Introduction

The estrous cycle of female rats consists of proestrus (P), estrus (E), metestrus (M), and diestrus (D) phases and last 4 to 5 d.³ Vaginal smear cytology in which the estrous phase is determined based on the proportions of leukocytes and epithelial and cornified cells is a commonly used method to identify specific stages of the estrous cycle in rodents. To obtain vaginal smears, the animal must be restrained for several minutes and requires habituation and a skilled investigator to minimize the stress.^1,6

Accurate assessment of phases of the estrous cycle is necessary for producing timed pregnancies for developmental studies, as ovulation in rodents occurs from the beginning of proestrus to the end of estrus when females are receptive to males;^5,11 thus, accurate identification of these phases is crucial. For that purpose, a fast, precise, and noninvasive technique of infrared thermography has recently emerged and intensively studied for measuring the surface temperature of different body parts of farm animals such as cattle, buffaloes,^2,7,9 and ewes.¹ The main advantage of this technique is that it requires relatively little handling of animals.⁹ Measurement of the ocular surface of ewes provides the most reliable temperature among all measured body parts. In addition, core body temperature can possibly be estimated from the ocular surface temperature because this is the only body part that is not covered by skin or fur.¹ Although the practical relevance of this measurement may be limited due to slight temperature changes during the estrous cycle in ewes, this method has not been evaluated rodents. We hypothesized that noninvasive thermal imaging of the ocular surface would allow identification of the estrous cycle phase in rats as an alternative to the traditional vaginal smear technique. If successful, this approach could simplify the reproductive management process of rodents and will also be consistent with the 3Rs (Replacement, Reduction and Refinement).

Materials and Methods

Core body and vaginal temperature measurement, and vaginal smear collection.

Estrous cycle determination was performed daily for 14 consecutive days, every morning between 0800 and 1000 h; assessment included measurement of ocular and vaginal temperature and collection of vaginal smears. Rats were moved to the procedure room and acclimated for at least 30 min (temperature: 25 °C; humidity: 55 ± 10%) before procedures were performed. During measurements, rats were evaluated in the same order of females every day. Ocular surface temperature¹³ was measured using a noncontact factory-calibrated handheld thermal imaging camera (Teledyne FLIR-E64501, Wilsonville, OR). Rats were always handled by the same individual to reduce stress. Each rat was gently restrained on a stable platform while 3 facial thermal images were acquired. The distance between the camera and the rat was constant (25 cm) during all measurements. Thermal images were analyzed using FLIR Tools software (FLIR Systems) by an individual who was blind regarding the temperature and estrous phase of the rats. The maximum temperature measured in the ocular area of both eyes was marked with a rectangle (Figure 1). The core body temperature was estimated as the average maximum temperature of the 3 measurements.¹³ Subsequently, the vaginal temperature was measured by inserting the tip of a digital thermometer (Microlife, Switzerland) into the vagina for 30 s. Next, 50 µL of saline (0.9% NaCl) was infused into the vaginal cavity, removed, deposited on a glass slide (Waldemar Knittel, Braunschweig, Germany), and immediately evaluated as an unstained wet mount preparation under a light microscope (Figure 2).⁶ Female rats were not synchronized; thus, the occurrence of the phases varied among the rats over the evaluation period. Therefore, an average temperature for each phase of the estrous cycle was calculated individually for rats.

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Results

The thermal-based estimated core body temperature (Figure 3A) measured in E (37.2 ± 0.6 °C) was significantly higher than in phases M (36.9 ± 0.6 °C, P < 0.05) and D (36.8 ± 0.5 °C, P < 0.01), but were not different from P (37.0 ± 0.5 °C, P = 0.28). Regarding the differences in thermal-based estimated core body temperature transitions between estrous cycle phases, a significant difference (P < 0.05) was found between P-E (0.2 ± 0.5 °C) and E-M transition phases (0.4 ± 0.5 °C), as well as between E-M (0.4 ± 0.5 °C) and D-P phases (0.2 ± 0.5 °C, P < 0.01, Figure 3C).

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Vaginal temperature (Figure 3B) was significantly higher in E (37.7 ± 0.6 °C) as compared with P (37.3 ± 0.6 °C, P < 0.01), M (37.1 ± 0.7 °C, P < 0.05), and D (37.0 °C ± 0.6, P < 0.01). A significant difference (P < 0.01) in the vaginal temperature was also found between the transition phases P-E (0.4 ± 0.5 °C) and E-M (0.5 ± 0.8 °C), and between E-M (0.5 ± 0.8 °C) and D-P (0.2 ± 0.7 °C, P < 0.05; Figure 3D).

The overall core body temperature measured with thermal imaging was positively correlated with vaginal temperature (r = 0.697, P < 0.001; Figure 4E) and with all phases of the estrous cycle (P: r = 0.516, P < 0.01, Figure 4A; E: r = 0.651, P < 0.001, Figure 4B; M: r = 0.702, P < 0.01, Figure 4C; D: r = 0.720, P < 0.001, Figure 4D).

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Discussion

Our results suggest that the use of a handheld thermal imaging camera is appropriate for estrous phase determination in rats, although our data indicate that at least a 12-d-long measurement period is adequate. During this time, approximately 3 estrous cycles are complete. With a thermal imaging camera, the increase of the core body temperature from proestrus to estrus is less apparent as compared with the vaginal temperature measured with a thermometer. Nevertheless, the measurement of ocular temperature is less invasive than the measurement of vaginal temperature. Our results are consistent with temperature fluctuation that occur during the menstrual cycle in women, in which an increase in vaginal temperature indicates ovulation.¹²

The estrus phase of rodents can be identified by using a vaginal wall impedance checker that measures differences in electrical resistance in the rat vagina.⁸ This method can be used to improve the breeding success in rats;⁴ however, as in the case of serial vaginal smear sampling in rats, it can also lead to pseudopregnancy.⁴ Long-term repeated stimulation of the vagina with the probe of impedance checker could have the same effect.¹⁰ Female rats in estrus have higher vaginal impedance values than nonestrous females, but vaginal impedance and estrous cycle stage determined by vaginal cytology are not correlated.¹⁰

In conclusion, the estimation of core body temperature using a thermal imaging camera provides a noninvasive approach to identifying estrus. However, this method is less accurate at determining phases of the estrous cycle as compared with vaginal smear collection. In addition, the determination of the estrus using thermography requires at least 12 d. Furthermore, our data may apply only to the outbred strain of rats we tested (Wistar). Use of this method in other outbred or inbred strains would require appropriate testing.

Data Access

The datasets generated during the current study are available from the corresponding author on request.

Conflict of Interest

The author(s) have no conflict(s) of interest to declare.

Funding

Funding for this study was provided by the Ministry of Education of Slovak Republic (VEGA1/0635/20 and VEGA 1/0341/23).