key: cord-0976562-wh5nwgqq authors: Seifi-Jamadi, Afshin; Zhandi, Mahdi; Kohram, Hamid; Luceño, Núria Llamas; Leemans, Bart; Henrotte, Emilie; Latour, Catherine; Demeyere, Kristel; Meyer, Evelyne; Van Soom, Ann title: Influence of seasonal differences on semen quality and subsequent embryo development of Belgian Blue bulls date: 2020-09-02 journal: Theriogenology DOI: 10.1016/j.theriogenology.2020.08.037 sha: 948596e121166cfc666075b6aeada33e83399e6b doc_id: 976562 cord_uid: wh5nwgqq Belgian Blue bulls are more susceptible to high temperature and humidity index (THI) than most other cattle breeds. Here, we investigated whether high ambient temperature during summer affected semen quality and subsequent embryo development in Belgian Blue cattle. For this purpose, semen samples were collected from six healthy mature Belgian Blue bulls in March (Low THI group; THI between 30.6 and 56.4) and August 2016 (High THI group; maximum THI of 83.7 during meiotic and spermiogenic stages of spermatogenesis; 14–28 days prior to semen collection) respectively. Motility, morphology, acrosome integrity, chromatin condensation, viability, and reactive oxygen species production were assessed for frozen-thawed semen. Moreover, the efficiency of blastocyst production from the frozen-thawed semen samples of two groups was determined in vitro. Blastocyst quality was determined by assessing inner cell mass ratio and apoptotic cell ratio. Fresh ejaculates showed a higher sperm concentration in low THI when compared to high THI group (P ≤ 0.05), whereas semen volume, subjective motility and total sperm output were not affected (P > 0.05). In frozen-thawed semen, total and progressive motility, viability and straight-line velocity were lower in high THI compared to low THI group (P < 0.05), while H(2)O(2) concentration, aberrant chromatin condensation and abnormal spermatozoa were higher in the high THI group (P < 0.05). Blastocyst rates were significantly higher when low THI samples were used (P < 0.05). Moreover, the total cell number and trophectoderm cells were significantly higher (P < 0.05) in blastocysts derived from low THI samples, whereas the apoptotic cell ratio was significantly higher (P < 0.01) in blastocysts derived from high THI spermatozoa. In summary, our data show that elevated ambient temperature and humidity during summer can decrease the quality of frozen-thawed spermatozoa in Belgian Blue bulls and also affect subsequent embryo development. on sperm quality: the effect becomes visible 14-42 days after high THI [23, 24] . Based on the 115 meteorological data [31] , three consecutive days were selected in July 2016, with maximum 116 temperature and maximum relative humidity (RH) more than 30 ˚C and 85% respectively, and a 117 THI oscillating between 63.5 and 83.7 ( Fig. 1 and 2 ). This range includes mild and severe heat 118 stress conditions for Belgian Blue bulls, taking into account that, the optimal environment for 119 bull semen production in European temperate climate conditions is estimated to range between 120 15 to 18 ˚C and THI ranging from 50 to 60 for the entire spermatogenesis period [6] . Moreover, 121 the semen samples of the low THI group were collected in March under a maximum temperature 122 and RH of 8 ˚C and 84% respectively; and the THI values were oscillating between 30.6 and 123 56.4. The meteorological data were provided for Ciney city by the Accuweather web site [31] 124 and THI indexes were calculated using the following equation where RH is relative humidity, T 125 is temperature and THI is Temperature Humidity Index [32] . 126 THI = (1.8 × T + 32) -(0.55 -0.0055 × RH) × (1.8 × T-26) 127 Within five minutes of semen collection, fresh semen quality parameters were determined 129 (five ejaculates/bull in each season, 30 samples for each treatment). The sperm concentration 130 was measured using an Accucell photometer (IMV Technologies, L'Aigle, France). Moreover, 131 individual motility of the spermatozoa was measured by phase-contrast microscopy (1:100 132 dilution in saline) and was expressed as the proportion of motile spermatozoa (percentage). spermatozoa/mL were further extended in Optidyl TM (Cryo-Vet, Québriac, France) extender and 136 cooled to 4°C for four hours in a fridge (0.125 °C/min). Then, the extended semen samples were 137 loaded into 0.25 mL straws (100 × 10 6 spermatozoa/straw) at 4°C, and freezing was performed 138 with a programmable freezing system (IMV, Technologies-Digitcool, L'Aigle, France). Briefly, 139 the straws were cooled to -10°C at -5°C/min, from -10°C to -140°C at -40°C/min, and were 140 subsequently plunged into liquid nitrogen until further analysis. divided into 4 quadrants to analyze the different subpopulations (Fig. 3) . with non-essential and essential amino acids (SOFaa), and ITS (5 µg/mL insulin; 5 µg/mL 252 transferrin; 5 ng/mL selenium). Medium droplets were covered with mineral oil and incubated at 253 38.5 ˚C in 5% CO 2 , 5% O 2 and 90% N. This experiment was repeated four times. 254 (P>0.05), whereas sperm concentration was negatively affected in high THI group, as the high 290 THI samples showed a lower (P<0.05) sperm concentration compared to low THI group. 291 Moreover, the bull effect on fresh semen parameters was significant. An interactive effect 292 between bull and season was statistically significant for both semen volume and sperm 293 concentration but not significant for subjective motility (Table 2) . 294 The impact of high THI on semen motility after thawing is presented in Table 3 . The frozen-297 thawed low THI spermatozoa had higher TM, PM, and VSL compared to high THI group 298 (P<0.05), but VCL, VAP and STR did not significantly differ between both groups. 299 Furthermore, interactive effect between bull and season was not statistically significant for all 300 parameters. 301 The effect of high THI on sperm morphology after freezing and thawing is shown in Table 303 4. The results show that high THI spermatozoa had higher percentage of abnormal spermatozoa 304 when compared to low THI (P<0.05). Moreover, the percentage of spermatozoa showing a bent 305 tail and a distal protoplasmic droplet was also significantly higher in the high THI samples, but 306 all other morphological parameters did not differ between the two groups (P>0.05). thawing are shown in Table 5 . The percentage of spermatozoa with aberrant chromatin 310 condensation was lower in low THI group (P<0.01), but the percentage of spermatozoa with 311 damaged acrosomes did not differ between both experimental groups (P>0.05). The effect of bull 312 (P=0.97) and the interaction between bull and treatment on acrosome status was also not 313 statistically significant. 314 Effect of high THI on sperm viability, O 2 and H 2 O 2 production after freezing and thawing is 316 presented in Table 5 . The elevated environmental temperature during summer was associated 317 with increased sperm H 2 O 2 production (P<0.05) which resulted in a decrease in sperm viability 318 (P<0.05) but the production of O 2 free radicals did not statistically differ between the two 319 groups. Moreover, the interactive effects between bull and treatment on viability, O 2 and H 2 O 2 320 production were not statistically significant. 321 Cleavage and blastocyst rates at Day 7 and 8 were higher in the group derived from low THI 323 spermatozoa (P<0.05). However, there was no significant difference in hatched/hatching rate 324 between treatments ( In the present study, the H 2 O 2 content was increased in semen samples collected and frozen 362 in summer. Furthermore, the elevated environmental temperature during summer was associated 363 with sperm O -* production, albeit not significantly. Hence, the decreased sperm quality and 364 subsequent embryo development may have been due to the increased production of ROS, 365 causing lower viability, higher morphological abnormalities and higher aberrant chromatin 366 condensation in the spermatozoa from high THI group. In general, increased testicular 367 temperature leads to increased metabolism in the testes and consequently to a higher need for 368 oxygen. Therefore, testes rely on increasing their blood flow to overcome heat stress-induced 369 deleterious effects [44] . However, if the testicular blood flow is not able to meet this increased 370 requirement, the testes became more hypoxic in response to the heat stress. Hypoxia can increase 371 the ROS production by affecting the sperm mitochondrial electron transport chain [45] . In a 372 recent review, Boni [10] stated that the deleterious effects of heat stress on reproductive 373 functions in mammals are caused by increased ROS concentration in cells and tissues 374 mitochondria according to a hypoxic condition. In addition, it is hypothesized that the ROS death and their overproduction is associated with sperm membrane lipid peroxidation [46, 47] . 377 The results of our study are in accordance with those of Hamilton et al [19] who observed that 378 the testicular insulation decreased motility and increased the percentage of damaged plasma 379 membrane, reacted acrosomes and spermatozoa with high mitochondrial membrane potential. 380 They concluded that this alteration may be due to higher ROS levels in ejaculated semen. 381 The present study showed that the percentage of total abnormal morphological spermatozoa The results of our study indicated that exposure of the bulls to high THI during summer can 404 also negatively affect subsequent in vitro embryo development. The sperm quality alteration 405 during spermatogenesis can affect quality of embryos as well as blastocyst development rates 406 [52] . Heat stress as a consequence of high THI can alter the integrity of paternal DNA by 407 protamine alteration [36] , and may lead to disrupted expression of key developmental genes 408 involved in formation of the blastocyst [53] . Similarly, in a previous study from our group, it has 409 been shown that the high THI during summer negatively affected the viability of spermatozoa in 410 Holstein bulls and led to a decrease in blastocyst development and delayed hatching [17] . 411 In conclusion, exposure of Belgian Blue bulls to elevated environmental temperature 413 negatively affected sperm quality. We observed an increased percentage of morphologically 414 abnormal spermatozoa and increased ROS generation after thawing. 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