Characterizing the acute heat stress response in gilts: II. Assessing repeatability and association with fertility

Abstract

Mitigating heat stress (HS) in swine production is important as it detrimentally affects multiple aspects of overall animal production efficiency. Study objectives were to determine if gilts characterized as tolerant (TOL) or susceptible (SUS) in response to HS maintain that phenotype later in life and if that phenotype influences reproductive ability during HS. Individual gilts identified as TOL (n = 50) or SUS (n = 50) from a prepubertal HS challenge were selected based on their rectal temperature (T_R) during acute HS. The study consisted of 4 experimental periods (P). During P0 (2 d), all pigs were exposed to thermoneutral (TN) conditions (21.1 °C). During P1 (14 d), all gilts received Matrix (15 mg altrenogest per day) to synchronize estrus, and were maintained in TN conditions. During P2 (9 d), Matrix supplementation was terminated and gilts were subjected to diurnal HS with ambient temperatures set at 35 °C from 1000 to 2200 h and 21 °C from 2200 to 1000 h. Also during P2 gilts underwent estrus detection and artificial insemination. During P3 gilts were housed in TN conditions for 41 d at which they were sacrificed and reproductive tracts were collected. During the last 2 d of P1 and throughout the entirety of P2, T_R and skin temperature (T_S) were recorded. During P2, SUS had increased T_R relative to TOL pigs during P2 (0.27 °C; P < 0.01). Overall, uterine wet weight, ovarian weight, corpora lutea (CL) count, and embryo survival were 5.6 ± 0.1 kg, 21.6 ± 0.3 g, 17.8 ± 0.3 CLs, and 79 ± 2%, respectively, and not influenced by prepubertal HS tolerance classification (P ≥ 0.37). Tolerant gilts had a longer return-to-estrus (6.1 vs. 5.5 d, respectively; P = 0.01) following altrenogest withdrawal and tended to have larger CL diameters (10.3 vs. 10.1 mm; P = 0.06) compared to SUS gilts. Fetal weight (25.4 vs. 23.6 g; P = 0.01) and fetal crown-rump length (74.8 vs. 72.8 mm; P < 0.01) were higher in gilts previously classified as SUS compared to those previously classified as TOL. Additionally, neither litter size nor the number of fetuses detected as a percentage of ovulations was influenced by classification. In summary, SUS gilts had a shorter return-to-estrus, increased fetus size, and tended to have smaller CL diameters compared to TOL gilts. Additionally, SUS gilts also retained their inability to maintain euthermia postpubertally relative to TOL gilts. In conclusion, there appeared to be little reproductive advantage of maintaining a lower T_R during HS.

INTRODUCTION

Seasonal infertility in the swine industry occurs annually when gilts and sows have decreased reproductive performance. This seasonal reproductive decline is characterized by anestrus, increased wean-to-estrus interval (WEI), decreased farrowing rate, and reduced litter size (Bertoldo et al., 2009); a scenario that places a substantial economic burden on pork production (Pollmann, 2010). Despite the reproducibility and predictability of seasonal infertility, knowledge of the underlying biological mechanisms contributing to reduced fertility during the warm summer months is scant (Hurtgen and Leman, 1980; Peltoniemi et al., 1999; Ross et al., 2017). Heat stress (HS) has long been shown to negatively impact reproduction in multiple species including pigs (Tompkins et al., 1967; Omtvedt et al., 1971). A meta-analysis of publications (1970–2009) revealed the effect of HS on feed intake and growth to be more pronounced in recent years suggesting a possible indirect and negative impact of genetic selection for growth and carcass traits on thermal sensitivity (Renaudeau et al., 2011). The negative economic impact of HS may intensify with predicted changes in climate patterns and continued selection for economically important phenotypes.

Heat stress compromises follicular and early embryo development in a variety of animal models and agriculturally important species (Dutt, 1963; Tompkins et al., 1967; Omtvedt et al., 1971; Ealy et al., 1993; Ozawa et al., 2005). Not only does HS have direct negative effects on the follicle and oocyte through impacting steroidogenesis and oocyte quality, secondary insults such as HS-induced endotoxin exposure and hyperinsulinemia also likely contribute to seasonal infertility (Baumgard and Rhoads, 2013; Ross et al., 2017).

There is large variability in the thermoregulatory and production responses to HS in gilts (Seibert et al., 2018). What remains unknown is whether the degree of heat tolerance in prepubertal gilts, based on rectal temperature (T_R) during acute HS, could predict future reproductive success during the warm summer months. Determining potential associations between prepubertal thermoregulatory responses with postpubertal reproductive outcomes may provide a management tool to identify gilts with greater fertility at an early age. Thus, study objectives were to determine if prepubertal gilts retrospectively classified as HS tolerant (TOL) or susceptible (SUS) based on their ability or inability, respectively, to maintain a safe T_R during an acute HS challenge respond differently to HS during the periconceptional period as assessed by reproductive outcomes.

MATERIALS AND METHODS

Animals and Experimental Design

Iowa State University Institutional Animal Care and Use Committee approved all animal procedures. One hundred gilts were selected from a previous study that identified gilts as being TOL (n = 50) or SUS (n = 50) to HS based on their ability or inability, respectively, to remain euthermic during a 24-h prepubertal HS period (Seibert et al., 2018). All gilts underwent estrus detection beginning at approximately 160 d of age and continued until 220 d of age to ensure the selected gilts had demonstrated at least 2 estrous cycles. At approximately 220 d of age, gilts were transported to an experimental facility where they were randomly assigned to individual stalls (n = 50 per room) with each retrospective classification (i.e., TOL and SUS) equally represented in both experimental rooms. Each crate was equipped with a stainless steel feeder and a nipple drinker. All gilts were limit fed 2.7 kg of feed per day and water was provided ad libitum during the entire experiment. One gilt was removed from the study for reasons unrelated to the experiment.

This study was divided into 4 experimental periods (P): P0, P1, P2, and P3. Period 0 (2 d in length) served as an acclimation period in which all pigs were housed individually and subjected to thermoneutral (TN) conditions. During P1 (14 d), all gilts were maintained in TN conditions and placed on an estrous synchronization program utilizing altrenogest, a progesterone receptor antagonist. For the duration for P1 6.8 mL of Matrix (15 mg altrenogest) was top-dressed on the 2.7 kg of feed in each individual feeder at 0700 h. Feed consumption was monitored on all animals to ensure all gilts effectively consumed the complete dose. Ambient temperature was controlled by the use of gas heaters within the room but humidity was not governed and both parameters were recorded every 30 min by 4 data loggers (Lascar EL-USB-2-LCD, Erie, PA) in each room and later condensed into averages. Fans were placed throughout the rooms to ensure equal distribution of the environmental conditions throughout the facility and verified by the distributed data loggers, which were located at the level of the animals. During the last 2 d of P1, T_R was determined using a calibrated and lubricated digital thermometer (Welch Allyn SureTemp Plus 690; accuracy: ± 0.1 °C; Skaneateles Falls, NY) and skin temperature (T_S) was measured using a calibrated infrared thermometer (HDE ST380A Infrared Thermometer; accuracy: ± 2.0 °C, HDE, Allentown, PA) at 0800, 1400, 1500, 1600, 1900, 2000, and 2100 h to represent each gilt’s TN average baseline for each thermoregulatory parameter. During P2 (9 d), Matrix supplementation was terminated and all gilts were subjected to diurnal HS (1000 to 2200 h) conditions. The intensity of the heat load was increased (humidity was not governed) incrementally during the first 3 d (28.9 °C, 31.1 °C, and 33.3 °C on day 1, 2, and 3, respectively) and then was held at 35 °C for the remaining 6 d of P2; the temperature from 2200 to 1000 each day was 21.1 °C during P2. Each thermoregulatory parameter was measured at the same 7 time points each day during P2 as during P1. Measurements recorded at 1400 to 2100 h during P2 were condensed into a single average, representing HS thermoregulatory parameters. A ΔT_R was also calculated by subtracting the average TN T_R from the average HS T_R. Thermoneutral T_R, HS T_R, and ΔT_R from the previous study (Seibert et al., 2018) were used to investigate relationships between prepubertal and postpubertal thermoregulatory responses.

During P2, each gilt underwent estrus detection prior to artificial insemination. Four boars were utilized via nose-to-nose exposure to facilitate identifying estrus behavior each morning after feeding and prior to HS resumption (i.e., between 0700 and 1000). Gilts were bred with a single dose of pooled semen (terminal Duroc) on the first day of standing estrus and received an additional insemination each day they continued to exhibit behavioral estrus. Additionally, on the first day of standing estrus the gilts T_R max thermoregulatory response (1400 to 2100 h) was recorded as their behavioral estrus T_R (bT_R). By the end of P2, all but one of the 99 gilts remaining on the study had demonstrated standing estrus and had received at least 1 dose of semen. During P3 (41 d; corresponding to day 43 to 48 of gestation with day of first service considered day 0), all gilts were subjected to the original TN conditions until the end of the study.

Harvesting and Fetal Analysis

Following P3 (gestation day 43 to 48 depending on the onset of estrus), all gilts were harvested in a commercial abattoir. The reproductive tract from each inseminated gilt was collected and immediately refrigerated. The entire reproductive tract from each gilt was weighed and then the fetuses and ovaries were removed. Fetuses were counted to determine litter size for each gilt and each fetus was individually weighed, then measured with Ultra Tech digital calipers (General Tools, Secaucus, NJ) to determine crown-rump length (CRL). For each ovary, whole-ovarian weight was recorded, the corpora lutea (CL) on each ovary were counted, and the diameter of each CL was measured with Ultra Tech digital calipers. Embryo survival was calculated for each gilt by dividing the number of fetuses by the number of CL on corresponding ovaries. Eight gilts were nonpregnant confirmed by the absence of fetuses.

Statistical Analysis

All data were statistically analyzed using SAS University Edition software, version 9.4 (SAS Institute Inc., Cary, NC). Relationships between thermoregulatory and reproductive performance data were analyzed using PROC CORR to generate Pearson’s correlation coefficients. Thermoregulatory and reproductive performance data between gilts classified as TOL or SUS were evaluated using PROC TTEST. Additionally, PROC MIXED was used to analyze T_R data with an autoregressive covariance structure with day of experiment as the repeated effect; the model included classification, day, and their interaction as fixed effects.

RESULTS

Thermoregulatory Response to HS

During P1, TN T_R did not differ between gilts retrospectively classified (Seibert et al., 2018) as TOL or SUS to a prepubertal heat challenge (P = 0.12; Table 1). However, during P2, SUS gilts had increased T_R (0.27 °C; P < 0.01; Table 1) compared to TOL gilts. Additionally, bT_R was greater (0.38 °C; P < 0.01; Table 1) in SUS compared to TOL gilts. Skin temperature did not differ (P ≥ 0.28) between TOL and SUS gilts during both P1 and P2 (Table 1). Rectal temperature increased steadily from day 1 to 5 of P2 and the severity of the increased T_R was influenced by prior classification, with SUS being greater than TOL for the duration of P2 (Fig. 1; P < 0.01).

Table 1.

Retrospective classification influence and effects of thermoneutral (TN) and heat stress (HS) conditions on thermoregulatory indices for postpubertal gilts classified as tolerant (TOL) or susceptible (SUS)

Parameter	TOL	SUS	SEM	P
TN TRa, °C	38.28	38.34	0.03	0.12
HS TRb, °C	38.74	39.01	0.03	<0.01
Behavioral estrus TRc, °C	38.91	39.29	0.05	<0.01
TN TSd, °C	28.95	28.72	0.11	0.28
HS TSe, °C	39.35	39.47	0.07	0.43

TR, rectal temperature; TS, skintemperature.

^aAverage rectal temperature during P1.

^bAverage rectal temperature during the HS phase of P2.

^cAverage rectal temperature during the HS phase of P2 on the day of behavioral estrus for each gilt.

^dAverage skin temperature during P1.

^eAverage skin temperature during the HS phase of P2.

Figure 1.

Postpubertal rectal temperature (T_R) is higher for SUS gilts compared to TOL gilts in response to the diurnal HS challenge during the follicular phase. A subset of gilts classified as SUS or TOL based on their T_R response to acute HS prepubertally (Seibert et al., 2018) were reassessed for thermoregulation during HS following the onset of puberty. The relative inability to maintain euthermia, as determined by T_R, was retained by SUS gilts compared to TOL gilts during postpubertal HS. Error bars represent SE by day and the dashed line separates period (P) 1 from P2. Rectal temperature did not differ between TOL and SUS gilts during P1 (P = 0.18).

Breeding and Reproductive Performance

Following estrus synchronization during P1, TOL gilts had increased time until behavioral estrus compared to SUS gilts (6.1 vs. 5.5 d, respectively; P = 0.01). All gilts that exhibited a standing estrus received 1 artificial insemination for each day that they exhibited behavioral estrus resulting in 2.4 ± 0.1 services per gilt and this variable was not influenced (P = 0.13) by classification.

Total uterine tract weight for the successfully bred gilts was 5.6 ± 0.1 kg and no difference was detected between TOL and SUS gilts (P = 0.37). The number of fetuses/gilt was 13.91 ± 0.34 and did not differ (P = 0.36) between classifications. Interestingly, fetal weight for SUS gilts was increased compared to TOL gilts (25.4 vs. 23.6 g; P = 0.01; Fig. 2A). A similar result was also detected for CRL as it was increased in SUS compared to TOL gilts (74.8 vs. 72.8 mm; P < 0.01; Fig. 2B). No classification differences were observed for total ovarian weight (21.62 ± 0.32; P = 0.90), CL number (17.83 ± 0.34; P = 0.54), or embryo survival (0.79 ± 0.02; P = 0.45). Interestingly, the CL diameter in TOL gilts tended to be increased compared to SUS gilts (10.3 vs. 10.1 mm; P = 0.06; Fig. 1C).

Figure 2.

Pregnant reproductive tract assessment of from HS TOL and SUS gilts during the follicular phase and breeding subsequently sacrificed during early gestation. A subset of gilts that were previously classified (Seibert et al., 2018) based on their rectal temperature (T_R) response to HS prepubertally as SUS had an increased fetal weight compared to gilts classified as TOL (A; P < 0.01). Additionally, fetuses from gilts classified as SUS had greater CRL compared to fetuses collected from TOL (B; P < 0.01). The number of CL on the ovaries was not different between gilt HS classifications, however, CL diameter of TOL gilts, those that maintained a lower T_R during HS tended to be greater SUS classified gilts (C; P = 0.06).

Relationship Between Maximum Rectal Temperature on Day of Insemination and Reproductive Outcome and Relationship of ΔT_R With Reproductive Performance

To examine the effects of HS on the specific day each gilt was bred, we compared the bT_R with each of the reproductive measurements (Table 2). Interestingly, the bT_R was correlated with both fetal weight (r = 0.26; P = 0.01) and fetal CRL (r = 0.26; P = 0.01) of the bred gilts (Table 2), but no relationships between bT_R and the remaining reproductive measurements were detected (Table 2). The ΔT_R of each gilt was also used to determine if the HS response was associated with reproductive performance and ability to tolerate HS during breeding. The average ΔT_R tended to be correlated with fetal CRL (r = 0.20; P = 0.06), but there were no correlations between ΔT_R and all other reproductive performance variables measured (Table 3).

Table 2.

Relationship of behavioral estrus rectal temperature (bT_R) and reproductive performance following HS during the follicular phase

Parameter	r	P
Uterine weight, kg	0.06	0.54
Fetal weight, g	0.26	0.01
Fetal CRL, mm	0.26	0.01
Fetal number	−0.11	0.32
Ovary weight, g	−0.01	0.89
CL number	−0.01	0.96
CL diameter, mm	−0.08	0.45
Embryo survivala	−0.11	0.31

^aNumber of fetuses/CL number.

Table 3.

Relationship of the change in rectal temperature (HS T_R − TN T_R; ΔT_R) and reproductive performance following HS during the follicular phase

Parameter	r	P
Uterine weight, kg	−0.05	0.64
Fetal weight, g	0.15	0.16
Fetal CRL, mm	0.20	0.06
Fetal number	0.05	0.63
Ovary weight, g	−0.04	0.68
CL number	−0.01	0.94
CL diameter, mm	−0.13	0.21
Embryo survivala	−0.09	0.42

^aNumber of viable fetuses divided by the number of CL.

Repeatability of HS Tolerance and Susceptibility

To determine if an early-life classification of TOL or SUS were predictive of the individual’s future HS response, we compared the prepubertal and postpubertal thermoregulatory parameters. While the postpubertal TN T_R was moderately associated with prepubertal TN T_R (Fig. 3A; r = 0.21; P = 0.04), the association between postpubertal HS T_R and the prepubertal TN T_R was stronger (Fig. 3B; r = 0.53; P < 0.01). Further, the postpubertal HS T_R was markedly related to the prepubertal HS T_R response (Fig. 3C; r = 0.63; P < 0.01) suggesting the ability to thermoregulate during HS is conserved throughout life and likely established through genetic regulatory mechanisms or is established early in life, if not prenatally. Consistently, the ΔT_R response to HS prepubertal was also strongly correlated to the HS ΔT_R postpubertal (r = 0.39; P < 0.01).

Figure 3.

Postpubertal rectal temperature (T_R) during TN and HS conditions during the follicular phase is associated with T_R control prior to puberty. Two hundred and thirty-five gilts were subjected to TN and acute HS conditions at approximately 3 to 4 mo of age (described in Seibert et al., 2018). A subset of gilts (n =98) were exposed to TN and HS conditions at approximately 8 mo of age during the follicular phase of a synchronized estrous cycle. The PROC CORR function of SAS was used to determine the relationship between the T_R responses before and after puberty in different environmental conditions. A positive correlation between the postpubertal and prepubertal TN T_R response was observed (A; r = 0.21, P = 0.04). Furthermore, the TN T_R response prior to puberty was associated with the HS T_R response following puberty (B; r = 0.53, P < 0.01). The HS T_R following puberty was strongly correlated to the HS T_R response experienced prior to puberty (C; r = 0.63, P < 0.01) suggesting that the HS T_R response may be intrinsically governed, perhaps through genetic regulation.

DISCUSSION

Seasonal infertility is a recurring problem that threatens agricultural economics and limits production of high quality protein for human consumption. Due to their lack of functional sweat glands, pigs are poor at dissipating body heat and rely on other strategies to maintain euthermia, such as panting, wallowing, reducing feed intake, and decreasing activity (Whittow, 1971). Heat stress may have a more severe effect on traditional production traits in modern genetic lines compared to commercial genetics used >40 yr ago, and this suggests selection for increased lean tissue accretion may reduce HS tolerance (Brown-Brandl et al., 2004; Renaudeau et al., 2011). Unfortunately, mitigating HS in the pig industry is difficult as the TN zone varies markedly depending upon animal size and physiological state (Ross et al., 2017).

Since reproductive efficiency represents the combined performance of several production phases, mitigating HS during all developmental stages is crucial in maintaining the reproductive vitality of the pork industry. The deleterious effects that HS has on reproductive efficiency include decreased litter size/farrowing rate, increased early embryonic death, and poor offspring performance (Hurtgen et al. 1980; Xue et al., 1994; Peltoniemi et al., 1999; Boddicker et al., 2014; Johnson et al., 2015). Heat stress can also increase nonproductive days due to prolonged WEIs and interruptions in ovarian follicular development (Prunier et al., 1996).

This study characterized whether gilts classified as TOL or SUS during prepubertal HS can maintain their retrospective classification after puberty and to identify the relationship between HS sensitivity following estrus synchronization and reproductive performance. Our hypothesis was that SUS gilts would remain unable to maintain euthermia postpubertally and have decreased reproductive success during HS. Understanding the repeatability of the HS thermoregulatory response, in addition to better defining the relationship between thermoregulation and reproductive efficiency, is imperative to overcoming the negative effects associated with seasonal infertility. Additionally, it would potentially provide a tool to determine which gilts should be selected for breeding or be identified for culling.

To determine how HS affected fecundity, reproductive performance was compared between gilts previously classified as TOL and SUS after being exposed to HS during the follicular phase of the estrous cycle. Unexpectedly, SUS gilts had a slightly shorter time (5.5 d) to estrus following altrenogest withdrawal than those previously classified as TOL (6.1 d). However, this may relate to better overall productivity as SUS gilts had better ADG during TN conditions compared to the TOL gilts earlier in life (Seibert et al., 2018).

The duration and robustness of behavioral estrus is thought to be impacted by HS (Bolocan, 2009). In this study, gilts were inseminated daily for the duration of behavioral estrus enabling the comparison of the number of services received per gilt to serve as a proxy for estrus duration. No difference was detected between the TOL and SUS gilts in the number of services received. However, the primary means to characterize the effects of HS on reproduction are through evaluation of the effect on pregnancy rate and litter characteristics since HS decreases both the number of piglets born alive and the total litter weight (Prunier et al., 1994; Xue et al., 1994). The overall lack of marked differences in reproductive success between TOL and SUS gilts could be related to the relatively small T_R differences (0.26 °C) between the classifications during HS. Although we did not detect differences in the number of fetuses, there was an unpredicted increase in fetal weight and CRL in SUS compared to TOL gilts, which could potentially be related to SUS gilts achieving estrus slightly earlier after altrenogest withdrawal. Further research to fully elucidate the mechanism for increased fetal size and its relationship with HS sensitivity is thus warranted.

A decrease in ovarian activity can cause early pregnancy loss and decreased farrowing rate, which are direct phenotypic manifestations of seasonal infertility (Love et al., 1993). This may pertain to decreased number of viable follicles during the summer and fall compared to the winter and spring (Lopes et al., 2014). Furthermore, sows culled for early pregnancy loss during the summer have fewer CLs compared to those culled during the spring (Bertoldo et al., 2011). In this study, there was no difference in viable CL number at harvest between TOL and SUS gilts, which may be due to differences in experimental design (namely, all animals used in this study were exposed to HS). However, TOL gilts demonstrated slightly larger (2%) CL diameters compared to SUS gilts. While this did not correspond to an increased gain in litter size, whether CL size is related to HS tolerance based on thermoregulation is of further interest.

The posit that genetic and biological underpinnings control an animal’s ability to thermoregulate is underscored by the correlation between prepubertal and postpubertal thermoregulatory parameters. In the current study, the prepubertal thermoregulatory indices (TN T_R, HS T_R, and ΔT_R) were compared to the same postpubertal measurements. All 3 measurements were positively correlated in prepubertal and postpubertal periods. Additionally, the prepubertal TN T_R was positively correlated to the postpubertal HS T_R (r = 0.53; P < 0.01). Taken together, females with higher prepubertal TN T_R are more likely to have an increased T_R during HS later in life.

CONCLUSION

Heat stress is a major contributor to seasonal infertility and mitigating HS-induced effects is crucial in maintaining reproductive performance. Gilts that are SUS, as described by their thermoregulatory response to HS prior to puberty, continue to be SUS to HS following puberty. However, the ability of prepubertal HS tolerance to predict future reproduction is not clear, as CL size was greater in TOL pigs but SUS gilts had increased fetal weights and fetal CRL. The consistency of the thermoregulatory phenotype during TN and HS conditions, as well as during prepubertal and postpubertal production stages, suggest that this phenotype is likely genetically regulated and represents an opportunity for future exploration.

Conflict of interest statement. This project was supported by the National Pork Board. Any opinion, findings, conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the National Pork Board. No conflicts of interest, financial or otherwise are declared by the author(s).