Keywords: cow, embryo transfer, heat stress, lactation, oocyte

Introduction: The Origins of Fertility Precede Fertilization

An embryo arises when the pronucleus of the newly-fertilized oocyte fuses with the pronucleus of the spermatozoa. The fate of the newly-formed embryo is an uncertain one, and many fail to survive to term. Whether an embryo can develop successfully depends in part on the envi-ronment of the maternal reproductive tract in which it resides and in part on its inheritance of genetic and non-genetic factors from the oocyte and sperm from which it was derived.

The process of formation of male and female gametes is prolonged. In cattle, the species focused on in this paper, spermatogenesis takes about 61 days. Oocyte growth is also a slow process. Once an ovarian follicle emerges from the pool of resting follicles and initiates growth, it takes ap-proximately 100 days to reach the point where ovulation can occur and the oocyte contained within the follicle is released (Figure 1).

Antecedents of mammalian fertility: Lessons from the heat-stressed cow regarding the importance of oocyte competence for fertilization and embryonic developmentPeter J. Hansen

Department of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program, and Genetics Institute, University of Florida, Gainesville, FL, USA

© Hansendoi:10.2527/af.2013-0031

Implications

• The process of formation of gametes in the male (spermatozoa) and female (oocyte) is prolonged, and errors early in the process can lead to reduced fertility many days later.

• The lactating dairy cow exposed to heat stress represents the most well studied case of environmental disruption of oocyte function. Heat stress results in an oocyte that has reduced competence to support development of the embryo after fertilization. Effects of heat stress are exerted over a broad physiological window that extends from as early as the initiation of growth of the follicle in which the oocyte develops until at least the penultimate stages of the life of the oocyte during the preovulatory period. As a result, female fertility can remain suboptimal for several weeks after the end of heat stress.

• Two potential strategies for reducing the impact of heat stress on fertility have emerged from understanding how heat stress com-prises oocyte function: hormonal-induced turnover of follicles and embryo transfer. The former has not yet been reduced to prac-tice, but the latter has been shown repeatedly to achieve preg-nancy rates in the summer that are equivalent to those seen with artificialinseminationincoolmonths.

• The possibility that other physiological and environmental factors that disrupt oocyte competence work through mechanisms similar to those engaged during heat stress implies that the heat-stressed cow may be a good model for understanding how to overcome disruptions in oocyte function.

Figure 1. Time course of follicular development in cattle. The timing of events, which is based on several sources (Lussier et al., 1987; Ginther et al., 2003; Her-nandez-Fonseca et al., 2005; Palhao et al., 2009), is approximate, particularly for events early in follicular development. Note that the time scale is not linear.

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It has long been known that insults to the testis can damage early stag-es of spermatogenesis and compromise sperm production many days later as the cells descending from the damaged germ cells enter the ejaculate. Thus, for example, effects of heat stress on motility of ejaculated sperm in bulls persist for six to eight weeks (Meyerhoeffer et al., 1985).

Oneofthefirstscientiststosuggestasimilarphenomenoncanoccurin the female was Jack Britt from North Carolina State University (Britt, 1991). He hypothesized that negative energy balance in dairy cows early in lactation affected the function of the preantral follicle so that fertility was compromised weeks later when the follicle ovulated. One implication of this concept, if true, is that management of females to achieve optimal fertility would require attention to events occurring long before insemina-tion.

Oocyte competence to support fertilization and development of the subsequent embryo is an important determinant of fertility in mammals, and there are a variety of environmental factors such as heat stress, low body condition score, low-fat or high-starch diets, and high concentrations of plasma urea nitrogen that have been implicated in affecting it (Hansen et al., 2010). In most cases, however, including for negative energy bal-ance, it is not known whether the environmental insult affects early or late stages of follicular development or whether there is a long-term carryover effect of the insult on oocyte competence.

The lactating dairy cow exposed to heat stress represents the most well-studied case of environmental disruption of oocyte competence. Heat stress can have catastrophic effects on fertility of lactating cows. As few as 10 to 20% of cows inseminated during heat stress are diagnosed as pregnant. The large amounts of internal heat production required for milk production makes the high-producing cow very sensitive to heat stress. A lactating cow can lose the ability to successfully regulate body tempera-ture at air temperatures as low as 25 to 29°C (Sartori et al., 2002; Dikmen and Hansen, 2009). As a result, infertility caused by heat stress affects dairy cows throughout much of the temperate and tropical areas of the world.Clarificationofthemechanismsbywhichheatstressdisruptfertil-ity is leading to new therapeutic approaches for improving reproductive function during heat stress.

As will be outlined here, one mechanism by which heat stress reduces reproductive function is by compromising the ability of a fertilized oo-cyte to develop into a blastocyst (the stage of development at which the embryo begins to differentiate into different cell types). Moreover, heat stress can affect oocyte function early in the process of follicular growth so that effects of heat stress on oocyte competence persist after the end of heat stress. This example of the Britt hypothesis is an important founda-tion for two possible reproductive management strategies to overcome infertility caused by heat stress: hormonal induced turnover of follicles and embryo transfer. The heat-stressed cow may, therefore, be a good model for understanding how to overcome actions of other environmen-tal factors that disrupt oocyte function.

Damage to the Oocyte Caused by Heat Stress

As shown in Figure 2, Holstein oocytes subject to in vitro maturation and fertilization are less capable of supporting development to the blasto-cyst stage during warm times of the year than during cool times. In many experiments, season of the year did not affect the rate at which oocytes werefertilized(asmeasuredbycompletionofthefirstcleavagedivision),but the embryos formed by fertilization were less likely to develop to the

blastocyst stage than when embryos were derived from oocytes during cool weather.

The fact that embryos formed from oocytes harvested in warm weather have reduced ability to develop indicates that heat stress disrupts one or more characteristics of the oocyte important for the subsequent embryo. The oocyte provides the embryo not only one-half of its chromosomes, but also virtually all of the organelles, mRNA, and other macromolecules required for embryonic development. For instance, the early embryo un-

Figure 2. Seasonal variation in competence of fertilized oocytes to develop to the blastocyst stage. Data are from experiments in Florida (Al-Katanani et al., 2002), Israel (Gendelman et al., 2010), and Brazil (Ferreira et al., 2011). The blue bars represent oocytes collected during cool seasons, and the red bars represent oocytes collected during warm seasons. Experiments in Florida and Israel used oocytes from Holstein ovaries collected at the slaughterhouse. The experiment in Brazil involved collection of Holstein oocytes by transvaginal, ultrasound-guided oocyte aspiration from non-lactating heifers, lactating cows at peak lactation, and repeat-breeder cows ( > three inseminations). In each experiment, oocytes were matured in vitro and fertilized with spermatozoa. The resultant embryos were cultured to the blastocyst stage.

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dergoes a period when its own genome is transcriptionally silent and it depends on mRNA inherited from the oocyte for protein translation. In the cow, extensive transcription is initiated about three to four days after fer-tilization, when the embryo is at the 8- to 16-cell stage (Memili and First, 2000). The embryo depends on mitochondria inherited from the oocyte for an even longer period; an increase in embryonic mitochondria in the bovine does not begin until the blastocyst stage at approximately seven days after fertilization (Smith et al., 2005).

Work from the laboratory of Zvi Roth at Hebrew University of Jerusa-lem has demonstrated that heat stress alters the embryo's inheritance from the oocyte. In germinal-vesicle stage oocytes (i.e., before resumption of meiosis is initiated coincident with ovulation), there was no difference between oocytes collected in hot and cold seasons in transcript abundance for four key genes (Gendelman and Roth, 2012a). After completion of meiosis I, however, oocytes collected in the hot season had fewer tran-scripts for all four genes than in the cold season. Possibly, heat stress changed the biochemical composition of the oocyte in a way that dis-rupted how mRNA was protected from degradation during resumption of meiosis. When oocytes were fertilized, differences in amounts of mRNA remained for some of the genes at the four-cell, eight-cell, and blasto-cyst stages of development. In another experiment, Gendelman and Roth (2012b) found that there was no effect of season on numbers of mitochon-dria in the oocyte. However, subcellular distribution of mitochondria was altered in oocytes in the summer compared with oocytes collected in the autumn and winter, and there was reduced expression of mitochondrial genes.

The mechanism by which heat stress leads to alterations in the oocyte is not clear. One possibility is that elevated body temperature can act di-rectly on the oocyte. In addition, endocrine changes associated with heat stress (for example, reduced follicular dominance and reduced circulating concentrations of progesterone) could disrupt follicular development and compromise the oocyte in the follicle destined to ovulate. There are ex-periments to support both mechanisms (see Hansen, 2013).

Timing of Actions of Heat Stress on the Oocyte

Oocytes from the experiments that demonstrate seasonal effects on oo-cyte function were collected largely from follicles that had not yet attained dominance (an event occurring about four days before ovulation). This fact implies that heat stress can affect the oocyte early in the process of folliculogenesis. Additional evidence for this idea is the observation that the end of summer heat stress is followed by a period in the autumn when oocyte competence (Roth et al., 2001a) and fertility (Al-Katanani et al., 1999; Huang et al., 2008) improve only gradually. Moreover, experimen-tal studies indicate that heat stress can affect function of the oocyte during a broad physiological window that extends from as early as the initiation of follicular growth until at least the penultimate stages of the life of the oocyte during completion of meiosis I.

Working with cattle of a Bos indicus breed (Gir), Torres-Júnior et al. (2008) provided evidence that heat stress at the earliest stages of follicular growth can lead to formation of oocytes with reduced competence. Cows were heat-stressed in environmental chambers for 28 days. Ultrasound-guided follicular aspiration was performed to collect oocytes for in vitro maturation and fertilization at intervals extending until 133 days after ini-tiation of heat stress. There was no effect of prior exposure to heat stress on cleavage rate after fertilization. However, oocytes recovered from pre-viously heat-stressed cows were less able to become a blastocyst after fertilization than control cows. This difference persisted to the end of the trial at a time that was 105 days after the end of heat stress (Figure 3). Given that it takes approximately 100 days for a follicle to complete its growth, the interpretation is that the oocyte is sensitive to heat stress for most of the period encompassing follicular growth.

Folliculogenesis is different in important respects between B. indicus and B. taurus, and it remains to be seen whether heat stress affects oocyte function in the same way in both types of cattle. There are indications that heat stress can affect follicular function in B. taurus well before ovulation.

Figure 3. Carryover effects of heat stress on oocyte competence. Gir cows were either heat-stressed for 28 days (HS) or were maintained in a control environment (control group, CG). Oocytes were harvested, matured, and fertilized in vitro, and the resultant embryos cultured to the blastocyst stage. Data are reproduced from Torres-Júnior et al. (2008) with permission.

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In particular, exposure of lactating Holsteins to heat stress for 12 hours af-fected follicular steroid production 20 to 26 days later (Roth et al., 2001b).

The oocyte remains sensitive to heat stress as late as the periovulatory period because exposure of superovulated cows to heat stress for 10 hours beginning at the onset of estrus decreased the proportion of embryos re-coveredatDay7afterestrusthatwereclassifiedasnormal(Putneyetal.,1989a). Among the mechanisms responsible for effects of heat stress on the oocyte during this time are actions of elevated temperature on nuclear maturation, spindle formation, cortical granule distribution, free radical formation, mitochondrial function, and apoptosis (see Hansen, 2013).

Implications for Management of Reproduction

Two potential strategies for reducing the impact of heat stress on fertil-ity have emerged from knowledge on how heat stress comprises oocyte function: hormonal-induced turnover of follicles and embryo transfer. The former has not yet been reduced to practice, but the latter has been shown repeatedly to achieve pregnancy rates in the summer that are equivalent to thoseseenwithartificialinseminationincoolmonths.

It takes several weeks for follicles compromised by heat stress to ovu-late or undergo atresia. Fertility will not be restored until all the heat-dam-aged follicles destined for ovulation have been removed from the ovary. This phenomenon is likely responsible for at least some of the delay in restoration of fertility in the autumn (Al-Katanani et al., 1999; Huang et al., 2008). Hastening the removal of follicles in the autumn would be expected, therefore, to improve fertility. That this may be the case was firstdemonstratedbyRothetal.(2001a).Follicleswereaspiratedforfourconsecutive estrous cycles in the autumn. Control cows were aspirated on Day 4 of each estrous cycle whereas treated cows were aspirated on Days 4, 7, 11, and 15 of each cycle to facilitate removal of follicles previously damaged by heat stress. Oocytes were matured in vitro and chemically activated to induce parthenogenetic embryo development. Oocyte cleav-age was low for all four cycles of the control cows but increased by cycle 3 in the treated cows. Embryonic development showed a similar pattern.

Follicular aspiration is not a very practical procedure for turning over follicle populations in commercial herds of cattle. Alternative approaches for doing so have also been examined. In particular, administration of either follicle-stimulating hormone (FSH) or somatotropin during one es-trous cycle in the autumn improved oocyte quality in the subsequent cycle (Roth et al., 2002). Treatment with FSH increased the number of 6- to 9-mm follicles on the ovaries while somatotropin increased the number of 3- to 5-mm follicles. In the subsequent cycle, FSH improved the pro-portionofaspiratedoocytesthatwereclassifiedashighqualityoocytesbased on morphology and also increased cleavage rate following chemi-cal activation. Treatment with somatotropin improved oocyte morphol-ogy but not cleavage rate. Another study indicated that pregnancy rate could be improved in the summer and autumn in primiparous cows by use of gonadotropin releasing hormone and prostaglandin F2α to generate three consecutive 9-day follicular waves beginning at 50-60 days in milk (Friedmanet al., 2011).Therewasnobenefit inmultiparouscowsandoptimization of such treatments to improve pregnancy rates is warranted.

Embryo transfer has already been shown to be effective at increasing fertility in heat-stressed cattle (Figure 4). This might seem counterintui-tive because transferred embryos develop in the uterus of a heat-stressed female. However, embryos are typically transferred at the blastocyst stage of development, well after the ~16-cell stage at which they become resis-

tant to elevated temperature (Hansen, 2013). Exposure to hyperthermia after that time has little effect on embryo survival. Embryo transfer also avoids heat stress effects on the oocyte. Even when produced during heat stress, embryos that are capable of becoming blastocysts are derived from oocytesthataresufficientlycompetenttobefertilizedandsupportdevel-opment. Additionally, use of cryopreservation could allow embryos to be produced in cool seasons.

The issue for embryo transfer is whether the technique can be imple-mented in a cost-effective manner. When oocytes recovered from an abat-toir are used, in vitro production of embryos can be a more inexpensive source of embryos than superovulation. However, the in-vitro-produced embryo is very susceptible to damage caused by cryopreservation (Han-sen et al., 2010). Embryo transfer improves pregnancy rate during heat stress regardless of method of embryo production when embryos are transferred fresh without cryopreservation. Conversely, improvements in pregnancy rate using cryopreserved embryos only occurs when embryos are produced by superovulation (Figure 4). Improvements in the systems for producing embryos are likely to increase the cost-effectiveness of em-bryo transfer as a tool for bypassing heat-stress effects on fertility.

Conclusions

Oneofthemostimportantdeterminantsintheenergeticefficiencyoflivestock species is reproductive function (Dickerson, 1978). While great progress has been made in development of pharmacological tools for regulation of estrous cyclicity in livestock, there are few tools at present for manipulating the female to increase pregnancy success, i.e., the likeli-hood that an ovulated oocyte will give rise to a healthy neonate. Key to development of such tools is developing a more detailed understanding of the processes controlling establishment and maintenance of pregnancy and of the errors in the reproductive process that limit fertility. Such in-sightisleadingtomodificationsoftimedartificialinseminationprotocolsto improve pregnancy rate (Bisinotto and Santos, 2011). As outlined in this paper, elucidation of the mechanism by which heat stress disrupts establishment of pregnancy has resulted in possible strategies for mini-mizing heat stress effects on reproductive function. Further advances in

Figure 4. Effectiveness of embryo transfer for improving fertility in lactating cows during the heat stress. Embryos were either transferred fresh, after cryopreserva-tion (Cryo.), or were a mix of fresh and cryopreserved embryos. Data are either pregnancy rate at 42 to 76 days of gestation or calving rate.

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our knowledge of the processes responsible for oocyte development are likely to result in new strategies for improvement of reproductive function in livestock species.

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About the AuthorPeter J. Hansen is L.E. “Red” Larson Pro-fessor of Animal Sciences in the Depart-ment of Animal Sciences at the University of Florida. His research interests center around the basic mechanisms controlling the establishment and maintenance of pregnancy. Particular emphasis is placed on elucidating effects of elevated tem-perature on early embryonic development and development of methods to overcome these effects, identifying genes control-ling embryonic survival, and characterizing interactions between the

immune system, the reproductive tract, and the embryo.Correspondence:Hansen@animal.ufl.edu

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