Effects of activation and assisted reproduction techniques on the composition, structure, and properties of the sauger (Sander Canadensis) spermatozoa plasma membrane.

The sperm plasma membrane is a multifunctional organelle essential to fertilization. However, assisted reproduction techniques often negatively affect this structure, resulting in reduced fertility. These reductions have been attributed to plasma membrane damage in a wide array of species, including fish. Considerable research has been conducted on the fish sperm membrane, but few have examined the effect of cryopreservation and other assisted reproduction techniques (ARTs) on not only membrane composition, but also specific characteristics (e.g., fluidity) and organization (e.g., lipid rafts). Herein, we determined the effects of three ARTs (testicular harvest, strip spawning, and cryopreservation) on the sperm plasma membrane, using Sauger (Sander canadensis) sperm as a model. To this end, a combination of fluorescent dyes (e.g., merocyanine 540, filipin III, cholera toxin subunit ß), liquid chromatography - mass spectroscopy (LC-MS) analysis of membrane lipids, and membrane ultracentrifugation coupled with plate assays and immunofluorescence were used to describe and compare sperm fluidity, membrane composition, as well as lipid raft composition and distribution among sperm types. Stripped sperm became more fluid following motility activation (40% increase in highly fluid cells characterized by a 2 × increase in fluorescence) and contained lipid rafts restricted to the midpiece. Testicular harvest yielded sperm with characteristics similar to stripped sperm. By contrast, cryopreservation impacted every aspect of membrane physiology. Two cell populations, one highly fluid and the other rigid, resulted from the freeze-thaw process. Cryopreservation reduced lipid raft cholesterol content by 44% and flotilin-2 (a lipid raft marker) was partially displaced owing to a decrease in buoyancy. Unlike stripped and testicular sperm, LC-MS analysis revealed increases in oxidative damage markers, membrane destabilization, and apoptotic signaling in cryopreserved sperm. Ultrastructural analysis also revealed widespread physical damage to the membrane following freeze-thaw. Sperm motility, however, was unrelated to any measure of membrane physiology used in this study. Our results demonstrate that ARTs have the potential to substantially affect the sperm plasma membrane, but not always detrimentally. These results provide multiple potential biomarkers of sperm quality as well as insight into sources of sub-fertility resulting from use of ARTs.

Effect of glucose concentration and cryopreservation on mitochondrial functions of bull spermatozoa and relationship with sire conception rate.

Mitochondrial function is essential for sperm viability, not only from a sperm metabolism perspective, but also for improvement of sperm storage in liquid and frozen states. Bull sperm have notable metabolic variability with energy production for motility and subsequently for fertilizing capacity resulting from both glycolysis and oxidative phosphorylation. The objective of this study was to determine mitochondrial function of sperm using high-throughput Seahorse Analyzer technology in fresh semen and subsequent to freezing-thawing when there was incubation in media commonly used for sperm storage (relatively large glucose concentration) and female tract (relatively small glucose concentration). Additionally, there were determinations whether there were differences in values for fertility variables by regressing sire conception rate on values for mitochondrial variables when there was evaluation of semen from bulls with varying fertility. Media with larger concentrations of glucose inhibited mitochondrial function in fresh sperm, as indicated by less maximal oxygen consumption, spare respiratory capacity and coupling efficiency when compared to sperm in the media containing less glucose. Furthermore, there was greater (P <  0.05) mitochondrial function in cryopreserved-thawed compared to fresh samples with there being no effect of incubation media. These results indicate that mitochondrial damage from cryopreservation cannot be simply overcome post-thawing with glucose supplementation of bull semen incubation media. The increase in mitochondrial function is likely due to "non-productive" oxygen consumption to maintain the mitochondrial proton gradient. Furthermore, there was a negative association of mitochondrial proton leakage with sire conception rate indicating this could be a potential biomarker of bull fertility.

Evaluation of Biofilm Production by Escherichia coli Isolated From Clinical Cases of Canine Pyometra.

Many Escherichia coli (E. coli) strains produce biofilm that confers antimicrobial resistance. However, studies of biofilm production by E. coli from canine pyometra are lacking. Objectives were to elucidate the role of biofilm production by E. coli in pyometra by: (1) assessing the ability of E. coli to produce biofilm in vitro, and (2) confirming biofilm in situ. Endometrial biopsies were obtained from bitches with pyometra and preserved for microscopic analysis (n = 25). An endometrial swab was submitted for aerobic culture. Samples with confirmed E. coli were evaluated further for biofilm production in vitro and in vivo. Seventy percent of cases (16/23) resulted in pure growth of 1 or 2 E. coli strains, totaling 20 isolates. Fifteen isolates (15/20, 75%) had higher optical densities then negative controls (P < .05). On histopathology, all tissues exhibited endometrial inflammation and mucus was located within endometrial glands and occasionally overlying epithelium on 14 slides (14/16, 88%). Bacteria was noted in 50% of slides (8/16). During FISH acellular debris within the uterine lumen consistent with biofilm was noted on 94% of samples (15/16) and E coli was positively identified on all samples (15/15). Areas suggestive of the presence of biofilm were observed on all samples on scanning electron microscopy; but, bacteria consistent with E. coli were only visualized in 9 samples (9/16, 56%). In conclusion, we demonstrated that relevant strains of E. coli produce biofilm in vitro and in vivo, which may be considered in the development of new pyometra treatments aimed at disrupting these E. coli biofilm.

Testicular collections as a technique to increase milt availability in sauger (sander canadensis).

This study was conducted to compare quality and quantity of sperm collected from sauger (S. canadensis) using two collection methods: stripping alone and testicular tissue collection combined with stripping. Sperm were collected from sauger broodstock (n = 20) during the breeding season. Fish were randomly assigned to two sperm collection groups: (1) stripping once or (2) stripping twice before testicular tissue collection for obtaining additional sperm. Sperm motility variables, morphology, total number produced, and fertilization (%) were compared using the two collection methods. Testicular sperm had greater total motility (70.1 ± 2.1% compared with 44.3 ± 5.7%) but there were fewer morphologically normal cells (76.4 ± 1.3% compared with 92.8 ± 1.0%) compared to sperm collected using the stripping procedure. Sperm collection regimen utilizing testicular collections and sperm extractions in combination with stripping resulted in a ∼ten fold increase in total number of motile and morphologically normal sperm (39.5 ± 4.1 × 10 9) compared with the currently utilized two sequential sperm stripping collection procedures alone (3.6 ± 4.1 × 10 9 sperm). In large-scale studies (150,000 eggs), fertilization, using sperm collected from testicular tissues (1.0 × 105 motile sperm/egg), was similar to sperm collected with only the stripping procedure (71.2 ± 5.5 %, 81.2 ± 5.5 %, P = 0.265). The results of this study indicate testicular collection combined with sperm extractions allows for collection of sperm of a quantity and quality to maximize fry production and reduce the problems with lack of broodstock availability for sperm collection.

Technical Note: The use of iSperm technology for on-farm measurement of equine sperm motility and concentration.

The iSperm is a newly released semen analysis tool from Aidmics Biotechnology Co. LTD, which allows an iPad Mini to be transformed into a handheld microscope with objective semen analysis software for equine available through the Apple Store (version 4.5.2). The aim of this study was to compare iSperm values for sperm motility and sperm concentration to current acceptable methods for semen analysis and to determine the agreement with these methods using statistical methods. Two ejaculates from each of five Standardbred stallions were used to compare sperm motility (computer-assisted semen analysis [CASA] vs. iSperm) and concentration (NucleoCounter SP-100 [NC] vs. hemocytometer vs. iSperm). Data were analyzed by first testing for the differences between the means of each method using a linear mixed-effects model. The agreement between the two continuous measurements for each method was then investigated by computing Lin's concordance correlation coefficient (CCC), with a value of 1 indicating perfect agreement between methods. Results are reported as the CCC with the associated 95% confidence interval in parentheses. Means for both total motility (TM) and progressive motility (PM) were equal between CASA and iSperm values (P = 0.0741 and P = 0.725, respectively). However, means for all velocity measurements were significantly different between CASA and iSperm readings (P < 0.001). For concentration, means were equal between NC and iSperm values (P = 0.748) and for hemocytometer and iSperm values (P = 0.953). The CCC for TM was 0.871 (0.788, 0.923) and for PM was 0.916 (0.847, 0.955) indicating good agreement between methods. Low levels of agreement were observed for all velocity measurements. Finally, the CCC for concentration compared by iSperm and NC was 0.970 (0.949, 0.982) and for iSperm and hemocytometer it was 0.962 (0.934, 0.978), both close to the line of perfect concordance. Although more work is needed to improve the iSperm software for velocity measurements to be acceptable by research standards, in its present form the iSperm will introduce a low-cost and affordable method for on-farm semen analysis (TM, PM, concentration) for breeders and veterinarians. As a result, more farms will have access to accurate sperm analysis tools which will help to standardize semen processing procedures leading to better overall quality of semen used for artificial insemination.