Damage to the oocyte during follicular growth has been demonstrated in experiments to evaluate seasonal variation in the competence of oocytes (harvested at slaughter or via ultrasound-guided aspiration) to be fertilized in vitro and develop into blastocysts. In these experiments, which used lactating Holstein cows, the proportion of oocytes that became blastocysts after in vitro fertilization^10–12 or chemical activation¹³ was lower in summer than winter (see Figure 64.1 for an example).

The seasonal effects on blastocyst formation largely represented decreased competence of cleaved embryos to develop rather than competence of the oocyte to cleave. One reason is that heat stress reduces accumulation of molecules in the oocyte important for early embryonic development. Actually, there are differences in transcript abundance between embryos produced by in vitro fertilization of oocytes collected in summer versus winter.¹⁴ Also, perhaps, oocytes in the summer are more likely to undergo abnormal chromosomal segregation so that subsequent embryos are chromosomally abnormal. Occurrence of chromosomal abnormalities can affect the rate of embryonic development.¹⁵

Appropriate follicular development is critical to the ovulation of an oocyte competent to be fertilized and able to transform into an embryo capable of successful development.^16,17 Heat stress can alter follicular dynamics by reducing growth of the dominant follicle and increasing the number of smaller-sized follicles.^3,18–20 Furthermore, heat stress can reduce follicular concentrations of estradiol-17β²¹ and insulin-like growth factor (IGF)-1,²⁰ while increasing follicular concentrations of androstenedione.²¹

The mechanism by which heat stress alters follicular development is not well understood. Experimentally, heat stress can reduce luteinizing hormone (LH) secretion.^22,23 There may also be direct effects of elevated body temperature on steroid synthesis. Culture of cumulus cells at 40.5 °C reduced estradiol-17β production in the fall but not in the summer or winter.²¹ In the same study, incubation at 40.5 °C reduced forskolin-stimulated androstenedione production by thecal cells when cells were collected in winter but not when cells were collected in summer or fall. Heat shock of 41 °C reduced basal and gonadotropin-stimulated synthesis of androstenedione and estradiol-17β by cultured follicular walls from the dominant follicle.²⁴

In some cases, heat stress can cause a reduction in circulating progesterone concentrations²⁵ and such a decline could conceivably contribute to a reduction in oocyte competence. This idea is based on the observation that fertility following ovulation of a first-wave dominant follicle, when circulating progesterone concentrations are low, is less than fertility following ovulation of a second-wave dominant follicle, when progesterone concentrations are high.^26,27 Moreover, supplementation with progesterone during the first follicular wave improved fertility.²⁷

Summer heat stress is followed by a gradual restoration of oocyte competence for fertility^28,29 and oocyte competence for chemically activated development.³⁰ These observations are symptomatic that heat stress can affect follicular development early in the 16-week period that is required for a primordial follicle to reach dominance.³¹ Indeed, there is experimental evidence for delayed effects of heat stress on function of the follicle and oocyte. In one study,³² exposure of lactating Holsteins to heat stress affected follicular steroid production 20–26 days later. An experiment in Gir cattle (Bos indicus) indicated that carry-over effects of heat stress can persist for up to 133 days after initiation of heat stress (i.e., 105 days after the end of heat stress).³ Cows were exposed to heat stress for 28 days and oocytes aspirated and subjected to in vitro maturation and fertilization at weekly intervals for up to 133 days after the beginning of heat stress. Heat stress did not affect follicular dynamics during the 28-day heat stress period but increased the diameter of the first and second largest follicles, as well as the number of follicles over 9 mm, from days 28 to 49 after initiation of heat stress. Moreover, the percent of oocytes that became a blastocyst was reduced by heat stress even at the end of the experiment at 133 days after initiation of heat stress (Figure 64.2).

Oocyte maturation

The oocyte remains susceptible to disruption by heat stress during the process of nuclear and cytoplasmic maturation coincident with ovulation. Heat stress of superovulated heifers for a 10-hour period beginning at the onset of estrus and before insemination at 15–20 hours after the beginning of estrus decreased the proportion of embryos recovered at day 7 after estrus that were classified as normal.³³ There was no effect of heat stress on fertilization rate so damage to the oocyte gave rise to an embryo with reduced competence for development.

Some of the effects of heat stress on oocyte function during maturation could be due to alterations in hormone secretion because heat stress lowered the magnitude of the preovulatory surge in LH and estradiol-17β.^23,34 In addition, exposure to elevated temperature can induce errors in oocyte maturation. Culture at elevated temperatures during maturation reduced the proportion of oocytes that completed nuclear maturation, increased the proportion with abnormal spindle formation, cortical granule distribution, depolarized mitochondria and apoptotic pronuclei, and decreased the proportion that cleaved and became blastocysts after insemination.^35–40

Apoptosis has been implicated in disruption of oocyte function during maturation. Inhibition of apoptosis with a caspase inhibitor,³⁶ sphingosine 1-phosphate,⁴¹ or inhibitor of ceramide synthesis⁴² reduced the effects of heat shock on developmental competence of the oocyte. There are also indications that the effects of heat shock involve acceleration of maturation and oocyte aging. Exposure of oocytes to 41 °C for the first 12 hours of maturation hastened both nuclear and cytoplasmic maturation.⁴³ Moreover, the deleterious effects of heat shock on development could be avoided if oocytes were fertilized at 19 hours of maturation instead of 24 hours⁴³ or if chemical activation was used to trigger development.⁴⁴

Mechanism for embryonic mortality caused by heat stress

The 2-cell embryo exposed to 41 °C experiences alterations in ultrastructure characterized by swelling of mitochondria and movement of organelles toward the center of the blastomere⁵³ (Figure 64.4). Organellar movement is caused by disruption of the cytoskeleton.⁵⁴ The early embryo exposed to heat shock also experiences a decrease in protein synthesis.⁵

Some of the damage caused by heat shock could be the result of increased production of free radicals as has been reported for embryos at days 0 and 2 after insemination.⁵⁵ Also, effects of heat shock were greater in 2-cell embryos cultured in a high oxygen atmosphere that would promote oxidative stress.⁵⁶ In contrast, the effects of heat shock were independent of oxygen tension in other studies.^51,57 Similarly, the antioxidant anthocyanin⁵⁸ and dithiothreitol⁵⁶ have been reported to mitigate the actions of heat shock whereas other antioxidants including vitamin E,⁵⁹ glutathione,⁶⁰ and glutathione ester⁶⁰ were reported to be without embryoprotective properties.

Effects of heat stress on embryonic survival in vivo could include actions on the reproductive tract as well as on the embryo directly. Heat stress can reduce the magnitude of the preovulatory surge in estradiol-17β²³ and this could conceivably change function of the oviduct or endometrium. The direct effects of heat shock on oviductal or endometrial function have not been examined at temperatures characteristic of those experienced by heat-stressed cows. However, it is likely that these tissues are resistant to heat shock because even a temperature as high as 43 °C did not have major effects on protein synthesis by endometrium or oviduct.⁶¹

Developmental acquisition of thermotolerance

Several hypotheses have arisen to explain the process by which the embryo transitions from a state in which it is very susceptible to heat shock, at the 1-cell to 2-cell stage, to one where it has acquired resistance to heat shock, by the 16-cell stage. One possibility is that the early embryo is very susceptible to heat shock because processes required for embryonic genome activation are easily disrupted by heat shock. If this was the case, embryos exposed to heat shock at the 2- or 4-cell stage should block in development between the 8-cell and 16-cell stages; embryonic genome activation is required for development past this stage.⁶² Such a preferential block at the 8-cell to 16-cell transition was seen in one experiment with 2-cell embryos⁵⁷ but not in another where embryos were heat-shocked at the 1-cell stage.⁵¹

Another possibility is that the acquisition of capacity for transcription after the 8-cell to 16-cell stage allows the embryo to engage a wider range of stress responses to stabilize cellular function during heat shock. However, steady-stage amounts of mRNA for some important stress proteins are higher in the 1-cell or 2-cell embryo than the morula^51,62 (Figure 64.5), probably because transcripts are inherited from the oocyte. Heat shock can induce synthesis of heat-shock protein 70 as early as the 2-cell stage.^5,63 Moreover, the morula-stage embryo undergoes a muted transcriptional response to heat shock, with only 4 of 68 genes associated with the heat-shock response showing an increase in response to elevated temperature.⁶⁴ Thus it might be that the damage to cellular macromolecules caused by heat shock that trigger stress protein responses is reduced in embryos at resistant stages of development. One reason might be a change in the balance between free radical generation and antioxidant protection. Heat shock caused increased free radical production at days 0 and 2 but not at days 4 and 6.⁵⁵ In addition, the cytoplasmic antioxidant glutathione is at its lowest during the 2- to 8-cell stages.⁶⁵