Rising temperatures caused by climate change have adverse effects on cattle physiology, welfare, health, and reproduction. Heat stress in cows affects the oocyte and embryo directly through heat shock on cellular function. Fewer data are available on the effect of high temperatures on male fertility. Temperature-humidity index (THI) is a measure for assessing the risk of heat stress that combines the effects of temperature and humidity. The aim of this study was to determine the relationship between THI and fresh or frozen-thawed sperm quality of Holstein bulls kept in temperate climates. Bull sperm data of 29,170 ejaculates from 933 bulls collected at 3 Dutch artificial insemination centers between 2015 and 2018 were evaluated. The assessed variables included total sperm motility and morphology of fresh semen, and total sperm motility, morphology, and progressive motility of frozen semen 0 and 3 h after thawing. In addition, 56-d nonreturn rates were analyzed. The assessed effects were season and THI on the day of semen collection and during spermatogenesis (30 d before collection), bull, age of bull, year, and location. Bulls were divided into 2 categories according to their age: young (36 mo). Overall sperm quality of young bulls improved as age increased. No effect of THI on fresh sperm variables was observed in either young or older bulls. However, high THI at spermatogenesis negatively affected the cryotolerance of sperm cells. Sperm cells from young and older bulls showed a pronounced decrease (14-18%) of the assessed variables 3 h after thawing after the increase of THI during spermatogenesis in autumn. Remarkably, older bulls were more sensitive to THI at spermatogenesis compared with semen collection, showing up to a 3.8 times higher negative effect on frozen sperm quality. However, an elevated THI at semen collection produced a tendency toward decreased 56-d nonreturn rates as the age of the bull increased. Although this decrease was up to 4%, rising temperatures may still cause important economic losses in the future. For the first time, the present study confirmed that climate compromises not only sperm quality, but also dairy bull fertility.

Semen motility is the most widely recognized semen quality parameter used by Artificial Insemination (AI) centers. With the increasing worldwide export of semen between AI centers there is an increasing need for standardized motility assessment methods. Computer-Assisted Sperm Analysis (CASA) technology is thought to provide an objective motility evaluation; however, results can still vary between laboratories. The aim of present study was to verify the impact of different setting values of the CASA IVOS II on motility, concentration, and morphology of bovine semen samples frozen in an extender with or without egg yolk and then decide on optimal settings for a further validation step across AI centers. Semen straws from 30 different bulls were analyzed using IVOS II with twelve modified settings. No significant changes were observed in semen concentration, percentage of motile sperm or kinetic results for either extender type. However, increasing settings for both STR and VAP progressive (%) from Low, Medium, and High cut-off values significantly (p<0.05) reduced the percentage of detected progressive spermatozoa, in egg yolk extender from 49.5±15.2, 37.2±11.9 to 11.9±5.3%, and in clear extender from 51.9±9.1, 35.8±7.3 to 10.0±2.4%, respectively. In clear extender only, the modification of droplet proximal head length significantly affected the detection of normal sperm percentages (88.0± 4.7 to 95.0±0.6 and 96.0±0.6%) and of the percentage of detected proximal droplets (12.2±4.7, 2.5±2.7 to 0.6±0.2%) for Low, Medium and High values respectively (p<0.05). The identification of sensitivity within the CASA system to changes in set parameters then led to the determination of an optimal IVOS II setting. The existing variability among centers for these phenotypes was reduced when the standardized settings were applied across different CASA units. The results clearly show the importance of applied settings for the final CASA results and emphasize the need for standardized settings to obtain comparable data.

Pinched-flow fractionation removes viruses from (porcine) semen, which can increase the biosecurity of artificial insemination.

Microfluidics and lab-on-chip technologies have been used in a wide range of biomedical applications. They are known as versatile, rapid, and low-cost alternatives for expensive equipment and time-intensive processing. The veterinary industry and human fertility clinics could greatly benefit from label-free and standardized methods for semen analysis. We developed a tool to determine the acrosome integrity of spermatozoa using microfluidic impedance cytometry. Spermatozoa from boars were treated with the calcium ionophore A23187 to induce acrosome reaction. The magnitude, phase and opacity of individual treated and non-treated (control) spermatozoa were analyzed and compared to conventional staining for acrosome integrity. The results show that the opacity at 19 MHz over 0.5 MHz is associated with acrosome integrity with a cut-off threshold at 0.86 (sensitivity 98%, specificity 97%). In short, we have demonstrated that acrosome integrity can be determined using opacity, illustrating that microfluidic impedance cytometers have the potential to become a versatile and efficient alternative in semen analysis and for fertility treatments in the veterinary industry and human fertility clinics.

The use of microfluidics in artificial reproductive technologies for manipulation or assessment of spermatozoa is unique in the sense that it is not always an end point measurement and the sample may be used afterward. During microfluidic processing, spermatozoa are exposed to shear stress, which may harm viability and functioning of spermatozoa. The shear stresses during general microfluidic processing steps were calculated and compared to estimated shear stresses during ejaculation. The viability of boar and bull spermatozoa after microfluidic processing was studied and compared to the typical handling method (centrifugation) and to a control (the sample in a tube at the same temperature). The boar spermatozoa showed a small but significant decrease in viability of 6% after microfluidic handling. Bull spermatozoa proved to be less susceptible to shear stress and were not significantly affected by microfluidic processing. These data indicate that the impact of microfluidic processing on the viability of boar and bull spermatozoa is less than the literature values reported for flow cytometry and comparable to the impact of centrifugation.