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Antiluteolytic strategies to improve fertility in cattle
ELSEVIER
ANTILUTEOLYTIC
STRATEGIES TO IMPROVE FERTILITY IN CATTLE
M. Binellii’L W.W. Thatcher? R. Matto? .
and P.S. Barusellil
‘Departamento de Reproducb Animal, FMVZ-USP, Pirassununga, 13630-000. Brazil *Department of Animal Sciences, University of Florida, Gainesville, FL 3261 l-0920
ABSTRACT During early pregnancy, a “critical period” may be defmed between Days 15 and 17. Embryonic mortality associated with this period causes significant economic losses to the cattle industry. During this period, the endometrium will follow a default program to release luteolytic pulses of PGFza, unless the conceptus sends appropriate antiluteolytic signals to block PGFz,, production. Maintenance of pregnancy is dependent on a successml blockage of endometrial PGFz, production. Biology of the critical period is complex and multifactorial. Endocrine, cellular and molecular factors, both from maternal and conceptus origins act in concert to determine whether luteolysis or maintenance of pregnancy will prevail. Understanclmg the influences of such factors in the biology of the critical period allowed researchers to produce a series of strategies aiming to favor maintenance of pregnancy in lieu of luteolysis. Strategies include hormonal and nutritional manipulations to decrease plasma concentrations of estradiol 17p (Ez) while increasing those of progesterone (Pd), and inhibiting the PGFra-synthesizing enzymatic machinery in the endometrium during the critical period. Experimental results indicate that use of such strategies has improved pregnancy rates following artificial insemination and embryo transfer programs. 0 2001 by Elsevier Science Inc. Key words: embryonic mortality, luteolysis, pregnancy recognition, PGFz,, cattle
INTRODUCTION:
DEFINING THE “CRITICAL PERIOD”
The interval comprised between two estrus is defined as an estrous cycle, which averages 21 days in cattle. There are 2 or 3 waves of follicular growth during an estrous cycle. Each wave is characterized by phases of recruitment, selection, dominance and atresia. The dominant follicle of the last wave escapes atresia and secretes increasing quantities of Ez. High E2 stimulates an LH surge, which induces ovulation. The ovulated follicle undergoes structural and functional changes, originating the CL. The CL grows quickly in size and secretes increasing quantities of Pd. At around Day 16 of the cycle (Day 0 = es&us), the uterine endometrium starts secreting pulses of PGFza, which causes both structural and functional demise of the CL (the process of luteolysis). Functional luteolysis results in a ready decrease in circulating Acknowledgments “Correspondence and reprint requests. Research was supported partially by FAPESP, Brazil. Authors would lie to thank Dr. Jose Luiz Moraes Vasconcelos, Dr. Ed Hofhnann Madureira e Mr. M&rcio de Oliveira Marques for critically revising this manuscript. Theriogenology 56:1451-1463,200l 0 2001 Elsevier Science Inc.
0093-691 X/O1 /$-see front matter PII: SOO93-691X(01)00646-X
Theriogenology
concentrations of Pa. Such low P4 concentrations allow the final growth of a dominant, preovulatory follicle and stimulate estrus behavior and ovulation. If the ovulated oocyte is not fertilized, this cycle will be repeated and luteolysis will occur again. However, if the oocyte is fertilized, the luteolytic process must be blocked (at around Day 16) to maintain pregnancy. Thus, a “critical period“ in the reproductive cycle of the cow could be defined at around Days 15 and 17 afler estrus. During this period, the cow should adjust its physiology properly, depending on whether it is pregnant or not. Changing corn a cyclic to a pregnant state (the process of pregnancy recognition) depends on an effective blockage of luteolysis. Blocking luteolysis is dependent on the ability of the conceptus to send effective antiluteolytic signals and on the capacity of the endometrium to respond to such signals, thus blocking PGFza production. Such communications between conceptus and maternal units are not always successful. In fact, embryonic mortality between days 8 and 16 of pregnancy approaches 30% (14). Such high mortality rate has obvious economic impacts in a cattle operation, increasing number of days open and retarding genetic progress. Therefore, a large quantity of research has been put forth to understanding the biology of the critical period, and several practical strategies have been proposed. Objectives of this paper are (1) to review local and endocrine regulation of luteolysis and pregnancy recognition and (2) to identify mechanisms that could be manipulated to decrease embryonic mortality associated with the critical period
REGULATION OF LUTEOLYSIS AND PREGNANCY RECOGNITION This topic is extremely complex and several excellent reviews are available for the reader (8,11,22,23,34,39,41,45). Discussion in this paper will be limited to concepts used in the formulation of antiluteolytic strategies discussed in the next section. In sheep, it has been proposed that initiation of luteolysis is dependent on E2-stimulated synthesis of endometrial oxytocin receptors during the critical period (24). Binding of oxytocin stimulates pulsatile release of PGF2,. In cattle, recent studies have demonstrated that oxytocin might not be essential for the initiation of luteolysis (16). However, presence of Ez is critical for luteolysis to occur. Indeed, removal of ovarian follicles by cauterization or X-irradiation delays luteolysis and increases duration of estrous cycles in cows (50). Moreover, injections of Ez stimulate PGFza secretion (46). Therefore, blocking of luteolysis during pregnancy recognition may involve inhibition of E2 production and its effects. For example, plasma concentrations of Ef are lower in pregnant animals than in cyclic animals (32). Furthermore, follicle growth and E2 production per follicle am less in pregnant compared to cyclic cows (42). Although cellular and molecular mechanisms by which E2 stimulates PGFza secretion are not completely elucidated, it is clear that it plays a central role on the process of luteolysis. Therefore, an important concept for developing antiluteolytic strategies is that decreasing circulating concentrations of EZ during the critical period should inhibit or delay luteolysis. Circulating concentrations of P4 seems to be positively correlated with pregnancy recognition in cattle. There is higher concentration of milk P4 in cows inseminated and pregnant compared to cows inseminated and not pregnant (IO, 17,19). This suggests that high P4 during the critical period is important for maintenance of pregnancy.
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It is well established that fertility and milk production are negatively associated in dairy cows (for discussion see 28,49). Vasconcelos (47) indicated that high-yielding dairy cows have lower circulating concentrations of Pq than low producers. This may be because higher-yielding cows have a greater rate of Pb catabolism. Perhaps lower circulating P4 could at least partly explain the lower fertility associated with high milk producers. One main regulator of pregnancy recognition is the conceptus-secreted bovine interferon-tau (bIFN-r). The bIFN-t is secreted in the uterine lumen during the critical period and interacts with the endometrium to block PGF2alpha synthesis. One possible mechanism through which P4 stimulates maintenance of pregnancy may be through stimulation of bIFN-r secretion. Indeed, Mann et al. (19) have shown that cows with a greater concentration of plasma P4 during the critical period produced conceptuses that were larger and produced more bIFN-2. These data imply that a second concept may be suggested: increasing concentrations of P4 during the luteal phase and the critical period should stimulate pregnancy recognition. Until the time of pregnancy recognition, uterine physiology is very similar between pregnant and cyclic animals, and a default PGF za synthetic program is in place in the endometrium. The practical outcome of this notion is that unless PGF2, synthesis is actively blocked, luteolysis will ensue regardless of the presence of a conceptus. Blocking PGFza production requires that the elongating conceptus occupy most of the uterine horn ipsilateral to the CL at the critical period. Thatcher and Hansen (43) demonstrated that Day 17 conceptuses varied in size from 15 to 250 mm. The implication of this observation is that luteolysis will occur even in the presence of conceptuses, if they are not large enough. Also, secretion of conceptus-derived paracrine factors, such as bIFN-r, is necessary for the inhibition of PGFza. Synthesis of PGF2, results horn the activation of a complex intracellular cascade of enzymatic reactions (11). The rate limiting reaction is the conversion of arachidonic acid to pmstaglandin Hz, catalyzed by the enzyme cyclooxygenase-2 (COX-2). To test the hypothesis that exposure to conceptus tissues down-regulated the PGFz,-synthesis cascade, Arnold and et al. (2) compared in vitro responsiveness of explants obtained from cyclic and pregnant cows on Day 15 after estrus to intracellular stimulators of PGFzu synthesis. Regardless of which enzymatic step was stimulated, pregnant tissue always yielded less PGFza compared to cyclic tissue. In summary, strategies that favor the presence of elongated and functional conceptuses and/or inhibit the endometrial PGF2,-synthesizing enzymatic machinery during the critical period should increase pregnancy rates in cattle. Altogether, this information allows a third concept to be stated: conceptus and endometrial functions could be manipulated to generate a microenvironment that is less conducive to luteolysis.
ANTILUTEOLYTIC
STRATEGIES
Based on the concepts stated above, a series of antiluteolytic strategies are proposed below. Figure 1 summarizes six possible strategies to favor pregnancy recognition in lieu of luteolysis.
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Strategy 1: Increase Size of Pre-ovulatory Follicle to Generate a Larger CL. Larger CLs will secrete more PJ and this may have a positive effect on pregnancy recognition, and consequently pregnancy rates both in artificial insemination (47, 48) and embryo transfer programs (5). For example, Baruselli et al. (5), synchronized embryo recipients with either a single PGF injection or following the “Ovsynch” protocol (33). Regardless of treatment, authors found a positive association between area of CL and Pq plasmatic concentration on Day 6 after estrus. Moreover, when recipients were classified according to CL size, heifers with larger CLs also showed higher conception rates (Table 1). In contrast, work by Battocchio et al. (7) demonstrated that CL volume detected by ultrasonography may not be a good criteria for characterizing the CL capacity to secrete Pa. In addition, Spell et al. (40) did not observe differences in CL size or plasma Pd concentrations between recipients that did and did not become pregnant atIer embryo transfer. In spite of those discrepant data, it is proposed that it could be advantageous to induce a large pre-ovulatory follicle aiming to obtain a larger CL in embryo recipients. The classical way to obtain a large follicle is to promote follicle growth under low, subluteal circulating concentrations of Pd or progestagens, which causes follicles to become persistent (36). Conception rates after artificial insemination associated with persistent dominant follicles are usually low, because of an altered oviductal environment (9), a premature maturation of the oocyte (26) or both. However, this would not be the case for embryo recipients, in which the oocyte coming from a persistent follicle would not be fertilized. Because dominant follicle size is associated with subsequent CL size (47, 48), a large persistent follicle could generate a large CL, which would provide high Pd concentration to favor development of the transferred embryo. To test this hypothesis, Moura et al. (27) treated crossbred recipient heifers with a norgestomet implant for 9 days. On Day 0, implants were administered and recipients were randomly assigned to receive 5 mg estradiol valerate and 3 mg norgestomet im (Group 1; s40) or 400 pg of delprostenate (a PGFr, analog; Group 2; n=42). On Day 9 all cows received a delprostenate injection and ovulation was induced by an estradiol benzoate injection (1 mg) on Day 10. It was expected that treatment administered to heifers in Group 1 should induce ovulation of a freshly recruited dominant follicle while treatment administered to heifers in Group 2 should induce the growth and ovulation of a pre-existent follicle (i.e., persistent). Ovaries were examined by ultrasonography on Day 10 (for dominant follicle measurement) and on Day 17 (for CL measurement). Results of this experiment indicated, in comparison to Group 1, heifers in Group 2 developed a larger size dominant follicle (1 .OOf 0.13 vs. 1.25 f 0.23cm) which resulted on CLs with larger diameter (1.54 f 0.17 vs. 1.81 f 0.12cm) and higher Pq concentrations on Day 17 (1.98 f 1.28 vs. 2.96 f 2.33ng/ml). Percent transferable recipients (i.e., cows presenting CL > 1.3cm) was also higher to cows in Group 2; (52.5 vs. 78.6) but pregnancy rates were not statistically different between Groups 1 (25.0%) and 2 (30.9%).
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-21 (ovul.)
10
15
:21
&.) ' Q,6
Figure 1. Summary of antiluteolytic strategies. Strategies are numbered 1 to 6 across an interval of time representing an estrous cycle followed by artificial insemination (AL) and Ovulations (ovul.) are indicated at Days -21 and 0. Progesterone pregnancy. concentrations are represented by dotted lines, dominant follicles by open circles, dominant ovulatory follicle by two superimposed circles (open and closed) and the critical period by a gray box. Strategies are: (1) increase size of pre-ovulatory follicle to generate larger CL, (2) increase rate of growth of CL, (3) increase luteal phase progesterone, (4) decrease effect of a dominant follicle on the critical period, (5) increase antiluteolytic stimulation-conceptus unit and (6) decrease luteolytic response-maternal unit.
Table 1. Progesterone concentration and conception rates according to CL area at Day 6 of the estrous cycle in crossbred recipient heifers (mean 2 standard deviation). CL area n Progesterone Average CL area Conception rate (ultrasonography) concentration (ng/mL) (cm*) -% 77 2.44 + 0.86’ CL1 (> 2.Ocm’) 2.66 + 0.51 47/77 (58.4 %)d CL2 (1.5 a2.0 cm’) 41 1.75 + 0.69b 1.74kO.10 17/41 (41.5 %)” 0.96,.z+ 0_:.-...-. 56’ CL3 1(<.l___.__l_l_ l.Scm*) 07/22 .-._22“__...,_ .-_,. ,._-- 1.19 + 0.20 __.-._“_l “_1(3 1.8 %>’ ‘*‘a6”Geanswithin columns with different superscripts differ sign&k&y (PC 0.01) defPercentages within columns with different superscripts differ significantly (PcO.05)
Strategy 2: Increase Pate of Growth of the CL. Optimal differentiation and rate of growth of the CL is related to duration and amplitude of the ovulatory LH surge (1). Induced LH surges, for example by injections of GnRH in
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Ovsynch protocols, are oflen of lower duration than endogenous, noninduced surges (12), which could limit optimal CL growth. Ambrose et al. (1) stimulated ovulation of nonlactating Holstein cows with a Deslorelin (a GnRH agonist) implant, as part of an Ovsynch program. They observed that implanted cows had a greater rate of CL growth and greater plasma Pq concentrations compared to a bolus injection of another G&I-I agonist (Buserelin). They concluded that the prolonged release of GnRH provided by the Deslorelin implant induced a LH surge of longer duration and thereby stimulated CL growth and function. Strategy 3: Increase Luteal Phase PJ. Several methods have been used aiming to increase conception rates through a greater plasma PI concentration during the luteal phase (see 44 for a review). Increased plasma Ps concentrations can be achieved by inducing an accessory CL. Injection of hCG stimulates formation of an accessory CL (20,35,37,38). Santos et al. (35) treated high producing lactating Holstein cows with either saline (n=203) or hCG (n=203) on Day 5 of a synchronized estrous cycle. Ultrasonography and blood sampling were performed from Days 11 to 16 after AI and presence of conceptuses was accessed by ultrasonography on Day 28 and by rectal palpation on Days 45 and 90. Treatment with hCG induced formation of accessory CLs in 86.2% of cows, compared to 23.2% in controls. Injection of hCG increased plasma P.r by 5 ng/mL and conception rates on Days 28 (45.8 vs. 38.7%), 45 (40.4 vs. 36.3%) and 90 (38.4 vs. 31.9%), compared to saline injection. Marques et al. (20) compared the efficacy of saline, LH, GnRH analog (Buserelin) or hCG injections on CL parameters and Pd plasmatic concentrations. Crossbred beef heifers were injected on Day 7 of a synchronized estrous cycle. Results on Table 2 indicate that all LH-, G&I-I- and hCG-treated heifers formed accessory CLs. However, main and accessory CL diameters and average plasma Pd concentration (Days 13 to 17) were higher for hCG treated cows compared to other treatments. Since hCG has a longer half-life than GnRH and LH, it can persist in the circulation longer. Therefore, besides inducing formation of accessory CLs, hCG may exert luteotropic effects by directly stimulating Pq production, which may increase pregnancy rates.
Table 2. Main and accessory CL diameter between Days 13 to 17 and plasma progesterone concentration in crossbred heifers treated with GnRH, hCG or LH at Day 7 of the estrous cycle (mean If standard deviation; D MainCL Main CL Progesterone Group n diameter (Days concentrations diameter diameter 13-17; cm) (Days 13 -17; cm) @ays 13 -17; ng/mL) (Day 6; cm) 1.55 + o.22a 4.79 _+2. lga 1.84 + 0.35 1.78 + 0.22b GnRH 8 8.42 t 3.64b 2.19&0.31’ 1.75 _+0.2gb hCG 8 1.79 + 0.32 6.07 2 3.09’ LH 7 1.69+0.17 1.79 + O.lSb 1.61 + 0.16’ . ._ .-.-_-____(___r ._ “._.I- ..,..____~-_-__.-lt-..-._“X_. .- -_.- _^-‘X’&ms within columns with different superscripts differ significantly (PC O.Gs)‘-“‘“- __ ..
Another possible way to stimulate higher plasma Pd concentrations during the luteal phase is through eCG injections. The eCG may exert both LH and FSH effects in cattle. Therefore, injecting eCG during the emergence phase of a synchronized follicular wave may: (1)
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increase the rate of growth of follicles, resulting on a larger pre-ovulatory follicle. Such larger follicle should result on a larger CL. (2) Stimulate simultaneous growth of multiple follicles that can be induced to ovulate and form multiple CLs. In both cases high Pq concentrations would be expected after ovulation. Baruselli et al. (6) synchronized recipient crossbred heifers at random stages of the estrous cycle with injections of estradiol benzoate and P4 and insertion of a CIDR device (Day 0). Five days later heifers were randomly assigned to receive either 800 IU of eCG or nothing. On Day 7, implants were removed and recipients received and injection of cloprostenol (PGF2alpha analog). Ovulations were induced by an injection of estradiol benzoate on Day 8, ovaries were examined by ultrasonography on Days 8 and 15 and embryos were transferred on Day 16. Results on Table 3 indicate that number of follicles greater than 8 mm on Day 8, total number of CLs, number of CLs greater than 13 mm (i.e., criteria for selecting recipients to receive embryos), Pd concentration on Day 15, conception and pregnancy rates were higher for cows treated with eCG compared to controls. Moreover, eCG-treated heifers bearing a single CL showed larger CL areas, Pd concentrations and conception rates than controls. Authors concluded that eCG treatment increased Pd plasmatic concentration and consequently pregnancy rates because: (1) it stimulated follicle growth and consequently number of CLs and (2) it stimulated size of individual CLs. Similar results presented by B6 et al. support such conclusions.
Table 3. Effects of eCG in follicle and CL size, CL function and reproductive effkiency of recipient heifers (mean + standard deviation). Controls eCG 0.6 If:0.5” Number of follicles >8rnm at Day 8 2.04 t 1.4b 0.5 k 0.51” Number of CLs at Day 15 2.58 5 2.93b 1.35 + 0.78’ Progesterone concentration at Day 15 (ng/mL) 4.17 * 3.73d % heifers with CLs >I3 mm at Day 8 17/50 (34.0%)’ 42/50 (84.0%~)~ Conception rates 5117 (29.4%) 21/38 (55.3%) Pregnancy rates 5/50 (1 o.O%)c 21/50 (42.0%)d “means within rows with different superscripts differ significantly (PC 0.01). “dMeans within rows with different superscripts differ significantly (PC 0.05). “bpercentages within rows with different superscripts differ significantly (PC 0.01). CdPercentages within rows with different superscripts differ significantly (PC 0.05).
A point for consideration regarding the use of hCG and eCG in antiluteolytic programs is that effectiveness of these hormones may decreases with multiple uses in the same cow. This is because they are antigenic molecules to cattle and may elicit antibody formation. Therefore, maximum effectiveness is achieved when animal is naive and may be reduced as antibody titers increase in the circulation. Strategy 4: Decrease the Effect of a Dominant Follicle on the Critical Period. Eliminating or decreasing the size of a follicle during the critical period should decrease circulating concentrations of Ez and consequently decrease the luteolytic stimulation associated with Er. For example, a strategic injection of GnRB could turn over a dominant follicle that may
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be present during the critical period. Follicle turnover will reduce Er concentrations and favor pregnancy recognition. Peters (3 1) summarized results of six studies that analyzed the effects of GnRH injections between Days 11 and 13 of the estrous cycle on pregnancy rates. Results ranged from -3.9 to 12% improvement in pregnancy rates for cows injected with GnRH compared to controls. In the study conducted by Ambrose et al. (described above; I), authors observed that the Deslorelin implant delayed appearance of Class III follicles (>9mm) to 14.2 days compared to 4.6 days, probably due to a down-regulation of LH secretion. Absence of large follicles during the critical period probably maintained low plasma E2 concentrations, which is favorable to pregnancy recognition. In fact, granted the number of cows used were small, treatment with Deslorelin significantly increased circulating concentrations of P4 and pregnancy rates (5/8) compared to Buserelin-treated cows (l/8). Besides stimulating luteal area and function, treatment with hCG on Day 5 also reprograms follicular growth to increase percentage of 3- compared to 2-wave cycles (13). This decreases the probability of cows having a large, highly estrogenic follicle during the critical period and may also contribute to higher conception rates observed by Santos et al. (35) for cows receiving hCG injections on Day 5. Strategy 5: Increase the Antiluteolytic Stimulation by the Conceptus Unit. During the critical period, conceptuses are undergoing a rapid increase in size. However, Thatcher and Hansen (43) found conceptuses horn 15 to 250 mm in Day 17 pregnant, lactating dairy cows. Smaller conceptuses will not be able to synthesize enough bIFN-r to block luteolysis. Therefore, pregnancies will be lost because of a transient inability of embryos to send antiluteolytic signals to the endometrium. One possible way to prevent such losses would be through administration of bIFN-z to cows during the critical period. Meyer et al. (25) administered twice daily intra-uterine infusions of recombinant bIF’N-z (rbIFN-r) to Holstein cows, from Days 14 to 24 of the estrous cycle. Infusions with rbIFN-r increased life span of the CL and duration of the estrous cycle compared to infusions of a control protein (bovine serum albumin). Unfortunately, there have not been any studies in which effects of rbIFN-t were tested in a large number of cows. Also, a more practical delivery route for administration of rbIFN-r (e.g., im injections) has not been available. Heat stress has been identified as one major cause of retardation of conceptus growth (43). High yielding lactating dairy cows in subtropical/tropical locations are especially susceptible to heat stress. In fact, pregnancy rates of such cows during the summer are drastically reduced (4). Therefore, management practices designed to reduce heat stress should result in increased conceptus size during the critical period and favor pregnancy recognition (15,43). Strategy 6: Decrease Luteolytic Response by the Maternal Unit. Specific inhibition of the PGFr,-synthesizing machinery in the uterus during the critical period should increase pregnancy recognition. Thatcher et al. (45) used a PGFr, synthesis
Theriogenology
inhibition assay to identify linoleic acid as an endometrial prostaglandin synthesis inhibitor. In vitro, linoleic acid inhibited the PGF*,-synthesizing capacity of a PGFza generator system through inhibiting COX-2 activity. Moreover, those authors demonstrated that there was a greater content of lmoleic acid and smaller content of arachidonic acid (a PGFza precursor) in the endometrium of Day 17 pregnant compared to Day 17 cyclic cows. Based on these findings, a possible strategy to decrease the luteolytic response of the uterus could be to change its lipid composition to increase the linoleic to arachidonic acid ratio in the uterus. Oldick et al. (30) administered abomasal infusions of yellow grease (high linoleic acid content) to lactating dairy cows. The linoleic acid-infused cows released less PGF2, in response to an oxytocin challenge given on Day 15 of a synchronized estrous cycle compared to cows receiving a control infusion. Feeding cows diets rich in linoleic acid constitute a practical alternative to inhibit PGFza synthesis in the uterus. However, lmoleic acid is biohydrogenated in the rurnen and special care must be taken with choosing feed ingredients. Wilkins et al. (51) fed beef cows with a diet containing forrnaline-treated cotton hulls, which is rich in protected linoleic acid (escapes biohydrogenation). Control cows received a palletized cotton hull meal diet. Conception rates to fast (61 vs. 46%) and second (71 vs. 56%) inseminations were higher to treated compared to control cows. Two other lipids with COX-2 inhibiting activity (18) and showing negligible ruminal biohydrogenation are eicosapentaenoic (EPA, C20:5, n-3) and docosahexaenoic (DHA; C22:6, n-3) acids (3). High concentrations of EPA and DHA are found in menhaden fishmeal. Thatcher et al. (41) supplemented lactating dairy cows with menhaden Ii&meal for 25 days and measured PGFM (a PGF2, metabolite easily measurable in plasma samples) plasma concentrations in response to an E2 injection followed by an oxytocin injection on Day 15 of a synchronized estrous cycle. The PGFM release was significantly reduced in cows fed fishrneal compared to cows fed control diets. Mattos et al. (21) fed diets containing 0, 2.6, 5.2 or 7.8% fishmeal for approximately 50 days. Similarly to results presented by Thatcher et al. (41), the PGFM response to an Ez-oxytocin challenge given on Day 15 of a synchronized estrous cycle was reduced for cows receiving fishmeal compared to control cows. Finally, another possible strategy to decrease uterine PGF2,qnthesizing ability is to treat cows with nonsteroidal anti-inflammatory drugs, such as flunixin meglumine (FM). Odensvik et al. (29) fed FM pellets to heifers twice, thrice or four times a day for 9 days, starting Day 14/15 of a synchronized estrous cycle. Compared to a previous, untreated estrous cycle of the same heifers, duration of estrous cycles increased from 18 to 22 to 20 to 24 days (heifers fed thrice daily) and from 18 to 21 to 25 to 27 days (heifers fed four times per day). For cows treated four times per day luteolysis only occurred after treatment was terminated. Therefore, administration of FM is an effective inhibitor of PGFza secretion during the critical period.
FINAL CONSIDERATIONS Embryonic mortality associated with the critical period is a complex and multifactorial issue. Understanding the biology of pregnancy recognition and luteolysis processes enabled animal scientists to come up with a series of antiluteolytic strategies with the objective of decreasing embryonic mortality associated with the critical period. Strategies presented here included
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endocrine and local manipulations to inhibit the maternal PGF2, production system while stimulating conceptus antiluteolytic capacity. Choice of most the appropriate strategy (or combination of strategie-) to be used depends on the physiologic state of the animal, resources available as well as economic considerations. Better antiluteolytic strategies will continue to emerge as long as more studies are performed to increase the understanding of physiology, cellular and molecular. biology of maternal-conceptus interactions during the critical period.
REFERENCES Ambrose JD, Pires MFA, Moreira F, Diaz T, Binelli M, Thatcher, WW Influence of Deslorelm (G&H-agonist) implant on plasma progesterone, first wave dominant follicle and pregnancy in dairy cattle. Theriogenology 1998; 50: 1157- 1170. 2. Arnold DA, Binelli M, Vonk J, Alexenco AP, Drost M, Thatcher WW. Intracellular regulation of endometrial PGFz, production in dairy cows during early pregnancy and following treatment with recombinant interferon-tau. Domest Anim Endocrinol 1999; 18:199-216. 3. Ashes JR, Siebert BD, Gulati SK, Cuthberston AZ, Scott TW. Incorporation of n-3 fatty acids of fish oil into tissue and serum lipids of ruminants. Lipids 1992; 27:629-63 1. 4. Badinga L, Collier RI, Thatcher WW, Wilcox CJ. Effects of climatic and management factors on conception rate of dairy cattle in sub-tropical environments, J Dairy Sci 1985; 68:78-85. 5. Baruselli PS, Marques MO, Carvalho NAT, Valentim R, Berber RCA, Carvalho Filho AF, Madureira EH, Costa Neto WP. Aumento da taxa de prenhez em receptoras de embri& bovino pela utiliza@o do protocolo “ovsynch” corn inovula@o em tempo furo. Arq. Fat. Vet. UFRGS 2000; 28:216 (abstr). 6. Baruselli P, Marques MO, Hothnann EM, Costa Neto WP, Grandinetti RR, B6 G. Increased pregnancy rates in embryo recipients treated with CIDR-B devices. Theriogenology 55:355 (abstr). 7. Battocchio M, Gabai G, Mollo A, Veronesi MC, Soldano F, Bono G, Cairoli F. Agreement between ultrasonographic classification of the CL and plasma progesterone concentrations in dairy cows. Theriogenology 1999; 5 1: 1059- 1069. 8. Bazer FW, Ott TL, Spencer TE. Pregnancy recognition in ruminants, pigs and horses. Signals from the trophoblast. Theriogenology 1994; 41:79-94. 9. Binelli M, Hampton, Buhi WC, Thatcher WW. Persistent dominant follicle alters pattern of oviductal secretory proteins from cows at estrus. Biol Reprod 1999; 61: 127- 134. 10. Bullman DC, Lamming GE. Milk progesterone levels in relation to conception, repeat breeding and factors influencing acyclicity in daii cows. J Reprod Fertil 1978; 54:447-458. Il. Burns PD, Graf GA Haynes SH, Silvia WJ. Cellular mechanisms by which oxytocin stimulates uterine PGFZalpha synthesis in bovine endometrium: roles of phospholipases C and A2. Domest Anim Endocrinol 1997; 14:181-191. 12. Chenault JR, Kratzer DD, Rzepkowsky RA, Goodwin MC. LH and FSH response of Holstein heifers to fertirelin acetate, gonadorelin and buserelin. Theriogenology 1990; 34:81-98. 1.
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13. Diaz T, Schmitt EJ-P, Thatcher M-J, Thatcher WW. Human chorionic gonadotropininduced alterations in ovarian follicular dynamics during the estrous cycle of heifers. J Anim Sci 1998; 761929-1936. 14. D&m MG, Sreenan JM. Fertilization and embryonic mortality rates in beef heifers after artificial insemination. J. Reprod Fertill980; 59:463-468. 15. Hansen PJ, Drost M, Rivera RM, Paula-Lopes FF, al-Katanani YM, Krininger CE 3’d, Chase CC Jr. Adverse impact of heat stress on embryo production: causes and strategies for mitigation. Theriogenology 2001; 55:91-103. 16. Kotwica J, Skarzynski D, Bogacki M, Melm P, Staroska B. The use of an oxytocin antagonist to study the function of ovarian oxytocin during luteolysis in cattle. Theriogenology 1997; 48:1287-1299. 17. Lamming GE, Darwash AO, Back HL. Corpus luteum timction in dairy cows and embryo mortality. J. Reprod Fertil 1989; 37:245-252. 18. Levine L, Worth N. Eicosapentaenoic acid-its effects on arachiconic acid metabolism by cells in culture. J Allergy Clin Immunol 1984; 74:430-436. 19. Mann GE, Lamming GE, Robinson RS, Wathes DC. The regulation of interferontau production and uterine receptors during early pregnancy. J. Reprod Fertil Suppl 1999; 54:317-328. 20. Marques MO, Madureira EH, Oliveira CA, Bo GA, Baruselli PS. Ovarian ultrasonography and plasma progesterone concentration in Bos taurus x Bos indicus heifers administered different treatments on Day 7 of the estrous cycle. Theriogenology 2002; submitted. 2 1. Mattos R, Staples CR Williams J, Amorocho A, McGuire MA, Thatcher WW. Uterine, ovarian and production responses of lactating dairy cows to increasing dietary concentrations of menhaden fish meal. J Dairy Sci 2001; submitted. 22. McCracken JA. Hormone receptor control of prostaglandin F2 alpha secretion by the ovine uterus. Adv Prostaglandin Thromboxane Res 1980; 8: 1329-l 344. 23. McCracken JA, Custer EE, Larnsa JC. Luteolysis: A neuroendocrine-mediated event. Physiological Reviews 1999; 79:263-324. 24. McCracken JA, Schramm W, Okulicz WC. Hormone receptor control of pulsatile secretion of PGFzo from ovine uterus during luteolysis and its abrogation in early pregnancy. Anim Reprod Sci 1984; 7:3 l-56. 25. Meyer MD, Hansen PJ, Thatcher WW, Drost M, Badinga L, Roberts RM, Li J, Ott TL, Bazer FW. Extension of corpus luteum life span and reduction of uterine secretion of prostaglandin Fza of cows in response to recombinant interferon-tau. J Dairy Sci 1995; 78:1921-1931. 26. Mihm M, Currant N, Hytell P, Boland MP, Roche JF. Resumption of meiosis in cattle oocytes from pre-ovulatory follicles with a short and long duration of dominance. J Reprod Fertil Abstr Ser 1994; 13-14. 27. Moura MT, Marques MO, Frare J, Madureira EH, B6 GA, Baruselli PS. Sincronizacb da ovula@o corn Crest& e CIDR@ para inovulacsI0 de embrioes bovinos em tempo fixo. 4’ Simposio International de Reproduction Animal 2001; 269 (abstr). 28. Nebel RL, McGilliard. Interactions of high milk yield and reproductive performance in dairy cows. J Dairy Sci 1993; 76:3257-3268. 29. Odensvik K, Gustafsson H, Kindahl H. The effect on luteolysis by intensive oral administration of flunixin granules in heifers. A&n Reprod Sci 1998; 50:35-44.
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30. Oldick BS, Staples CR, Thatcher WW, Gyawu P. Abomasal infusion of glucose and fateffect on digestion, production, and ovarian and uterine functions of cows. J Dairy Sci 1997; 80:1315-1328. 3 1. Peters AR. Embryo mortality in the cow. Anim Breed Abstr 1996; 64:587-598. 32. Pritchard JY, Shriek FN, Inskeep EK. Relationship of pregnancy rate to peripheral concentrations of progesterone and estradiol in beef cows. Theriogenology 1994; 42:247259. 33. Pursley JR, Mee MO, Wiltbank MC. Synchronization of ovulation in dairy cows using PGF20 and GnRH. Theriogenology 1995; 44:915-923. 34. Roberts RM, Xie S, Mathialagan N. Maternal recognition of pregnancy. Biol Reprod 1996; 54: 294-302. 35. Santos JEP, Thatcher WW, Pool L, Overton MW. Effect of human chorionic gonadotropin on luteal function and reproductive performance of high producing lactating Holstein dairy cows. J. Anim. Sci. 2001; in press. 36. Savio JD, Thatcher WW, Badinga L, de la Sota RL, Wolfeson D. Regulation of dominant follicle turnover during the estrous cycle in cows. J Reprod Fertil 1993; 97: 197-203. 37. Schmitt E J-P, Diaz T, Barros CM, de la Sota RL, Drost M, Fredriksson EW, Staples CR, Thomer R, Thatcher WW. Differential response of the luteal phase and fertility in cattle following ovulation of the fust-wave follicle with human chorionic gonadotropin or an agonist of gonadotropin-releasing hormone. J Anim Sci 1996; 74: 1074- 1083. 38. Sianangama PC, Rajamahendram R. Effect of human chorionic gonadotropin administered at specific times following breeding on milk progesterone and pregnancy in cows. Theriogenology 1992; 38:85-96. 39. Silvia WJ, Lewis GS, McCracken JA, Thatcher WW, Wilson L Jr. Hormonal regulation of uterine secretion of prostaglandin F2alpha during luteolysis in ruminants. Biol Reprod 1991; 45: 655-663. 40. Spell AR, Beal WE, Corah LR, Lamb GC. Evaluating recipient and embryo factors that affect pregnancy rates of embryo transfer in beef cattle. Theriogenology 2001; 56:287-297. 41. Thatcher WW, Binelli M, Burke J, Staples CR, Ambrose JD, CoeJho S; Antiluteolytic signal between the conceptus and endometrium. Theriogenology 1997; 47: 13 l-140. 42. Thatcher WW, Driancomt MA, Terqui M, Badmga L. Dynamics of ovarian follicular development in cattle following hysterectomy and during early pregnancy. Domestical Animal Endocrinology 1991; 8:223-234. 43. Thatcher WW, Hansen PJ. Systems to alter embryo survival. In: Van Horn HH, Wilcox CJ (eds), Large Dairy Herd Management. Champaign: Am. Dairy Sci. Assoc, 1992; 16-30. 44. Thatcher WW, Moreira F, Santos JEP, Mattos RC, Lopes FL, Pancarci SM, Risco CA. Effects of hormonal treatments on reproductive performance and embryo production. Theriogenology 2001; 55:75-89. 45. Thatcher WW, Staples CR Danet-Desnoyers G, Oldick B, Schmitt E-P. Embryo health and mortality in sheep and cattle. J. Anim. Sci. 1994; 72: 16-30. 46. Thatcher WW, Terqui M, Thimonier J, Mauleon P. Effect of estradiol-17t3 on peripheral plasma concentration of 15-keto-13,lCdihydro PGF2a and luteolysis in cyclic cattle. Prostaglandms 1986; 3 1:745-756.
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47. Vasconcelos JLM. Controle do estro e da ovula@o visando na insemina@o artificial em tempo fixo em bovinos de leite. In: Marques MO (ed), Controle kmacologico do ciclo estral em ruminantes. Sb Paulo: Funda@o da Faculdade de Medicina Veterinkia e Zoom&a, USP, 2000; 115-157. 48. Vasconcelos JLM, Sartori R, Oliveira HN, Guenther JG, Wiltbank M. Reduction is size of the ovulatory follicle reduces subsequent luteal size and pregnancy rate. Theriogenology 2001; 56:307-314. 49. Vasconcelos JLM, Silcox RW, Rosa GJM, Pursley JR, Wiltbank MC. 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 1999; 52:1067-1078. 50. Villa-Godoy A, Ireland JJ, Wortman JA, Ames NK, Hughes TL, Fogwel RL. Effect of ovarian follicles on luteal regression in heifers. J. Anim. Sci. 1985; 60:519-527. 51. Wilkins JF, Hoffman WD, Larsson SK, Hamilton BA, Hemressy DW, Hillard MA. Protected lipid/protein supplement improves synchrony of estrous and conception rate in beef cows. 13* Intern Congr Anim Reprod, Sydney, Australia, 1996;3: 19-29.
ANTILUTEOLYTIC
STRATEGIES TO IMPROVE FERTILITY IN CATTLE
M. Binellii’L W.W. Thatcher? R. Matto? .
and P.S. Barusellil
‘Departamento de Reproducb Animal, FMVZ-USP, Pirassununga, 13630-000. Brazil *Department of Animal Sciences, University of Florida, Gainesville, FL 3261 l-0920
ABSTRACT During early pregnancy, a “critical period” may be defmed between Days 15 and 17. Embryonic mortality associated with this period causes significant economic losses to the cattle industry. During this period, the endometrium will follow a default program to release luteolytic pulses of PGFza, unless the conceptus sends appropriate antiluteolytic signals to block PGFz,, production. Maintenance of pregnancy is dependent on a successml blockage of endometrial PGFz, production. Biology of the critical period is complex and multifactorial. Endocrine, cellular and molecular factors, both from maternal and conceptus origins act in concert to determine whether luteolysis or maintenance of pregnancy will prevail. Understanclmg the influences of such factors in the biology of the critical period allowed researchers to produce a series of strategies aiming to favor maintenance of pregnancy in lieu of luteolysis. Strategies include hormonal and nutritional manipulations to decrease plasma concentrations of estradiol 17p (Ez) while increasing those of progesterone (Pd), and inhibiting the PGFra-synthesizing enzymatic machinery in the endometrium during the critical period. Experimental results indicate that use of such strategies has improved pregnancy rates following artificial insemination and embryo transfer programs. 0 2001 by Elsevier Science Inc. Key words: embryonic mortality, luteolysis, pregnancy recognition, PGFz,, cattle
INTRODUCTION:
DEFINING THE “CRITICAL PERIOD”
The interval comprised between two estrus is defined as an estrous cycle, which averages 21 days in cattle. There are 2 or 3 waves of follicular growth during an estrous cycle. Each wave is characterized by phases of recruitment, selection, dominance and atresia. The dominant follicle of the last wave escapes atresia and secretes increasing quantities of Ez. High E2 stimulates an LH surge, which induces ovulation. The ovulated follicle undergoes structural and functional changes, originating the CL. The CL grows quickly in size and secretes increasing quantities of Pd. At around Day 16 of the cycle (Day 0 = es&us), the uterine endometrium starts secreting pulses of PGFza, which causes both structural and functional demise of the CL (the process of luteolysis). Functional luteolysis results in a ready decrease in circulating Acknowledgments “Correspondence and reprint requests. Research was supported partially by FAPESP, Brazil. Authors would lie to thank Dr. Jose Luiz Moraes Vasconcelos, Dr. Ed Hofhnann Madureira e Mr. M&rcio de Oliveira Marques for critically revising this manuscript. Theriogenology 56:1451-1463,200l 0 2001 Elsevier Science Inc.
0093-691 X/O1 /$-see front matter PII: SOO93-691X(01)00646-X
Theriogenology
concentrations of Pa. Such low P4 concentrations allow the final growth of a dominant, preovulatory follicle and stimulate estrus behavior and ovulation. If the ovulated oocyte is not fertilized, this cycle will be repeated and luteolysis will occur again. However, if the oocyte is fertilized, the luteolytic process must be blocked (at around Day 16) to maintain pregnancy. Thus, a “critical period“ in the reproductive cycle of the cow could be defined at around Days 15 and 17 afler estrus. During this period, the cow should adjust its physiology properly, depending on whether it is pregnant or not. Changing corn a cyclic to a pregnant state (the process of pregnancy recognition) depends on an effective blockage of luteolysis. Blocking luteolysis is dependent on the ability of the conceptus to send effective antiluteolytic signals and on the capacity of the endometrium to respond to such signals, thus blocking PGFza production. Such communications between conceptus and maternal units are not always successful. In fact, embryonic mortality between days 8 and 16 of pregnancy approaches 30% (14). Such high mortality rate has obvious economic impacts in a cattle operation, increasing number of days open and retarding genetic progress. Therefore, a large quantity of research has been put forth to understanding the biology of the critical period, and several practical strategies have been proposed. Objectives of this paper are (1) to review local and endocrine regulation of luteolysis and pregnancy recognition and (2) to identify mechanisms that could be manipulated to decrease embryonic mortality associated with the critical period
REGULATION OF LUTEOLYSIS AND PREGNANCY RECOGNITION This topic is extremely complex and several excellent reviews are available for the reader (8,11,22,23,34,39,41,45). Discussion in this paper will be limited to concepts used in the formulation of antiluteolytic strategies discussed in the next section. In sheep, it has been proposed that initiation of luteolysis is dependent on E2-stimulated synthesis of endometrial oxytocin receptors during the critical period (24). Binding of oxytocin stimulates pulsatile release of PGF2,. In cattle, recent studies have demonstrated that oxytocin might not be essential for the initiation of luteolysis (16). However, presence of Ez is critical for luteolysis to occur. Indeed, removal of ovarian follicles by cauterization or X-irradiation delays luteolysis and increases duration of estrous cycles in cows (50). Moreover, injections of Ez stimulate PGFza secretion (46). Therefore, blocking of luteolysis during pregnancy recognition may involve inhibition of E2 production and its effects. For example, plasma concentrations of Ef are lower in pregnant animals than in cyclic animals (32). Furthermore, follicle growth and E2 production per follicle am less in pregnant compared to cyclic cows (42). Although cellular and molecular mechanisms by which E2 stimulates PGFza secretion are not completely elucidated, it is clear that it plays a central role on the process of luteolysis. Therefore, an important concept for developing antiluteolytic strategies is that decreasing circulating concentrations of EZ during the critical period should inhibit or delay luteolysis. Circulating concentrations of P4 seems to be positively correlated with pregnancy recognition in cattle. There is higher concentration of milk P4 in cows inseminated and pregnant compared to cows inseminated and not pregnant (IO, 17,19). This suggests that high P4 during the critical period is important for maintenance of pregnancy.
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It is well established that fertility and milk production are negatively associated in dairy cows (for discussion see 28,49). Vasconcelos (47) indicated that high-yielding dairy cows have lower circulating concentrations of Pq than low producers. This may be because higher-yielding cows have a greater rate of Pb catabolism. Perhaps lower circulating P4 could at least partly explain the lower fertility associated with high milk producers. One main regulator of pregnancy recognition is the conceptus-secreted bovine interferon-tau (bIFN-r). The bIFN-t is secreted in the uterine lumen during the critical period and interacts with the endometrium to block PGF2alpha synthesis. One possible mechanism through which P4 stimulates maintenance of pregnancy may be through stimulation of bIFN-r secretion. Indeed, Mann et al. (19) have shown that cows with a greater concentration of plasma P4 during the critical period produced conceptuses that were larger and produced more bIFN-2. These data imply that a second concept may be suggested: increasing concentrations of P4 during the luteal phase and the critical period should stimulate pregnancy recognition. Until the time of pregnancy recognition, uterine physiology is very similar between pregnant and cyclic animals, and a default PGF za synthetic program is in place in the endometrium. The practical outcome of this notion is that unless PGF2, synthesis is actively blocked, luteolysis will ensue regardless of the presence of a conceptus. Blocking PGFza production requires that the elongating conceptus occupy most of the uterine horn ipsilateral to the CL at the critical period. Thatcher and Hansen (43) demonstrated that Day 17 conceptuses varied in size from 15 to 250 mm. The implication of this observation is that luteolysis will occur even in the presence of conceptuses, if they are not large enough. Also, secretion of conceptus-derived paracrine factors, such as bIFN-r, is necessary for the inhibition of PGFza. Synthesis of PGF2, results horn the activation of a complex intracellular cascade of enzymatic reactions (11). The rate limiting reaction is the conversion of arachidonic acid to pmstaglandin Hz, catalyzed by the enzyme cyclooxygenase-2 (COX-2). To test the hypothesis that exposure to conceptus tissues down-regulated the PGFz,-synthesis cascade, Arnold and et al. (2) compared in vitro responsiveness of explants obtained from cyclic and pregnant cows on Day 15 after estrus to intracellular stimulators of PGFzu synthesis. Regardless of which enzymatic step was stimulated, pregnant tissue always yielded less PGFza compared to cyclic tissue. In summary, strategies that favor the presence of elongated and functional conceptuses and/or inhibit the endometrial PGF2,-synthesizing enzymatic machinery during the critical period should increase pregnancy rates in cattle. Altogether, this information allows a third concept to be stated: conceptus and endometrial functions could be manipulated to generate a microenvironment that is less conducive to luteolysis.
ANTILUTEOLYTIC
STRATEGIES
Based on the concepts stated above, a series of antiluteolytic strategies are proposed below. Figure 1 summarizes six possible strategies to favor pregnancy recognition in lieu of luteolysis.
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Strategy 1: Increase Size of Pre-ovulatory Follicle to Generate a Larger CL. Larger CLs will secrete more PJ and this may have a positive effect on pregnancy recognition, and consequently pregnancy rates both in artificial insemination (47, 48) and embryo transfer programs (5). For example, Baruselli et al. (5), synchronized embryo recipients with either a single PGF injection or following the “Ovsynch” protocol (33). Regardless of treatment, authors found a positive association between area of CL and Pq plasmatic concentration on Day 6 after estrus. Moreover, when recipients were classified according to CL size, heifers with larger CLs also showed higher conception rates (Table 1). In contrast, work by Battocchio et al. (7) demonstrated that CL volume detected by ultrasonography may not be a good criteria for characterizing the CL capacity to secrete Pa. In addition, Spell et al. (40) did not observe differences in CL size or plasma Pd concentrations between recipients that did and did not become pregnant atIer embryo transfer. In spite of those discrepant data, it is proposed that it could be advantageous to induce a large pre-ovulatory follicle aiming to obtain a larger CL in embryo recipients. The classical way to obtain a large follicle is to promote follicle growth under low, subluteal circulating concentrations of Pd or progestagens, which causes follicles to become persistent (36). Conception rates after artificial insemination associated with persistent dominant follicles are usually low, because of an altered oviductal environment (9), a premature maturation of the oocyte (26) or both. However, this would not be the case for embryo recipients, in which the oocyte coming from a persistent follicle would not be fertilized. Because dominant follicle size is associated with subsequent CL size (47, 48), a large persistent follicle could generate a large CL, which would provide high Pd concentration to favor development of the transferred embryo. To test this hypothesis, Moura et al. (27) treated crossbred recipient heifers with a norgestomet implant for 9 days. On Day 0, implants were administered and recipients were randomly assigned to receive 5 mg estradiol valerate and 3 mg norgestomet im (Group 1; s40) or 400 pg of delprostenate (a PGFr, analog; Group 2; n=42). On Day 9 all cows received a delprostenate injection and ovulation was induced by an estradiol benzoate injection (1 mg) on Day 10. It was expected that treatment administered to heifers in Group 1 should induce ovulation of a freshly recruited dominant follicle while treatment administered to heifers in Group 2 should induce the growth and ovulation of a pre-existent follicle (i.e., persistent). Ovaries were examined by ultrasonography on Day 10 (for dominant follicle measurement) and on Day 17 (for CL measurement). Results of this experiment indicated, in comparison to Group 1, heifers in Group 2 developed a larger size dominant follicle (1 .OOf 0.13 vs. 1.25 f 0.23cm) which resulted on CLs with larger diameter (1.54 f 0.17 vs. 1.81 f 0.12cm) and higher Pq concentrations on Day 17 (1.98 f 1.28 vs. 2.96 f 2.33ng/ml). Percent transferable recipients (i.e., cows presenting CL > 1.3cm) was also higher to cows in Group 2; (52.5 vs. 78.6) but pregnancy rates were not statistically different between Groups 1 (25.0%) and 2 (30.9%).
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-21 (ovul.)
10
15
:21
&.) ' Q,6
Figure 1. Summary of antiluteolytic strategies. Strategies are numbered 1 to 6 across an interval of time representing an estrous cycle followed by artificial insemination (AL) and Ovulations (ovul.) are indicated at Days -21 and 0. Progesterone pregnancy. concentrations are represented by dotted lines, dominant follicles by open circles, dominant ovulatory follicle by two superimposed circles (open and closed) and the critical period by a gray box. Strategies are: (1) increase size of pre-ovulatory follicle to generate larger CL, (2) increase rate of growth of CL, (3) increase luteal phase progesterone, (4) decrease effect of a dominant follicle on the critical period, (5) increase antiluteolytic stimulation-conceptus unit and (6) decrease luteolytic response-maternal unit.
Table 1. Progesterone concentration and conception rates according to CL area at Day 6 of the estrous cycle in crossbred recipient heifers (mean 2 standard deviation). CL area n Progesterone Average CL area Conception rate (ultrasonography) concentration (ng/mL) (cm*) -% 77 2.44 + 0.86’ CL1 (> 2.Ocm’) 2.66 + 0.51 47/77 (58.4 %)d CL2 (1.5 a2.0 cm’) 41 1.75 + 0.69b 1.74kO.10 17/41 (41.5 %)” 0.96,.z+ 0_:.-...-. 56’ CL3 1(<.l___.__l_l_ l.Scm*) 07/22 .-._22“__...,_ .-_,. ,._-- 1.19 + 0.20 __.-._“_l “_1(3 1.8 %>’ ‘*‘a6”Geanswithin columns with different superscripts differ sign&k&y (PC 0.01) defPercentages within columns with different superscripts differ significantly (PcO.05)
Strategy 2: Increase Pate of Growth of the CL. Optimal differentiation and rate of growth of the CL is related to duration and amplitude of the ovulatory LH surge (1). Induced LH surges, for example by injections of GnRH in
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Ovsynch protocols, are oflen of lower duration than endogenous, noninduced surges (12), which could limit optimal CL growth. Ambrose et al. (1) stimulated ovulation of nonlactating Holstein cows with a Deslorelin (a GnRH agonist) implant, as part of an Ovsynch program. They observed that implanted cows had a greater rate of CL growth and greater plasma Pq concentrations compared to a bolus injection of another G&I-I agonist (Buserelin). They concluded that the prolonged release of GnRH provided by the Deslorelin implant induced a LH surge of longer duration and thereby stimulated CL growth and function. Strategy 3: Increase Luteal Phase PJ. Several methods have been used aiming to increase conception rates through a greater plasma PI concentration during the luteal phase (see 44 for a review). Increased plasma Ps concentrations can be achieved by inducing an accessory CL. Injection of hCG stimulates formation of an accessory CL (20,35,37,38). Santos et al. (35) treated high producing lactating Holstein cows with either saline (n=203) or hCG (n=203) on Day 5 of a synchronized estrous cycle. Ultrasonography and blood sampling were performed from Days 11 to 16 after AI and presence of conceptuses was accessed by ultrasonography on Day 28 and by rectal palpation on Days 45 and 90. Treatment with hCG induced formation of accessory CLs in 86.2% of cows, compared to 23.2% in controls. Injection of hCG increased plasma P.r by 5 ng/mL and conception rates on Days 28 (45.8 vs. 38.7%), 45 (40.4 vs. 36.3%) and 90 (38.4 vs. 31.9%), compared to saline injection. Marques et al. (20) compared the efficacy of saline, LH, GnRH analog (Buserelin) or hCG injections on CL parameters and Pd plasmatic concentrations. Crossbred beef heifers were injected on Day 7 of a synchronized estrous cycle. Results on Table 2 indicate that all LH-, G&I-I- and hCG-treated heifers formed accessory CLs. However, main and accessory CL diameters and average plasma Pd concentration (Days 13 to 17) were higher for hCG treated cows compared to other treatments. Since hCG has a longer half-life than GnRH and LH, it can persist in the circulation longer. Therefore, besides inducing formation of accessory CLs, hCG may exert luteotropic effects by directly stimulating Pq production, which may increase pregnancy rates.
Table 2. Main and accessory CL diameter between Days 13 to 17 and plasma progesterone concentration in crossbred heifers treated with GnRH, hCG or LH at Day 7 of the estrous cycle (mean If standard deviation; D MainCL Main CL Progesterone Group n diameter (Days concentrations diameter diameter 13-17; cm) (Days 13 -17; cm) @ays 13 -17; ng/mL) (Day 6; cm) 1.55 + o.22a 4.79 _+2. lga 1.84 + 0.35 1.78 + 0.22b GnRH 8 8.42 t 3.64b 2.19&0.31’ 1.75 _+0.2gb hCG 8 1.79 + 0.32 6.07 2 3.09’ LH 7 1.69+0.17 1.79 + O.lSb 1.61 + 0.16’ . ._ .-.-_-____(___r ._ “._.I- ..,..____~-_-__.-lt-..-._“X_. .- -_.- _^-‘X’&ms within columns with different superscripts differ significantly (PC O.Gs)‘-“‘“- __ ..
Another possible way to stimulate higher plasma Pd concentrations during the luteal phase is through eCG injections. The eCG may exert both LH and FSH effects in cattle. Therefore, injecting eCG during the emergence phase of a synchronized follicular wave may: (1)
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1457
increase the rate of growth of follicles, resulting on a larger pre-ovulatory follicle. Such larger follicle should result on a larger CL. (2) Stimulate simultaneous growth of multiple follicles that can be induced to ovulate and form multiple CLs. In both cases high Pq concentrations would be expected after ovulation. Baruselli et al. (6) synchronized recipient crossbred heifers at random stages of the estrous cycle with injections of estradiol benzoate and P4 and insertion of a CIDR device (Day 0). Five days later heifers were randomly assigned to receive either 800 IU of eCG or nothing. On Day 7, implants were removed and recipients received and injection of cloprostenol (PGF2alpha analog). Ovulations were induced by an injection of estradiol benzoate on Day 8, ovaries were examined by ultrasonography on Days 8 and 15 and embryos were transferred on Day 16. Results on Table 3 indicate that number of follicles greater than 8 mm on Day 8, total number of CLs, number of CLs greater than 13 mm (i.e., criteria for selecting recipients to receive embryos), Pd concentration on Day 15, conception and pregnancy rates were higher for cows treated with eCG compared to controls. Moreover, eCG-treated heifers bearing a single CL showed larger CL areas, Pd concentrations and conception rates than controls. Authors concluded that eCG treatment increased Pd plasmatic concentration and consequently pregnancy rates because: (1) it stimulated follicle growth and consequently number of CLs and (2) it stimulated size of individual CLs. Similar results presented by B6 et al. support such conclusions.
Table 3. Effects of eCG in follicle and CL size, CL function and reproductive effkiency of recipient heifers (mean + standard deviation). Controls eCG 0.6 If:0.5” Number of follicles >8rnm at Day 8 2.04 t 1.4b 0.5 k 0.51” Number of CLs at Day 15 2.58 5 2.93b 1.35 + 0.78’ Progesterone concentration at Day 15 (ng/mL) 4.17 * 3.73d % heifers with CLs >I3 mm at Day 8 17/50 (34.0%)’ 42/50 (84.0%~)~ Conception rates 5117 (29.4%) 21/38 (55.3%) Pregnancy rates 5/50 (1 o.O%)c 21/50 (42.0%)d “means within rows with different superscripts differ significantly (PC 0.01). “dMeans within rows with different superscripts differ significantly (PC 0.05). “bpercentages within rows with different superscripts differ significantly (PC 0.01). CdPercentages within rows with different superscripts differ significantly (PC 0.05).
A point for consideration regarding the use of hCG and eCG in antiluteolytic programs is that effectiveness of these hormones may decreases with multiple uses in the same cow. This is because they are antigenic molecules to cattle and may elicit antibody formation. Therefore, maximum effectiveness is achieved when animal is naive and may be reduced as antibody titers increase in the circulation. Strategy 4: Decrease the Effect of a Dominant Follicle on the Critical Period. Eliminating or decreasing the size of a follicle during the critical period should decrease circulating concentrations of Ez and consequently decrease the luteolytic stimulation associated with Er. For example, a strategic injection of GnRB could turn over a dominant follicle that may
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be present during the critical period. Follicle turnover will reduce Er concentrations and favor pregnancy recognition. Peters (3 1) summarized results of six studies that analyzed the effects of GnRH injections between Days 11 and 13 of the estrous cycle on pregnancy rates. Results ranged from -3.9 to 12% improvement in pregnancy rates for cows injected with GnRH compared to controls. In the study conducted by Ambrose et al. (described above; I), authors observed that the Deslorelin implant delayed appearance of Class III follicles (>9mm) to 14.2 days compared to 4.6 days, probably due to a down-regulation of LH secretion. Absence of large follicles during the critical period probably maintained low plasma E2 concentrations, which is favorable to pregnancy recognition. In fact, granted the number of cows used were small, treatment with Deslorelin significantly increased circulating concentrations of P4 and pregnancy rates (5/8) compared to Buserelin-treated cows (l/8). Besides stimulating luteal area and function, treatment with hCG on Day 5 also reprograms follicular growth to increase percentage of 3- compared to 2-wave cycles (13). This decreases the probability of cows having a large, highly estrogenic follicle during the critical period and may also contribute to higher conception rates observed by Santos et al. (35) for cows receiving hCG injections on Day 5. Strategy 5: Increase the Antiluteolytic Stimulation by the Conceptus Unit. During the critical period, conceptuses are undergoing a rapid increase in size. However, Thatcher and Hansen (43) found conceptuses horn 15 to 250 mm in Day 17 pregnant, lactating dairy cows. Smaller conceptuses will not be able to synthesize enough bIFN-r to block luteolysis. Therefore, pregnancies will be lost because of a transient inability of embryos to send antiluteolytic signals to the endometrium. One possible way to prevent such losses would be through administration of bIFN-z to cows during the critical period. Meyer et al. (25) administered twice daily intra-uterine infusions of recombinant bIF’N-z (rbIFN-r) to Holstein cows, from Days 14 to 24 of the estrous cycle. Infusions with rbIFN-r increased life span of the CL and duration of the estrous cycle compared to infusions of a control protein (bovine serum albumin). Unfortunately, there have not been any studies in which effects of rbIFN-t were tested in a large number of cows. Also, a more practical delivery route for administration of rbIFN-r (e.g., im injections) has not been available. Heat stress has been identified as one major cause of retardation of conceptus growth (43). High yielding lactating dairy cows in subtropical/tropical locations are especially susceptible to heat stress. In fact, pregnancy rates of such cows during the summer are drastically reduced (4). Therefore, management practices designed to reduce heat stress should result in increased conceptus size during the critical period and favor pregnancy recognition (15,43). Strategy 6: Decrease Luteolytic Response by the Maternal Unit. Specific inhibition of the PGFr,-synthesizing machinery in the uterus during the critical period should increase pregnancy recognition. Thatcher et al. (45) used a PGFr, synthesis
Theriogenology
inhibition assay to identify linoleic acid as an endometrial prostaglandin synthesis inhibitor. In vitro, linoleic acid inhibited the PGF*,-synthesizing capacity of a PGFza generator system through inhibiting COX-2 activity. Moreover, those authors demonstrated that there was a greater content of lmoleic acid and smaller content of arachidonic acid (a PGFza precursor) in the endometrium of Day 17 pregnant compared to Day 17 cyclic cows. Based on these findings, a possible strategy to decrease the luteolytic response of the uterus could be to change its lipid composition to increase the linoleic to arachidonic acid ratio in the uterus. Oldick et al. (30) administered abomasal infusions of yellow grease (high linoleic acid content) to lactating dairy cows. The linoleic acid-infused cows released less PGF2, in response to an oxytocin challenge given on Day 15 of a synchronized estrous cycle compared to cows receiving a control infusion. Feeding cows diets rich in linoleic acid constitute a practical alternative to inhibit PGFza synthesis in the uterus. However, lmoleic acid is biohydrogenated in the rurnen and special care must be taken with choosing feed ingredients. Wilkins et al. (51) fed beef cows with a diet containing forrnaline-treated cotton hulls, which is rich in protected linoleic acid (escapes biohydrogenation). Control cows received a palletized cotton hull meal diet. Conception rates to fast (61 vs. 46%) and second (71 vs. 56%) inseminations were higher to treated compared to control cows. Two other lipids with COX-2 inhibiting activity (18) and showing negligible ruminal biohydrogenation are eicosapentaenoic (EPA, C20:5, n-3) and docosahexaenoic (DHA; C22:6, n-3) acids (3). High concentrations of EPA and DHA are found in menhaden fishmeal. Thatcher et al. (41) supplemented lactating dairy cows with menhaden Ii&meal for 25 days and measured PGFM (a PGF2, metabolite easily measurable in plasma samples) plasma concentrations in response to an E2 injection followed by an oxytocin injection on Day 15 of a synchronized estrous cycle. The PGFM release was significantly reduced in cows fed fishrneal compared to cows fed control diets. Mattos et al. (21) fed diets containing 0, 2.6, 5.2 or 7.8% fishmeal for approximately 50 days. Similarly to results presented by Thatcher et al. (41), the PGFM response to an Ez-oxytocin challenge given on Day 15 of a synchronized estrous cycle was reduced for cows receiving fishmeal compared to control cows. Finally, another possible strategy to decrease uterine PGF2,qnthesizing ability is to treat cows with nonsteroidal anti-inflammatory drugs, such as flunixin meglumine (FM). Odensvik et al. (29) fed FM pellets to heifers twice, thrice or four times a day for 9 days, starting Day 14/15 of a synchronized estrous cycle. Compared to a previous, untreated estrous cycle of the same heifers, duration of estrous cycles increased from 18 to 22 to 20 to 24 days (heifers fed thrice daily) and from 18 to 21 to 25 to 27 days (heifers fed four times per day). For cows treated four times per day luteolysis only occurred after treatment was terminated. Therefore, administration of FM is an effective inhibitor of PGFza secretion during the critical period.
FINAL CONSIDERATIONS Embryonic mortality associated with the critical period is a complex and multifactorial issue. Understanding the biology of pregnancy recognition and luteolysis processes enabled animal scientists to come up with a series of antiluteolytic strategies with the objective of decreasing embryonic mortality associated with the critical period. Strategies presented here included
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endocrine and local manipulations to inhibit the maternal PGF2, production system while stimulating conceptus antiluteolytic capacity. Choice of most the appropriate strategy (or combination of strategie-) to be used depends on the physiologic state of the animal, resources available as well as economic considerations. Better antiluteolytic strategies will continue to emerge as long as more studies are performed to increase the understanding of physiology, cellular and molecular. biology of maternal-conceptus interactions during the critical period.
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