Impact of various cryo-preservation steps on sperm rheotaxis and sperm kinematics in bull.

Semen cryopreservation is an important tool that has massively contributed to the progression of animal reproduction, especially in cattle. Nonetheless, a large part of the sperm population suffers from cryostress and loses fertility during the process. Although bovine semen cryopreservation is more advanced than any other species, there are still some missing links in the technology knowledge. The aim of the current study was to detect the effect of cryopreservation steps on sperm rheotaxis. Semen samples were collected from sex bulls and analyzed inside a microfluidic platform with CASA after each step of cryopreservation, including control, dilution with yolk citrate, cryoprotectant addition, and cooling or freezing. The results showed that positive rheotaxis % (PR) was not affected during cryopreservation. On the contrary, the sperm kinematics of the positive rheotactic sperm undergo significant changes, as velocity parameters (VCL, VSL, and VAP) were lower in both the cryoprotectant adding and cooling/freezing steps than in the control and yolk citrate dilution steps, while progression parameters (LIN and BCF) were higher in the cryoprotectant and cooling/freezing steps than in the control and yolk citrate dilution steps. Beside these results, an interesting phenomenon of sperm backward positive rheotaxis has been observed. The results of backward sperm rheotaxis samples revealed a significant decrease in PR%, while all sperm kinematics except BCF were significantly higher than normal rheotaxis samples. Based on these results, we conclude that positive rheotactic sperm cells are the elite of the sperm population; however, they still get some sublethal cryodamage, as shown by alterations in sperm kinematics. We also suggest that the sperm-positive rheotaxis mechanism is a mixture of an active and passive process rather than a passive physical one.

Sperm tendency to agglutinate in motile bundles in relation to sperm competition and fertility duration in chickens.

A unique sperm behavior was observed in Egyptian chickens. Sperm showed a tendency to agglutinate forming motile thread-like bundles. Sperm agglutination behavior, kinematics, and some morphometric measures were studied in relation to sperm competition and fertility duration in Sharkasi and Dandarawi chickens. Sperm tendency to agglutinate was assessed by examining sperm morphology using scanning electron microscopy, Acridine orange-stained semen smears using fluorescence microscopy, and recording videos of sperm under phase contrast microscope. Sperm velocity and morphometric measures were evaluated using image-J software. To assess sperm competition, Sharkasi and Dandarawi hens were artificially inseminated by semen pools possessing equal number of Sharaksi and Dandarawi sperm. Artificial insemination was repeated ten times. The eggs obtained were incubated, and the hatchlings were discriminated as descending from Sharkasi or Dandarawi fathers according to their phenotype. To assess the fertility duration, Sharkasi and Dandarawi hens were inseminated by semen collected from roosters of the same strain. Eggs were collected for a period of 28 days post-insemination and incubated. Sharkasi spermatozoa showed higher tendency to agglutinate forming longer and thicker motile bundles. No significant differences were observed in sperm curvilinear and straight line velocity and in sperm morphometric measures between Sharkasi and Dandarawi chickens. Sharkasi roosters fathered 81.6% and 67.7% of the hatchlings produced by Sharkasi and Dandarawi mothers, respectively. The fertility period in Sharkasi and Dandarawi was 22 and 14 days, respectively. We suggest that the differences seen in sperm competitiveness and fertility duration can be attributed to sperm agglutination behavior.

New insights into sperm rheotaxis, agglutination and bundle formation in Sharkasi chickens based on an in vitro study.

Fertility in birds is dependent on their ability to store adequate populations of viable sperm for extended durations in sperm storage tubules (SSTs). The exact mechanisms by which sperm enter, reside, and egress from the SSTs are still controversial. Sharkasi chicken sperm showed a high tendency to agglutinate, forming motile thread-like bundles comprising many cells. Since it is difficult to observe sperm motility and behavior inside the opaque oviduct, we employed a microfluidic device with a microchannel cross-section resembling close to that of sperm glands allowing for the study of sperm agglutination and motility behavior. This study discusses how sperm bundles are formed, how they move, and what role they may have in extending sperm residency inside the SSTs. We investigated sperm velocity and rheotaxis behavior when a fluid flow was generated inside a microfluidic channel by hydrostatic pressure (flow velocity = 33 µm/s). Spermatozoa tended to swim against the flow (positive rheotaxis) and sperm bundles had significantly lower velocity compared to lonesome sperm. Sperm bundles were observed to swim in a spiral-like motion and to grow in length and thickness as more lonesome sperm are recruited. Sperm bundles were observed approaching and adhering to the sidewalls of the microfluidic channels to avoid being swept with fluid flow velocity > 33 µm/s. Scanning and transmission electron microscopy revealed that sperm bundles were supported by a copious dense substance. The findings show the distinct motility of Sharkasi chicken sperm, as well as sperm's capacity to agglutinate and form motile bundles, which provides a better understanding of long-term sperm storage in the SSTs.

Development of computer-assisted sperm analysis plugin for analyzing sperm motion in microfluidic environments using Image-J.

We modified a previously reported computer-assisted sperm analysis (CASA) plugin for Image-J to enable analyzing motion of sperm cells in microfluidic environments. Microfluidics is increasingly being used in sperm-related applications such as sperm selection, IVF, and sperm motion behavior. Current CASA systems are not capable of analyzing motion of sperm cells in microfluidic devices where both sperm cells and the liquid itself are constantly moving, contrary to the conventional situation of sperm cells moving in a stationary liquid. We resolved this deficiency in the modified plugin reported here and built an image processing pipeline to enhance object detection, which increased CASA accuracy considerably. More importantly, particle tracking was improved and modified to accommodate sperm cells going out of focus for short periods during swimming on the same track. This last feature is particularly important in microfluidics where height of the microchannel is larger than that of CASA custom chambers to avoid channel blockage; this increased height causes sperm cells to frequently come in and out of focus. New parameters were introduced to allow studying new aspects of sperm motion behavior such as rheotaxis and wall tracking. The new plugin was able to detect and analyze motion of human, bull, and chicken sperm. A preliminary study using this tool agreed well with previously reported studies on rheotaxis and wall tracking behavior of sperm.

Characterization of rheotaxis of bull sperm using microfluidics.

We study rheotaxis of bull sperm inside microchannels to characterize the effects of flow and wall shape on sperm swimming behavior. We found that a large percentage of sperm cells, 80 to 84%, exhibited positive rheotaxis (sperm cells swimming against the flow) within flow velocities of 33 to 134 µm s(-1). Sperm cells were also found to reverse their swimming direction when the liquid flow direction was reversed. Time taken by sperm cells to reverse their swimming direction was inversely proportional to the flow velocity. Sperm behavior was significantly affected by the sperm position with respect to the channel wall. Sperm cells close to the channel wall moved upstream faster than sperm cells moving along the channel centerline. Shear stress, which is an indicator of velocity distribution, was found to play an important role in regulating rheotactic behavior of sperm cells. Side pockets were added to some microchannels to mimic storage sites in mucosal folds and pockets in the fallopian tube of the female reproductive system and sperm interaction with these pockets was monitored. We found that sperm cells tend to follow channel walls and enter these pockets without any chemical binding, which further confirms the wall tracking behavior of mammalian sperm cells. Our results confirm that sperm rheotaxis is a strong mechanism for guiding sperm cells towards the oocyte along the female genital tract.

Effect of dinoprost and cloprostenol on serum nitric oxide and corpus luteum blood flow during luteolysis in ewes.

In this study we compared the effect of dinoprost and cloprostenol on changes of corpus luteum blood flow during luteolysis. Ten nonlactating cyclic ewes were synchronized with double PGF2α injections 11 days apart. At Day 10, the animals were classified into 2 groups and received the third dose of PGF2α after confirmation of the presence of a mature CL. The first group received (12.5 mg/im) dinoprost and the second group received (250 µg/im) cloprostenol. A color Doppler ultrasound scan was performed by the same operator according to the following timeline: 0, 0.5, 1, 2, 4, 6, 12, and 24 hours, then every 24 hours until Day 4). The size, morphology, and blood flow of the CL was evaluated during the regression. The results showed that regression of the CL did not differ between the dinoprost and cloprostenol groups. There was no significant effect on diameter of the CL in both groups, though the size of the CL decreased gradually and slowly. Pretreatment progesterone concentration did not differ between groups. The results showed that the nitric oxide level was significantly increased within half an hour after the dinoprost treatment, and was significantly decreased in the cloprostenol group after half an hour. The blood velocity was increased significantly half an hour after the dinoprost treatment and it was decreased in the cloprostenol-treated group. In conclusion, both cloprostenol and dinoprost affect CL by controlling the nitric oxide level and blood supply of the CL via different mechanisms to induce luteolysis.