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December – January, 2019, vol. 9, no. 3
pages: 639-642
Article type: Biotechnology of Biotechnology
DOI: 10.15414/jmbfs.2019/
Abstract: The aim of this study was to evaluate the effect of mild temperature stress on ovarian activity (corpus luteum count; transferable embryos; morulae and blastocysts) in cows. The evaluation included 54 Holstein cows superovulated and flushed at one farm in the Czech Republic in three different temperature periods in flushing day (mild hot season THI = 75.66; season with optimal temperature THI = 68.85; season with lower temperature THI = 45.56). The data were analysed using the PROC GLM of Statistica®. The statistical model included the effect of season and other factors (age; condition; breeding value) on the studied reproduction traits. The results showed significantly higher ovarian activity in the cooler period of the year than in periods of mild temperature stress. There was also a non-significantly better reproduction result in younger animals under optimal condition.
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Bezdíček, J., Stádník, L., Makarevich, A., Kubovičová, E., Louda, F., Hegedüšová, Z., Holásek, R.


The aim of this study was to evaluate the effect of mild temperature stress on ovarian activity (corpus luteum count; transferable embryos; morulae and blastocysts) in cows. The evaluation included 54 Holstein cows superovulated and flushed at one farm in the Czech Republic in three different temperature periods in flushing day (mild hot season THI = 75.66; season with optimal temperature THI = 68.85; season with lower temperature THI = 45.56). The data were analysed using the PROC GLM of Statistica®. The statistical model included the effect of season and other factors (age; condition; breeding value) on the studied reproduction traits. The results showed significantly higher ovarian activity in the cooler period of the year than in periods of mild temperature stress. There was also a non-significantly better reproduction result in younger animals under optimal condition.


temperature stress; corpus luteum; embryo transfer


In cattle breeding, there is generally maximum effort exerted to avoid stress in the animals. One of important stress factors is a temperature proven to have a negative effect on a number of physiological traits. For this reason, concerted effort is made to ensure cooling for the animals in the hot summer months, for example using cattle showers and shades. The reduction in the body temperature using cattle showers was reported by Legrand et al. (2011) and the importance of shade and its relationship to reducing temperature stress was described by Schütz et al. (2011).

The influence of temperature stress has been demonstrated for a number of physiological functions, e.g. reproduction (AlKatanani et al., 1999; De Rensis et al., 2002; Schüller et al., 2014), milk production (André et al., 2011; Bernabucci et al., 2002), milk components (Bernabucci et al., 2014) and in different animal species (Olexikova et al., 2007, 2008; 2010; 2013; Makarevich et al., 2007). In the area of reproduction, temperature stress was described at different levels, for example from the point of view gametogenesis (Nichi et al., 2006), early embryo mortality (Ryan et al., 1993), service period (Oseni et al., 2004; Ray et al., 1992) etc.

Temperature stress was also investigated in the area of gametogenesis, in both male and female animals. In bulls, the influence of temperature stress on the quality of the ejaculate was studied. This relationship has also been demonstrated, for example by Mathevon et al. (1998), Nichi et al. (2006) and other authors.

In female animals temperature stress has been the subject of number of studies. AlKatanani et al. 2002 found a lower proportion of embryos developed to blastocysts during the warm season compared with the cool season. Similar findings are reported by Rocha et al. (1998). These authors found higher production of normal oocytes during the cool season (75.9%) than in the hot season (41.0%). They also determined a higher percentage of oocytes developing to the 8-cell and morula stage (65.4 and 46.6 %) than in the hot season (21.2 and 6.0 %). Similar conclusions were drawn in our previous study Bezdíček et al. (2015) on embryo transfer. We found a significantly lower number of obtained embryos in the hot months of the year than the cold period and also a lower proportion of transferable embryos in the hot months. Similar findings were reported by Ferreira et al. (2011), Putney et al. (1988), Ryan et al. (1993) and other authors.

The effect of temperature stress was also confirmed by comparing the development of heat-stressed and non-heat-stressed bovine ova. Edwards et al. (2009) found a reduced development of the blastocyst, when morulae were cultured under heat-stress (41.0 °C) in comparison with non-heat-stressed morulae (38.5 °C). These conclusions were reported by other authors too (Sakatanet et al., 2015; Edwards and Hansen, 1997). Research in this area also focuses on efforts to ameliorate the situation and improve reproduction in heat-stressed cows, for example, by vitamin A (Lawrence et al., 2004) and not only in cows, during oocyte maturation. Lawrence et al. (2004) reported that heat-stressed oocytes (at 41.0 °C) with 5 μM retinol showed no increase in activation of parthenogenesis. The authors concluded that retinol may protect oocytes from the heat stress (Lawrence et al., 2004).

From the viewpoint of the progesterone production in heat-stressed cows, the results of many studies are often contradictory (Rensis and Scaramuzz, 2003). These authors showed some studies that found a decrease in progesterone level in the heat-stress period (Howell et al., 1994; Ronchi et al., 2001), and others that showed an increased or unchanged concentration of progesterone. Some studies show that the delayed effect of heat stress is also an important factor (Roth et al., 2001a, b).

In tropical and subtropical areas it is important to take into consideration temperature stress. The upper critical temperatures for Holstein cattle in the subtropical climate is 25-26 °C (Berman et al., 1985). However, temperature stress also refers to other factors, such as humidity, low airflow and direct sunlight (rev. by Scholtz et al., 2013). Therefore, temperature stress is often defined as temperature humidity index (THI), which includes air temperature and relative humidity. Different models for the calculation of temperature stress were reviewed by Dash et al. (2016). Armstrong (1994) categorized the level of THI into five classes: no stress (THI <72), mild stress (THI = 72-78), moderate stress (THI = 79-88), severe stress (THI = 89-98) and danger stress (THI >98).

Temperature stress is a significant factor for reproduction and production traits in livestock. Concerning reproduction, the effect of temperature stress was proved not only in terms of animal husbandry characteristics (service-period, NR rate and others), but also in terms of gametogenesis (males and females) and subsequent embryo development. The aim of this study was to evaluate reproduction in cows (embryo donors) at the beginning of temperature stress (end of spring and early summer). The evaluation was performed by sonographic examination of the ovaries after superovulation treatments (corpus luteum count) and subsequent flushing of embryos.

Material and methods

The samples were collected from 54 Holstein cows, superovulated at the farm located in the lowlands of the Czech Republic during one year. The average milk production for the 1st lactation at the time of the experiment was as follows: 7 843 kg of milk; 4.01% of fat; 315 kg of fat; 3.30% of protein; 259 kg of protein.

Oestrus of embryo donors was synchronized by prostaglandin F2α analogue – OESTROPHAN (Bioveta a.s., Ivanovice na Hane, Czech Republic). Superovulation was performed by injection of porcine pituitary gonadotropin (Pluset ®- FSHp-LHp, Laboratorios Callier, Barcelona, Spain) twice daily for 5 days, at 08:00 and 20:00 h, given in decreasing doses starting with doses of 150 IU of follicle stimulating hormone (FSH) +150 IU luteal hormone (LH) in the morning on days 11 to 50 IU FSH+50 IU LH in the evening on day 15 of the oestrous cycle. Luteolysis was induced on day 13 by the injection of OESTROPHAN. Insemination was carried out four times with frozen-thawed semen at 12 h intervals starting at 12 h after detection of the standing oestrous. Embryo recovery was performed by flushing out the uterine horns on the day 7 after the first insemination. Uterine flushing was done with a complete flush solution (Bioniche, Belleville, Ontario, Canada) using a silicone two-way Foley catheter (Minitüb GmbH, Tiefenbach, Germany). Flushed ova and embryos were transferred to the holding medium (phosphate-buffered solution with 20% of foetal calf serum – FCS, Gibco BRL, USA). The embryos were evaluated as transferable or non-transferable (degenerated and unfertilized) according to their stage of development using a stereomicroscope. Number of corpus luteum (CL) on both ovaries was determined by a Tringa Linear sonograph (Canmedical, Canada).

Data were analysed using the PROC GLM of Statistica®. The effects of season and other factors were estimated from the model as follows:

Yijkl = µ + Seasoni + Agej + BVk + Conditionl + eijkl

where :

Yijkl = corrected value (dependent variable) = corpus luteum; transferable embryos; morulae; blastocysts count;

μ = mean value

Seasoni = mild hot temperature (THI in flushing days = 75.66); optimal temperature (THI in flushing days = 65.85); lower temperature (THI in flushing days = 45.56)

Agej = age (1 = to 30 months; 2 = 31 – 45 months; 3 = above 46 months)

BVk = breeding value (1 = to 100; 2 = 101-600; 3 = above 601)

Condition = body condition of animal (1 = optimal condition = 2.5-3.5 points; 2 = inappropriate condition = under 2.5; above 3.5 points)

eijkl = residual error.

Differences between the estimated variables were tested at the level of significance P < 0.05 (a; b). Breeding values of milk production were based on the actual results from January 2015.

In this study, the temperature and humidity relationship was calculated according to the following equation:

Temperature-humidity index (THI) = 0.8×ambient temperature+ [(% relative humidity÷100)×(ambient temperature−14.4)] +46.4 (Buffington et al., 1981).

Maximal day temperature and day humidity were obtained from the Czech Hydro meteorological Institute in Prague. The THI index was calculated as the average of four days (day of flushing of embryos and the three preceding days). In a period of mild temperature stress, the maximum THI was 79.89 (Table 1).

Table 1 Weather conditions in days of embryo flushing.

Season Month             of flushing THI in flushing days Weather conditions (THI and °C) in 4 days* Count of donors
Average THI Max. THI Max. temperature (°C)
lower temperature March 44.82 45.56 53.42 59.5 15.3 8
November 46.29 46.7 49.87 9.9 11
optimal temparature April 65.27 65.85 62.25 65.27 18.7 10
October 66.42 61.44 66.42 19.2 8
mild hot May 75.49 75.66 72.96 78.27 26.3 9
Jun 75.83 72.13 79.89 27.4 9

* Weather conditions in 4 days = flushing day and three preceding days

Results and Discussion

Test of homogeneity (Cochran-Hartley-Bartlett test) and residual plots (Figure 1) were the first step in the statistical analysis. These tests indicate the suitability of the following GLM analyses.

The influence of different temperature periods of the year (mild hot, optimal, optimal lower) was studied in superovulated cows in terms of number of corpus luteum and number of transferable embryos (morulae and blastocysts). The results showed a lower number of corpus luteum in the mild warm period than in the optimal or optimal lower ambient temperature (6.05 vs. 8.15; 8.19), but the differences were insignificant (table 2). A similar trend was found for transferable embryos (1.94; 2.41; 3.73) and morulae count (1.45 vs. 2.96; 3.17). At this comparison, the differences between mild warm and optimal lower were significant. The tendency to better ovarian response in cooler periods is also evident from the graphic representation of number of corpus luteum, transferable embryos and the number of morulae (Figures 2, 3 and 4). For the effect “age at flushing” greater numbers of corpus luteum and transferable embryos were found in younger animals (to 30 months), compared to the group of 31 – 45 or over 46 months (corpus luteum: 8.38 vs. 7.37, 6.63, transferable embryos: 2.59, 3.26, 2.23, morulae: 2.24, 3.00, 2.35). The effect of “animal condition” was more prominent in the animals with an optimal condition than in animals tending to over- or underweight (corpus luteum: 7.63 vs. 7.29; transferable embryos: 3.18; 2.20; morulae 3.04 vs. 2.02) but the differences were not significant. For the factor “breeding value” we found slightly poorer (insignificant) results in animals with higher breeding value (BV > 600). The difference was manifested mainly in corpus luteum count (lower breeding value = 9.07; high breeding value = 5.64), but this was not significant. Table 2 shows the numbers of blastocysts, but in this case there were fewer donor cows and, therefore, this is only supplementary information to the values ​​of transferable embryos (transferable embryos = morulae + blastocysts). Table 3 presents the statistics significance of various factors included in the model. Season appears to be the most important factor (according to the F- ratio).

Figure 1 Residual plots: count of corpus luteum.

Table 2 Influence of different factors on ovarian activity in superovulated holstein cows.

Count of curpus luteum (LSM) Cows             (N) Count of transferable Embryos (LSM) Cows              (N) Transferable Embryos
Count of Morulae (LSM) Cows                    (N) Count of Blastocysts (LSM) Cows                    (N)
Season mild hot                                  (THI = 75.66)* 6.05±1.0353 17 1.94±0.5922a 17 1.45±0.5134a 13 3.66±0.6339 3
optimal temperature                             (THI = 65.85)* 8.15±1.0961 18 2.41±0.4134 18 2.96±0.5255 13 1.64±0.6002 4
lower temperature                            (THI = 45.56)* 8.19±1.0297 18 3.73±0.6105a 16 3.17±0.4716a 15 1.61±0.44275 8
Age to 30 months 8.38±1.3380 13 2.59±0.7911 12 2.24±0.6397 11 2.04±1.0868 2
31-45 months 7.37±0.9408 20 3.26±0.5517 19 3.00±0.4949 15 2.78±0.4311 9
above 46 6.63±1.0023 20 2.23±0.5732 20 2.35±0.4944 15 2.10±0.6204 4
Breeding value to 100 9.07±1.20 15 3.65±0.6894 15 2.68±0.5620 13 2.14±0.5726 7
101-600 7.67±0.9971 22 2.26±0.5985 20 2.32±0.5073 16 1.83±0.7910 5
above 600 5.64±1.1078 16 2.17±0.6340 16 2.58±0.5846 12 2.94±0.8225 3
Condition optimal condition 7.63±0.7797 32 3.18±0.4530 31 3.04±0.3656 26 2.06±0.3723 9
 inappropriate condition 7.29±0.9610 21 2.20±0.5647 20 2.02±0.4788 15 2.56±0.4800 6
Adjusted R2 0.0392 0.1057 0.1048 0.2652

P < 0.05 (a)

* THI = Temperature-humidity index in flushing days

Table 3 Significance of various factors included in the model.

Corpus Luteum Transferable embryos Morulae Blastocysts
SS F p SS F p SS F p SS F p
Season 48.35 1.48 0.2536 24.96 2.56 0.1192 22.1 3.96 0.0419 7.57 4.75 0.0617
Age 16.11 0.4715 0.6271 9.96 0.8921 0.4172 4.7 0.6477 0.5297 1.26 0.7100 0.5239
Breeding Value 66.36 1.20 0.1552 17.77 1.13 0.2154 0.86 0.1362 0.8731 0.54 0.3068 0.7452
Condition 1.21 0.0706 0.7917 9.79 1.30 0.1925 8.55 2.91 0.1086 0.73 0.8160 0.3964

2a                                                                                                    2b                                                                                             2c  

Figure 2 Ovarian activity (2a corpus luteum; 2b transferable embryos; 2c morulae as count) in different seasons of the year.

Al-Katanani et al. (2002) investigated heat stress in Holstein cows using in vitro methodology. The authors found that the percentage of embryos that developed into blastocysts (8 days after fertilization) was lower in the warm season than in the cold season. A comparison of results in the season with heat stress and winter season showed a lower number of collected oocytes (19.3 vs. 25.1), lower percent of blastocysts at d 8 (11.4 vs. 29.9 out of total oocytes) and lower total cell number (68.8 vs. 82.4)

Rocha et al. (1998) also found lower oocyte production in the warm season of a year. In superovulated cows, the authors described a significantly lower percentage of normal oocytes (24.4 vs. 80.0) and lower percentage of fertilized oocytes developing to the 8-cell, morula and blastocyst stage during the hot season, than in the cool season.

Putney et al. (1988) found an effect of heat stress on the proportion of degenerate embryos. In stressed Holstein heifers there was a lower percentage of normal embryos than in thermoneutral animals (20.7 vs. 51.5 %). The authors concluded that stressed Holstein heifers had a higher incidence of impaired (abnormal and retarded) embryos with degenerate, nonviable blastomers than heifers in a thermoneutral climate. Wilson et al. (1998) also reported the effect of heat-stress on ovarian activity as smaller follicle size and decrease in serum estradiol concentrations in heat-stressed heifers. They concluded that heat stress inhibited growth and function of the dominant follicle. Similar findings are described by López-Gatius et al. (2010), who found unimportant relationship between corpus luteum and response of embryos to stress factors in the early foetal period. Increasing heat stress (THI ≥74) is also connected with decreased serum progesterone concentration (< 1 ng/ml) on the day of oestrus (Schüller et al., 2017). Authors also found, that increasing temperature stress (for each THI point) is associated with decreased follicular size by 0.1 mm.


These results show the significant effect of the mild hot season on cattle reproduction. Ovarian activity in superovulated cows, represented by the number of corpus luteum, transferable embryos and morulae count, was significantly higher in the cooler period of the year than in periods of moderate temperature stress. Although better results were found in younger animals in optimum condition, the differences between groups were insignificant. Conclusions show, that also mild temperature stress can bring a slightly worse ovarian response in superovulated cows.

Acknowledgement: This work was supported by “S” grant of MSMT CR and the research project NAZV QI91A061.


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