Effects of the temperature-humidity index on conception rates in Holstein heifers and cows receiving in vitro-produced Japanese Black cattle embryos

Abstract

We investigated the effect of the temperature-humidity index (THI) on the conception rate (CR) in Holstein heifers and cows receiving in vitro-produced (IVP) Japanese Black cattle fresh embryos. IVP embryos were transferred to Holstein heifers (n = 1,407) and cows (n = 3,189) on 245 commercial farms. The monthly average ambient temperature (AT) and THI ranged from 4.7 to 29°C and 41 to 81, respectively; both were the highest in August. The monthly CR ranged from 16.3% to 46.7% in cows and 23.8% to 74.1% in heifers. The CR of heifers was unaffected by THI, AT, or the month of embryo transfer. However, these parameters affected the CR of cows. The CR at THI values of 61–65 and 71–75 was greater than that at THI > 75, whereas other THI values had no effect. The CR at temperatures > 25°C was lower (P = 0.008) than that at temperatures of 15–20°C and 20–25°C. Moreover, the CR was lowest (P = 0.003) in July. THI and parity (P = 0.057 and P = 0.001, respectively) and AT and parity (P = 0.019 and P = 0.001, respectively) showed significant effects on CR; however, there was no interaction between these two factors. In conclusion, AT > 25°C and THI > 75 adversely affect the CR outcome in cows but not in heifers.

The season has a significant impact on the conception rate following artificial insemination (AI) in dairy cows. In particular, the pregnancy rate of high-producing lactating dairy cows reportedly decreases in summer owing to heat stress [1]. Extreme heat or cold stress in the days preceding or following insemination, especially high temperatures 3 days prior or 1 day after insemination, reduces the conception rate in dairy cows [2]. Reduced pregnancy rates result in substantial economic losses for dairy production systems [3].

Factors such as temperature, humidity, and wind speed affect the impact of heat stress on pregnancy in cattle [1, 2, 4]. The temperature-humidity index (THI) is an indicator of heat stress [1, 4, 5], and its impact on pregnancy rate following in vitro-produced (IVP) embryo transfer in dairy cows is largely unexplored [6].

Heat stress reduces the viability of oocytes and sperm, consequently reducing fertilization and embryonic developmental competence [1, 2, 7]. In addition, early embryonic development in the oviduct is inhibited by heat stress [3]. Blastocyst formation decreases by approximately 25% on the first day following AI in heat-stressed cows [8]. The detrimental effects of high lactation levels on oocyte quality, fertilization, and early embryonic development in dairy cows are exacerbated by heat stress [9]. Heat stress primarily impacts embryos during the early developmental stage, 1–3 days after fertilization [8]. Therefore, 7-day-old IVP embryos may mitigate this problem. The pregnancy rate following embryo transfer during periods of heat stress has been reported to be higher than that following AI [9, 10]. Embryo transfer from superovulated donors without heat stress can considerably increase pregnancy rates in heat-stressed dairy cattle [11]. As the IVP embryo transfer process circumvents the heat stress susceptibility period [9], it can be employed to increase the pregnancy rates in cows by alleviating the effects of heat stress [12, 13].

Intense summer heat in the southern Kyushu region of Japan necessitates an improvement in the summer pregnancy rate of dairy cows. The embryo transfer technique can meet the high demand for milk production during the summer. Therefore, we investigated the effect of THI on the day of embryo transfer on the conception rate of Holstein recipients of Japanese Black cattle IVP embryos under field conditions. We used an extensive dataset of more than 4,500 embryo transfer cases from 245 commercial farms over a 3-year period to test the hypothesis that a high THI adversely affects the conception rate in lactating Holstein cows to a greater extent than that in heifers following the transfer of IVP fresh blastocysts.

Materials and Methods

Animals and housing

In this study, we included records from the Kumamoto Dairy Cooperative Association in Kumamoto, Japan, covering a period of more than 3 years (January 2012 to December 2014) regarding Japanese Black cattle fresh IVP embryo transfer to Holstein cows and heifers (Kumamoto Dairy Cooperative Association Document, number 246, September 12, 2002). Holstein cows (n = 3,189) and heifers (n = 1,407) from 245 commercial farms (cows, 229 farms; heifers, 163 farms) in Kumamoto Prefecture, southern Kyushu region, Japan (32°06–33°42 N and 130°26–131°10 E) were used as recipients of IVP embryos. The majority of cows were kept in tie-stall or free-stall barns, while some were kept in loose barns. The heifers were kept in free-stall or loose barns.

Embryo transfer

The IVP Japanese Black embryos, provided by the Livestock Improvement Association of Japan, were produced utilizing oocytes collected from ovaries obtained from abattoirs and sperm from seven proven bulls [14]. Fresh IVP embryos (IETS quality code 1) [15] that had reached the blastocyst stage after 7–8 days (insemination on Day 0) were suspended in TCM199 medium (12340030; Gibco, NY, USA), supplemented with 20% calf serum, and placed in 0.25 ml straws (IMV Technologies, L’Aigle, France). The straws were placed in an embryo transfer container (FHK, Tokyo, Japan), stored at 35°C, and transferred to the farms by air for 2 h, followed by road travel for 1.5 h. The embryos were non-surgically transferred ipsilateral to the corpus luteum using an embryo transfer gun to the recipients between 7 to 9 days after natural estrus. Pregnancy was diagnosed by rectal palpation 40–60 days after estrus detection. The conception rate was defined as the number of detected pregnant animals / number of embryo transfers.

Temperature-humidity index

The monthly mean THI was calculated using air temperature and relative humidity data obtained from the Kumamoto Local Meteorological Observatory of the Japan Meteorological Agency. Moreover, the following formula was used for this calculation [16]: THI = (0.8 × T) + [(RH / 100) × (T − 14.4)] + 46.4, where T represents the ambient temperature (°C), and RH denotes the relative humidity (%). The THI values were categorized into seven groups, namely THI 1–7 (≤ 50, 51–55, 56–60, 61–65, 66–70, 71–75, and > 75) [17]. The ambient temperature was classified into six categories: < 5, 5–10, 10–15, 15–20, 20–25, and > 25°C, whereas the months of embryo transfer data were categorized according to the calendar months (January to December, 12 categories). Cows and heifers were assigned to the THI category, ambient temperature group, and month according to the conditions on the day of each embryo transfer.

Statistical analysis

Statistical analyses were performed using the GLIMMIX procedure in SAS Enterprise Guide 6.1 (SAS version 9.4; SAS Institute Inc., Cary, NY, USA), considering pregnancy status as a binary response variable (0 = nonpregnant, 1 = pregnant). Initially, the cow and heifer datasets were independently analyzed to determine the effect of one of three fixed factors: THI (seven levels), ambient temperature (six levels), or month (12 levels). The model incorporated the farm identification number and year of embryo transfer as random factors. Conception rate data were then analyzed by merging the heifer and cow datasets in order to determine the effects of THI, ambient temperature, or month (fixed factor 1), parity (cow versus heifer; fixed factor 2), and their interaction; the model included the farm identification number and year as random factors. Multiple comparisons of least-squares means were performed using Tukey’s adjustment when the p-value for the effects or interactions was < 0.05. Data were expressed as percentage conception rates using the least square means and standard error of the mean output of the SAS GLIMMIX procedure.

Results

The average ambient temperature during this study ranged from 4.7 to 29°C; it was highest in July (26.8–28.6°C) and August (26.8–29°C) and lowest in December (6.2–6.6°C) and January (4.7–6.2°C) (Fig. 1). The calculated THI values ranged from 43.6 to 80.6, and they were then converted to whole numbers for categorization into the seven levels. The THI values were > 70 in June and peaked in August (78–81). The THI was highest at the highest temperature and lowest at the lowest temperature. The monthly conception rates following embryo transfer ranged from 16.3% to 46.7% in cows and from 23.8% to 74.1% in heifers. The THI, ambient temperature, and monthly conception rate data (least-squares mean and standard error of the mean) are summarized in Tables 1, 2, 3. Regardless of the month of embryo transfer, ambient temperature, THI level, or farm location, the conception rate after the transfer of fresh IVP embryos was higher in Holstein heifers (722/1407, 51.3%) than that in lactating Holstein cows (1135/3189, 35.6%; chi-square P < 0.0001).

Fig. 1.

Monthly mean ambient temperature, temperature-humidity index (THI), and conception rates of Holstein heifers and cows receiving in vitro-produced Japanese Black cattle embryos, from January 2012 to December 2014.

Table 1. Effects of THI on conception rates after transfer of in vitro-produced fresh blastocysts in lactating Holstein cows and heifers.

THI Levels	Heifers			Cows			All
	n	LSM (%)	SDEM (%)	n	LSM (%)	SDEM (%)	n	LSM (%)	SDEM (%)
THI 1	322	49.9	3.0	724	34.4	1.9	1046	41.8	1.8
THI 2	162	52.7	3.3	321	34.1	2.2	483	43.0	2.0
THI 3	139	53.0	4.3	345	35.8	2.8	484	44.4	2.6
THI 4	137	59.4	5.7	280	42.9 a	4.1	417	50.7 a	3.6
THI 5	207	55.3	4.3	462	38.2	2.7	669	46.3	2.6
THI 6	199	50.8	3.8	517	38.2 a	2.3	716	44.1	2.2
THI 7	241	50.1	3.4	540	28.9 b	2.1	781	38.7 b	2.0

THI: temperature–humidity index (THI 1: ≤ 50, THI 2: 51–55, THI 3: 56–60, THI 4: 61–65, THI 5: 66–70, THI 6: 71–75, and THI 7: > 75); LSM, least squares mean; SDEM, standard error of the mean. ^{a, b} P < 0.05 within the same column.

Table 2. Effects of ambient temperature and parity on conception rate after transfer of in vitro-produced fresh blastocysts in lactating Holstein cows and heifers.

Ambient Temperature Categories (°C)	Heifers			Cows			All
	n	LSM (%)	SDEM (%)	n	LSM (%)	SDEM (%)	n	LSM (%)	SDEM (%)
< 5	28	68.7	9.0	49	26.0	6.4	77	46.5	6.6
5–10	294	48.1	3.1	675	35.0	2.0	969	41.3	1.8
10–15	301	52.8	3.1	666	35.8	2.0	967	44.1	1.9
15–20	180	55.4	3.9	412	39.1 a	2.6	592	47.0 a	2.4
20–25	336	54.1	2.9	745	37.7 a	1.9	1081	45.5	1.8
> 25	268	48.8	3.3	642	29.3 b	1.9	910	38.2 b	1.9

LSM, least-squares mean; SDEM, standard error of the mean. ^{a, b} P < 0.05 within the same column.

Table 3. Conception rates of Holstein heifers and cows receiving in vitro-produced Japanese Black embryos 7 to 9 days after natural estrus, categorized by month.

Month of embryo transfer	Heifers			Cows			All
	n	LSM (%)	SDEM (%)	n	LSM (%)	SDEM (%)	n	LSM (%)	SDEM (%)
January	90	53.4	5.5	170	28.1	3.6	260	40.0	3.4
February	110	44.8	5.0	245	32.6	3.1	355	38.4	2.9
March	124	52.9	4.7	232	36.0 a	3.3	356	44.3	2.9
April	140	55.9	4.4	278	35.7 a	3.0	418	45.4 a	2.7
May	104	53.8	5.1	221	35.5	3.4	325	44.1	3.1
June	84	55.3	5.7	206	38.3 a	3.5	290	46.4 a	3.4
July	100	44.9	5.2	218	21.5 b	2.9	318	31.9 b	2.9
August	141	53.8	4.4	322	34.1	2.8	463	43.4	2.6
September	115	47.5	4.9	311	38.1 a	2.9	426	42.5	2.8
October	143	58.9	4.3	331	40.3 a	2.8	474	49.4 a	2.7
November	134	48.9	4.5	346	34.5	2.7	480	41.5	2.6
December	122	51.8	4.8	309	39.2 a	2.9	431	45.3 a	2.8

Conception rates are expressed as LSM ± standard error derived from GLIMMIX analysis. LSM, least-squares mean; SDEM, standard error of mean; reported using the SAS GLIMMIX procedure. ^{a, b} P < 0.05 within the same column.

Heifers

When the pregnancy status (binary response variable) of heifers (n = 1,407) was examined with one independent fixed factor (THI, ambient temperature, or month of embryo transfer) and adjusted for two random factors (163 farms and 3 years), THI (P = 0.765), ambient temperature (P = 0.218), and the month of embryo transfer (P = 0.574) had no effect on the conception rate.

Cows

A comparable analysis was conducted on cows (n = 3189, 229 farms, and 3 years), which indicated that THI (P = 0.013), ambient temperature (P = 0.008), and the month of embryo transfer (P = 0.003) affected the conception rate. Conception rates (%) of THI 4 and THI 6 (42.9 ± 4.1 and 38.2 ± 2.3, respectively) were greater than those of THI 7 (28.9 ± 2.1), whereas other levels had no significant effect on conception rates. Conception rates (%) after embryo transfer at > 25°C (29.3 ± 1.9) were lower than those at 15–20°C and 20–25°C (39.1 ± 2.6 and 37.7 ± 1.9, respectively). The conception rates for the embryos transferred in July were lower than those transferred in March, April, June, September, October, and December.

All cattle (heifers and cows combined)

When the conception status of heifers and cows (n = 4,596) was included in the analysis using a factorial design (THI × parity and interaction) and adjusted for the random effects of farm numbers (n = 245) and years (n = 3), THI (P = 0.057) and parity (P < 0.001; 52.5 ± 1.6 for heifers, 36.0 ± 1.1 for cows) had significant effects on conception rate; however, there was no interaction (P = 0.806) between these two factors. According to the Tukey–Kramer test for post-hoc analysis, THI 4 and THI 7 had different conception rates (%) (50.7 ± 3.6 and 38.7 ± 2.0, respectively), whereas those of other groups did not differ.

In the combined analysis, ambient temperature (P = 0.019) and parity (P < 0.001) (6 × 2 factorial) as well as the month of embryo transfer (P = 0.007) and parity (P < 0.001) (12 × 2 factorial) showed differences in conception rates (including all heifers and cows). The interactions between the groups were not statistically significant. The conception rate (%) was lower for embryos transferred at > 25°C (38.2 ± 1.9) than that for embryos transferred at 15–20°C (47.0 ± 2.4). Likewise, conception rates (%) after embryo transfer were lower in July (31.9 ± 2.9) than those in April (45.4 ± 2.7), June (46.4 ± 3.4), October (49.4 ± 2.6), and December (45.3 ± 2.8). The other ambient temperature ranges and months of embryo transfer had intermediate effects on the conception rate.

Discussion

Our hypothesis that high THI and ambient temperature adversely affect the conception rate in lactating Holstein cows to a greater extent than that in heifers following the transfer of IVP fresh blastocysts was confirmed. Although we had an extensive dataset (> 4,500 animals from 245 commercial farms), no effect of THI could be detected in dairy heifers with conception rates of 50–59% at different THI levels. Furthermore, conception rates in lactating animals declined by 8–10%, indicating a marked biological impact; however, even under less favorable environmental conditions, overall conception rates were > 28%.

Previous studies have demonstrated that heat stress, caused by increased ambient temperature and humidity, negatively impacts conception rates and milk production in dairy cows following AI, resulting in economic losses [2, 4]. In addition, evidence revealed that at low THI levels, dairy cows were more mobile during estrus (i.e., increased number of steps), indicating less physical stress. Garcia et al. [16] observed that estrus detection rates decreased when THI > 75. In lactating cows, milk yield and food intake decrease with increasing THI [1, 4, 5]. Moreover, the conception rate of dairy cows following AI is reduced by heat stress [11, 13, 18]. Consequently, appropriate strategies are necessary to improve conception rates during heat stress. An important clinical finding of our study was that an acceptable conception rate can be obtained using fresh IVP embryos, even at high ambient temperatures and THI levels up to 80. In contrast to heifers, the conception rate in cows was affected by THI, ambient temperature, and the month of embryo transfer by approximately 10%.

In the present study, heifers had a higher overall conception rate (51%) than that of cows (36%). Our data on the effect of THI on conception rate are comparable to those of a previous study from the south-east region of the USA [6], which reported significantly (P < 0.05) lower conception rates in cows at THI values of 72–79 and 80–89 than those at a THI < 72. In contrast to our findings, the USA study indicated that embryo transfer at a THI of 80–89 in heifers was also significantly lower than that at THI > 72 and 72–79. This disparity between the two studies could be due to the higher THI (i.e., 89) in the south-east USA than that in Kumamoto Prefecture, situated in southern Japan (i.e., 81). The highest THI detected in our study was < 81, which did not appear to affect the conception rate of heifers [4, 6, 19, 20]. Considering the findings of previous studies [6] and those of our study, we inferred that the post-embryo transfer conception rate in cows decreased at THI > 75 but remained unaffected in heifers at a THI of 81.

Schüller et al. [21] reported that heat stress on estrous days considerably reduced the intensity of external estrous signs and that the size of estrous follicles decreased with increasing THI. Furthermore, conception rates in lactating dairy cows decrease at THI > 72 [19]. In addition, they demonstrated that exposure to heat stress for 3–5 weeks prior to insemination or natural mating reduced conception rates, with the effect being most pronounced 6–7 days following insemination. Schüller et al. [20] demonstrated that the THI threshold for affecting fertility in moderate climatic conditions (Germany) was 73. The greatest negative effect of heat stress on conception rate occurred 21 to 1 day(s) prior to breeding, and the conception rate decreased from 31% to 12% when the average THI during this period was greater than 73. In our study, the conception rate at the time of embryo transfer in cows at a THI > 75 was significantly lower than that in cows at a THI of 61–65 or 71–75. Therefore, a high THI adversely affected both lactation and fertility in dairy cows. The conception rates of cows in this study were in accordance with those observed in prior research [19, 20].

The thermal comfort temperature range for dairy cows is 4–25°C [22]. When the temperature exceeds 25°C, the physiological functioning and reproductive performance of dairy cows are adversely affected. This effect was evident in high-producing lactating cows. The conception rate decreases in cows when the temperature before and after AI exceeds 20°C [7, 16]. In the present study, the conception rate of cows at ambient temperatures above 25°C was significantly lower than that of cows exposed to temperatures of 15–25°C. Sakatani et al. [23] indicated that high ambient temperatures during summer increase body temperature and intracellular oxidative stress and reduce signs of estrus in Japanese Black cows. Another study in Japan [22] found that the optimum temperature for livestock is approximately 20°C and that the thermoregulatory mechanism functions at higher temperatures; however, it breaks down, and body temperature rises when the critical temperature is exceeded. In addition, the upper limit of the critical temperature is regarded as 25°C for dairy cattle and approximately 30°C for other domestic animals; when this threshold is exceeded, the body temperature rises rapidly [22].

It is believed that a high THI environment affects estrous behavior [4,21] and follicular growth [21]. The conception rates of cows and heifers begin to decrease when the THI exceeds 73 and 80, respectively [6, 16, 19, 20]. The THI threshold is influenced by the climate of the region in which cattle are raised and managed. Moreover, the effect of high THI on conception rate is influenced by THI values for several weeks or days before estrus, at the time of AI or embryo transfer [19, 20], and approximately one week following AI. In this study, THI and temperature values were not significantly different between July (78.6 and 27.6°C, respectively) and August (79.4 and 28.2°C, respectively), although July had the lowest conception rate for cows. Therefore, the increase in the average THI to ≥ 72 in June resulted in a decrease in the conception rate in July, and a similar trend has been reported previously [19]. The authors of the present study hypothesized that cows were unable to adapt to heat stress at a THI > 70 in June, which affected the conception rate until July. The subsequent gradual adaptation to a high THI restored the conception rate in August.

Nabenishi et al. [24] investigated the relationship between THI and conception rates in lactating dairy cows following AI. They found that the average conception rate during a high-THI period from July to September (29.5%) was significantly lower than that from October to June (38.2%) when the THI was low and stable [24]. In addition, the conception rate declined when cows experienced the highest THI (≥ 80) 2 days prior to and on the day of AI [24]. The reason for the low conception rate following AI in the presence of a high THI is that the increase in body temperature inhibits the competence of oocytes in the follicle [24]. In contrast, the results of the present study indicated that a high conception rate could be achieved using IVP embryo transfer, even if the recipients were subjected to heat stress. Additionally, recipients that adapted to heat stress had a higher chance of conceiving following this method.

Conception rates following embryo transfer are significantly lower at THI > 72 [6]. In the present study, the conception rate was significantly lower at THI values of 75–80 than that at THI values of 61–75. We postulated that at THI > 75, an increase in body temperature could inhibit conception, even if blastocyst-stage heat-resistant embryos were transferred. Therefore, reducing heat stress during the summer is essential for protecting the health of lactating dairy cows. Large fans and fine fog-generating devices have been used to reduce heat stress. Building methods, including the calcification of roofs and the strengthening of insulation materials and cheesecloth, have also been used, particularly during seasons of high temperatures and humidity [4]. Dairy cows lose their thermoregulatory ability at THI > 72 [24, 25]. The vaginal temperature starts to increase above a THI of 67 [24]. In recent years, thermoregulation in lactating cows has become more difficult because of the nutritional demands associated with high production rates [26]. In the present study, the THI ranged between 66 and 67 in May and 71 and 73 in June, indicating that interventions to mitigate heat stress should be implemented in May to improve conception rates in July and August. Fixed-time AI protocols improve fertility during heat stress [5]. Conception rates may be further improved using fixed-time embryo transfer during periods of heat stress [2, 27, 28].

In summary, the analyses of the embryo transfer data of >4,500 dairy cattle at 245 commercial farms in southern Japan were unable to reveal a connection between THI or ambient temperature (4.7 to 29°C) and conception rates in heifers. The conception rates in heifers ranged from 50% to 59%, with THI levels ranging from 44 to 81. In contrast, conception rates in lactating Holstein cows declined by 8–10% at THI values of 75–81 compared to THI values of 61–65 and 71–75. Similarly, conception rates in lactating cows at ambient temperatures above 25°C were lower than those at 15–20°C and 20–25°C, indicating a marked biological impact. However, even under less favorable environmental conditions, the overall conception rates remained above 28%. Our hypothesis that high THI and ambient temperatures should adversely affect the conception rate in lactating Holstein cows to a greater extent than that in heifers post-IVP fresh blastocyst transfer was supported by the fact that we were able to detect the deleterious effects of THI and ambient temperature in lactating cows but not in heifers. From a practical perspective, the transfer of IVP fresh Japanese Black cattle embryos is an effective strategy for increasing the conception rates of Holstein cattle under heat stress. This technique may be suitable for application in tropical and temperate regions, where high temperatures cause declines in cow fertility.

Conflict of interests

Authors declare no conflicts of interest for this article.