Characterization of early embryonic death and prevention of pregnancy wastage

Up to one third of fertilized eggs may not result in live calves due to early embryonic death losses (within 42 days of insemination). Metabolic and hormonal effects appear to be involved in embryonic losses Use of timed insemination protocols may reduce embryonic losses.

Introduction

In high producing dairy cows, herd pregnancy rates are often reduced due to poor estrus expression and/or detection, anestrus, low conception rates and increased embryo mortality. Furthermore, these problems are exacerbated under stressful environmental conditions such as heat stress, which is even more detrimental in higher milk producing cows. Reproductive performance has decreased in North America, Europe, Israel and Australia. There are a number of reasons for this decline and not all are due to increases in milk production (1).

Epidemiological studies indicate that factors such as reproductive diseases (i.e., retained placenta, metritis and ovarian cysts) or season of calving had a bigger effect than milk yield on reproductive performance (1, 2). In fact, higher producing herds can have better reproductive performance because of better nutrition, reproductive management and healthier cows. Nevertheless, the physiological state of lactation is associated with a lower reproductive rate compared to heifers (3). The challenge to characterize the factors compromising embryo development and developing strategies to improve embryo survival is complex involving steroidogenesis, cell proliferation, follicle development, ovulation, fertilization, corpus luteum development and maintenance, oviductal and uterine functions, embryo development and function, implantation and subsequent fetal growth. Indeed, our current day production and reproductive management systems impact on all of these coordinated events which need to be optimized to improve reproductive efficiency of lactating dairy cows.

Objectives of this presentation are to characterize embryo development and losses, to identify physiological windows that may be impaired and associated with embryo wastage, and to identify strategies to improve pregnancy rates.

Embryo Development and Losses

Estimates of fertilization rates in dairy heifers fall within a range of 97-100%. Estimates in dairy cows are more variable within a range of 85 to 100%, but these estimates were determined over 25 years ago, and it was not always clear whether the cows were lactating or not and, if so, what was the level of production (4).

Sreenan et al., (4) summarized the literature for staged estimates of embryo losses from the earlier literature (i.e., > 21 years ago). Fertilization rates were estimated to be 90% and average calving rates of about 55%. This suggests an embryonic and fetal mortality rate of about 39%. Very few embryos are lost in the days immediately after fertilization and up to day 8 of gestation. A significant increment of the total losses (27 - 31%) occurred between days 8 and 16 after insemination, 3.8% of the total occurred between days 16 and 42, and a further 1.9 to 3.1% occurred between day 42 and parturition. A major question is whether the temporal pattern of embryonic and fetal losses has changed in the current populations of high producing, lactating Holstein dairy cows.

Several recent reports have evaluated fertilization rates in modern day, high producing dairy cows whose overall fertility is low. A recent report (6) vividly demonstrates the negative impact of lactation on early embryo development compared to nonlactating dairy cows. In a temperate environment, fertilization rates, estimated at day 5 after ovulation, were 87.8 and 89.5% for lactating and nonlactating cows, respectively. However, day 5 embryos from lactating dairy cows were detectably inferior (lower quality embryo score, and lower percentage of excellent-good-fair embryos [52.8%] compared to embryos from nonlactating cows [82.3%]).

From a practical perspective, we can divide embryonic losses (EL) into early (EEL) and late (LEL) embryonic losses. Each can be monitored with such techniques as milk progesterone (P4), pregnancy specific protein B (PSPB; a protein secreted by the binucleate cells of the trophoectoderm), ultrasound (US) and rectal palpation.

Humblot (8) proposed that luteolysis within 24 days after AI could be associated with either a lack of fertilization or to early embryonic mortality (EEM) that did not allow the CL to be maintained. In contrast, extended CL maintenance and return to estrus after 24 days could be associated with late embryonic mortality (LEM) occurring at or more than 16 days after AI.

In a study involving 44 dairy herds (1395 Holstein cows) of low fertility in a temperate environment of France, pregnancy rate, EEM and LEM rates after first AI were 42.9 (599/1395), 31.6 (441/1395) and 14.7% (209/1395), respectively (8). Several factors were associated with pregnancy rate (interval to first AI: >90 day, 46.6% vs < 90 days, 41.5%; parity: primiparous, 47.5% vs lactation 2+3, 42.7% vs > 4 lactations, 34.6%; milk yield: > 39 kg/d, 34.9%, < 39 kg/d, 45.5%). Interestingly, LEM was greater in high producing dairy cows with BCS > 2.5 but there was no association with BCS in lower producing cows.

On three commercial dairy farms in California, cows inseminated either following estrus detection or at fixed time with an OvSynch protocol had similar losses of pregnancy (13.7 vs 11.7%). Estimates in two other studies (13,15), involving AI following estrus detection or TAI with Heatsynch or OvSynch protocols, reported that pregnancy losses between 31 and 45 days did not differ. Controlled breeding programs that are properly implemented do not seem to affect pregnancy losses when compared to insemination following estrus detection. This does not mean that the current systems can not be improved. However, TAI does not seem to result in increased pregnancy loss.

In a study in Ireland (17) pregnancy losses between 28 and 84 d after AI were similar for grazing lactating cows producing 7247 kg of milk/year compared to heifers (7.2% vs 6.1%, respectively). Of the losses detected, 47.5% occurred between days 28 and 42 of gestation. It is interesting that in this production system of grazing and lower absolute milk production, that embryonic losses are considerably lower than reported above in intensely managed dairy herds. The extent of embryonic loss was greater in cows that lost body condition between days 28 and 56 of pregnancy compared to cows maintaining or gaining in body condition.

Physiological Problems That May Contribute To Pregnancy Losses

In current production systems, many factors and interactions may influence reproductive performance.

Cycle prior to insemination and periovulatory period:

Several reports indicate that low plasma progesterone concentrations during the luteal phase of the estrous cycle preceding AI was associated with lower fertility than cows with high plasma progesterone concentrations. Progesterone concentrations can influence several physiological events such as ovarian follicular dynamics and subsequent uterine function.

Higher rates of liver blood flow and steroid metabolism in lactating dairy cows may reflect the chronic effects of higher feed intakes leading to lower steroid concentrations. The lower concentrations of progesterone and estradiol in lactating dairy cows compared to nonlactating dairy cows (19, 20) appear to influence ovarian follicular dynamics. Evidence across these reports support the concept that lactating dairy cows have a larger number of larger follicles, a larger ovulatory follicle, lower concentrations of estradiol and a longer interval to ovulation. Of critical importance is the subsequent ability of the oocyte arising from such follicles to form normal embryos.

Day 5 embryo quality has been shown to be reduced in lactating dairy cows (6). Greater clearance of estradiol in high producing lactating dairy cows may result in less inhibition of FSH secretion that would alter follicular deviation leading to a greater occurrence of double ovulations (21). Furthermore, lower concentrations of progesterone may influence LH pulsatility and lead to longer persistence of the dominant follicle.

Israeli investigators (22) proposed that low progesterone concentrations during an estrous cycle may have a delayed stimulatory effect on uterine responsiveness to oxytocin during the late luteal phase of the subsequent cycle that approaches the time when the embryo initiates maintenance of the CL. It is clear that the preovulatory/periovulatory changes in steroids influence subsequent functions of the oviduct, uterus and ovary.

An additional factor associated with the preovulatory period that reduces fertility is the development of persistent follicles. Again this is coupled with the lower concentrations of progesterone, which predisposes the cow to a higher LH pulse frequency and maintenance of the dominant follicle. When a persistent follicle ovulates, the oocyte is at a later stage of maturation (24). The oocyte undergoes fertilization but early embryonic death occurs. It is important to recognize this phenomenon when we deal with types of synchronization systems. We need to use synchronization and/or ovulatory control systems that do not entail long periods of low progesterone exposure and promote induction of a new dominant follicle that is induced to ovulate before expressing dominance beyond a 5-day period. Period of follicular dominance longer than 8 days is associated with reduced fertility (25).

Normally cows have two or three wave cycles. A recent report (26) indicated that fertility was greater in lactating cows inseminated after ovulation of the third-wave follicle that had developed for fewer days of the estrus cycle (81% pregnancy rate) as compared with two-wave cows (63% pregnancy rate). The longer developed ovulatory follicle of the second wave group should not be considered a persistent follicle, but it does emphasize the potential importance of recruiting a fresh follicle as part of a synchronization or timedinsemination program.

Post ovulatory cycle:

Elevation of progesterone soon after ovulation may advance maturation of the uterine endometrium and accelerate growth of the developing embryo (27). It is clear that lactation is reducing luteal phase progesterone concentrations. The reduced postovulatory concentrations in progesterone of lactating dairy cows may reduce embryo development (29) and thereby reduce interferon t production by the subsequent filamentous embryo that would contribute to both EEL and LEL.

An additional factor influencing both EEL and/or LEL is the occurrence of mastitis. Cows that had clinical mastitis during the first 45 d of gestation were 2.7 times at higher risk of abortion within the next 90 d (32). Both days open and services per pregnancy were increased in cows with clinical mastitis that occurred between first service and establishment of pregnancy (33).

Strategies to Improve Pregnancy Rates

Development and Optimization of Timed Insemination Programs:

Effective estrus synchronization programs provide a number of advantages: cows or heifers are in estrus at a predicted time which facilitates AI, and embryo transfer; time and labor expense for detection of estrus are reduced; AI becomes more practical under extensive conditions; and timed insemination may occur without need for detection of estrus.

The ability to control the time of ovulation precisely permits a timed insemination, following a period in which follicular development and CL regression have been programmed sequentially. With the implementation of fixed timed inseminations, specific timed treatments to improve embryo survival can be implemented effectively. Such programs are essential in high producing dairy cows that experience a reduction in estrus intensity that contributes to undetected heats, reoccurring luteal phases without estrus expression, or re-occurring waves of follicles that fail to ovulate.

OvSynch:

One program that has been extremely successful for insemination of cows at a fixed time for first service without the need for detection of estrus is the Ovynch program in which injections of GnRH are given 7 d before and 48 h after an injection of PGF2á, and cows are inseminated 12 to 16 h after the second injection of GnRH. This system synchronizes follicle maturation with regression of the corpus luteum before the GnRH-induced ovulation and timed insemination. Numerous studies indicate that pregnancy rates (proportion of all treated cows that were pregnant) to the OvSynch program were comparable and in some studies greater than the appropriate control group (see review 34).

There are several stages of the estrous cycle when initiation of the OvSynch program causes reduced pregnancy rates. Initiation of the program between days 13 to 17 of the cycle will produce asynchronized cows that may ovulate prior to the time of insemination and insemination will be to late for the cow to conceive. During the early stages of the cycle (e.g., days 2 to 4), the recruited dominant follicle is not sufficiently developed to ovulate in response to GnRH. Collectively, these findings indicate that presynchronization of cows prior to implementation of the OvSynch program should improve pregnancy rates if cows enter the OvSynch program at the most favorable period of the estrous cycle (i.e., days 5 to 12 of the cycle).

Presynch-OvSynch:

A program defined as Presynch-OvSynch was developed in which pre-synchronization is achieved with a standard estrous synchronization protocol (PGF2á given twice at a 14-day interval) with the OvSynch program initiated 12 days after the second injection of PGF2á (10). A Presynch-OvSynch program increased pregnancy rates 18 percentage units (i.e., 25% to 43%;) in lactating cyclic cows (10) . This stimulation in pregnancy rates was attributed to manipulation of the estrous cycle such that the OvSynch, timed insemination program was initiated at the most favorable stages of the estrous cycle. Success of the OvSynch program is dependent on whether lactating dairy cows are anestrus or cycling. Pregnancy rates were less in cows that were not cycling at the time the OvSynch program was initiated (e.g., 22% versus 42%). If anestrous cows ovulate to the first and second GnRH injections of the OvSynch program then pregnancy rates appeared to be normal (e.g., 39%).

Heatsynch:

An alternative strategy to control the time of ovulation is the ability of exogenous estradiol to induce a LH surge during late diestrus and proestrus. An estradiol induced LH surge lasts for approximately 10 h, which is comparable to a spontaneous LH surge and longer than the LH surge induced by GnRH. Estradiol cypionate (ECP) is used to replace the second GnRH injection of an OvSynch program and is called Heatsynch (34, 35).

Based on synchronization of ovulation and pregnancy rates, ECP can be utilized as an alternative to induce ovulation in place of GnRH for a timed insemination. Since lactating dairy cows have reduced concentrations of plasma estradiol in the preovulatory period and reduced intensity of estrus, the elevation of estradiol following ECP injection supplements for a lactational induced deficiency, and our experience indicates that cows expressing estrus are fertile.

If cows are anovulatory (e.g., anestrus or have not developed positive estradiol feedback), the Heatsynch program may not be as effective as the GnRH-based OvSynch program. This is because the GnRH causes the direct secretion of LH. Greater uterine tone, ease of insemination and occurrence of estrus with the use of the Heatsynch program are well received by inseminators. Alternatively, in facilities with concrete flooring, the reduced estrous expression associated with the OvSynch program may be preferred.

Bovine Somatortopin (bST) to Improve Embryo Development and Pregnancy rates:

In cycling lactating dairy cows, injection of bovine Somatotropin (500 mg Posilac, Monsanto Co, St. Louis, Missouri) at the time of the first GnRH injection or at insemination in cows of a Presynch-OvSynch program increased pregnancy rates (57% > 42.6%; 10). Since bST was effective at insemination, it is likely that bST stimulated embryonic development and survival following insemination in lactating dairy cows. There was no evidence that bST given at the 9th week of lactation is detrimental to fertility when used with a timed breeding protocol such as OvSynch. A study in Mexico reported (36) that, in cows identified as having three or more prior services, bST given at estrus and again 10 days later stimulated pregnancy rates.

Administration of bST at AI to superovulated donor cows decreased the number of unfertilized ova, increased the percentage of transferable embryos, and stimulated embryonic development to the blastocyst stage. Moreover, bST affected both early embryonic development and recipient components to increase pregnancy rates following embryo transfer (37). Both bST and IGF-1 stimulated embryo development in vitro (38). Our recent studies indicate that the beneficial effect of bST may be restricted to lactating dairy cows in contrast to non-lactating dairy cows. Metabolic and physiological differences between these two physiological states appear to make lactating cows more sensitive and responsive to bST and IGF-1 to improve embryo development and survival.

HCG induction of Accessory CL, three wave cycles and pregnancy rate:

The opportunity to regulate ovarian function after insemination to improve pregnancy rates is an additional production management strategy. The administration of hCG induces ovulation with the subsequent formation of a functional accessory CL. hCG induction of an accessory CL with increased progesterone may enhance embryo survival. Since a greater number of cows conceived that had three follicular waves after insemination compared with cows having two follicular waves, hCG induction of three-wave cycles also may contribute to higher pregnancy rates.

A study was designed to determine the effects of hCG (3,300 IU i.m.; Chorulon, Intervet Inc., Millsboro, DE) administered on d 5 after AI on accessory CL formation, plasma progesterone concentration, conception rate, and pregnancy loss in high producing Holstein dairy cows (41). This study supported the concept that increased progesterone during the luteal phase increases embryo survival. However, this effect was not evident in the heat stress period where early embryo losses probably precluded any subsequent increases in embryo survival. The effect of hCG to improve pregnancy rates through possible increases in early embryo development and survival, in the non-heat stress season are encouraging.

Effect of Lipids on Reproductive Performance:

Several experiments performed in vivo (43, 45) and in vitro (43, 44) indicate that the omega-3 fatty acids are able to decrease secretion of PGF2a. Animal trials with natural sources of omega-3 fatty acids such as EPA, DHA, and a-linolenic acids have shown apparent improvement in pregnancy rates (45, 46, 47, 48). These fatty acids are capable of decreasing the secretion of PGF2a and compliment the antiluteolytic action of interferon-t. EPA and DHA are known to have distinct anti-inflammatory and immunosuppressive effects that compliment the normal immunosuppressive and anti-inflammatory effects of progesterone and interferon-t in early pregnancy. It would be interesting to determine if cows fed anti-inflammatory lipid diets would reduce the incidence of EEM and LEM associated with the occurrence of mastitis.

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(This is a summary of an article by the authors)
(This is a summary of an article by the authors)

Authors

University of Florida

University of Florida