Garverick, G. W. Smith, M. F. Smith, B. E. Salfen, and R. S. Youngquist. 1997b. Expression of messenger ribonucleic acid (mrna) encoding 3beta-hydroxysteroid dehydrogenase delta4, delta5 isomerase (3beta-hsd) during recruitment and selection of bovine ovarian follicles: Identification of dominant follicles by expression of 3beta-hsd mrna within the granulosa cell layer. Biol. Reprod. 56: Bedford, J. M Sperm transport, capacitation and fertilization. Page 338 in Reproductive Biology. H. Balin and S. Glasser, ed. Excerpta Medica, Amsterdam, the Netherlands. Christen, R., R. W. Schackmann, and B. M. Shapiro Elevation of the intracellular ph activates respiration and motility of sperm of the sea urchin, Strongylocentrotus purpuratus. J. Biol. Chem. 257: Elrod, C. C., and W. R. Butler Reduction of fertility and alteration of uterine ph in heifers fed excess ruminally degradable protein. J. Anim. Sci. 71: Evans, A. C., P. O Keeffe, M. Mihm, J. F. Roche, K. L. Macmillan, and M. P. Boland Effect of oestradiol benzoate given after prostaglandin at two stages of follicle wave development on oestrus synchronisation, the LH surge and ovulation in heifers. Anim. Reprod. Sci. 76: Ginther, O. J., D. R. Bergfelt, L. J. Kulick, and K. Kot Selection of the dominant follicle in cattle: Role of estradiol. Biol. Reprod. 63: Goltz, J. S., T. K. Gardner, K. S. Kanous, and C. B. Lindemann The interaction of ph and cyclic adenosine 3,5 -monophosphate on activation of motility in Triton X-100 extracted bull sperm. Biol. Reprod. 39: Hammerstedt, R. H Maintenance of bioenergetic balance in sperm and prevention of lipid peroxidation: A review of the effect on design of storage preservation systems. Reprod. Fertil. Dev. 5: Hawk, H. W Sperm survival and transport in the female reproductive tract. J. Dairy Sci. 66: Inskeep, E. K., L. P. Stevens, and C. R. Rudy Fertility in ewes receiving low doses of estradiol during synchronized estrus. J. Anim. Sci. 48: Johnson, C. H., D. L. Clapper, M. M. Winkler, H. C. Lee, and D. Epel A volatile inhibitor immobilizes sea urchin sperm in semen by depressing the intracellular ph. Dev. Biol. 98:

8 Effects of estrus and estradiol on uterine ph 2935 Jones, J. M., and B. D. Bavister Acidification of intracellular ph in bovine spermatozoa suppresses motility and extends viable life. J. Androl. 21: Kulick, L. J., K. Kot, M. C. Wiltbank, and O. J. Ginther Follicular and hormonal dynamics during the first follicular wave in heifers. Theriogenology 52: Lane, E. A., E. J. Austin, J. F. Roche, and M. A. Crowe The effect of estradiol benzoate on synchrony of estrus and fertility in cattle after removal of a progesterone-releasing intravaginal device. Theriogenology 55: Lee, H. C., C. Johnson, and D. Epel Changes in internal ph associated with initiation of motility and acrosome reaction of sea urchin sperm. Dev. Biol. 95: Littell, R. C., P. R. Henry, and C. B. Ammerman Statistical analysis of repeated measures data using SAS procedures. J. Anim. Sci. 76: Martin, T. L., R. L. Fogwell, and J. J. Ireland Concentrations of inhibins and steroids in follicular fluid during development of dominant follicules in heifers. Biol. Reprod. 44: Miller, B. G., and N. W. Moore Effect of progesterone and oestradiol on endometrial metabolism and embryo survival in the ovariectomized ewe. Theriogenology 6:636. Noyes, R. W., C. E. Adams, and A. Walton The passage of spermatozoa through the genital tract of female rabbits after ovariectomy and oestrogen treatment. J. Endocrinol. 18: Perry, G. A., and B. L. Perry Effect of preovulatory concentrations of estradiol and initiation of standing estrus on uterine ph in beef cows. Domest. Anim. Endocrinol. 34: Perry, G. A., M. F. Smith, M. C. Lucy, J. A. Green, T. E. Parks, M. D. Macneil, A. J. Roberts, and T. W. Geary Relationship between follicle size at insemination and pregnancy success. Proc. Natl. Acad. Sci. USA 102: Perry, G. A., M. F. Smith, A. J. Roberts, M. D. MacNeil, and T. W. Geary Relationship between size of ovulatory follicle and pregnancy success in beef heifers. J. Anim. Sci. 85: Pursley, J. R., M. O. Mee, and M. C. Wiltbank Synchronization of ovulation in dairy cows using PGF 2α and GnRH. Theriogenology 44: Santos, J. E., W. W. Thatcher, R. C. Chebel, R. L. Cerri, and K. N. Galvao The effect of embryonic death rates in cattle on the efficacy of estrus synchronization programs. Anim. Reprod. Sci : Schackmann, R. W., R. Christen, and B. M. Shapiro Membrane potential depolarization and increased intracellular ph accompany the acrosome reaction of sea urchin sperm. Proc. Natl. Acad. Sci. USA 78: Snedecor, G. W., and W. G. Cochran Statistical Methods. 8th ed. Iowa State University Press, Ames. Souza, A. H., A. Gumen, E. P. Silva, A. P. Cunha, J. N. Guenther, C. M. Peto, D. Z. Caraviello, and M. C. Wiltbank Supplementation with estradiol-17β before the last gonadotropinreleasing hormone injection of the Ovsynch protocol in lactating dairy cows. J. Dairy Sci. 90: Vasconcelos, J. L., R. W. Silcox, G. J. Rosa, J. R. Pursley, and M. C. Wiltbank Synchronization rate, size of the ovulatory follicle, and pregnancy rate after synchronization of ovulation beginning on different days of the estrous cycle in lactating dairy cows. Theriogenology 52: Wong, P. Y., W. M. Lee, and A. Y. Tsang The effects of extracellular sodium on acid release and motility initiation in rat caudal epididymal spermatozoa in vitro. Exp. Cell Res. 131: Xu, Z., H. A. Garverick, G. W. Smith, M. F. Smith, S. A. Hamilton, and R. S. Youngquist Expression of messenger ribonucleic acid encoding cytochrome P450 side-chain cleavage, cytochrome P450 17α-hydroxylase, and cytochrome P450 aromatase in bovine follicles during the first follicular wave. Endocrinology 136: