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.

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.

Using Estrous Behavior to Time Initiation of Oxytocin Administration to Prolong Luteal Function in Mares.

The objective of this study was to use estrous behavior alone to determine the appropriate time for beginning an oxytocin treatment protocol for estrus suppression. We hypothesized that administration of oxytocin beginning 8 days after the onset of estrus will prolong the luteal phase in mares. Twenty-three light breed mares (aged 4-20 years) were exposed to a stallion and observed for signs of sexual receptivity. Mares not displaying signs received 250 µg of cloprostenol intramuscularly (IM) and were teased again 3-4 days later. On the day that estrous behavior was observed (Day 0), mares were randomly divided into two groups: oxytocin (n = 11): oxytocin (60 IU, IM) was administered once daily from Day 8-17; control (n = 12): did not receive treatment. Blood was collected from all mares every 4 days throughout Day 17, and every 7 days thereafter until Day 45. Serum progesterone concentrations >1.0 ng/mL were indicative of a functioning corpus luteum. Interestrus interval was defined as the period between Day 0 and the day when progesterone next reached <1.0 ng/mL. The average interestrus interval was higher for treated mares compared with control mares (32.4 ± 4.2 vs. 21.8 ± 1.5 days, respectively, P = .01). In the oxytocin group, the interestrus interval was longer than 31 days in 6 of 11 (54.5%) mares and up to 45 days in 5 of 11 mares (45.5%). We conclude that luteal maintenance beyond 30 days was attained by once-daily oxytocin administration beginning 8 days following behavioral estrus in a majority of mares.

The sperm mitochondrion: Organelle of many functions.

This review summarizes current research in sperm mitochondrial function with specific emphasis on mitochondrial metabolism, reactive oxygen species production and mitochondrial genomics. This organelle is key in many crucial sperm functions including motility, hyperactivation, capacitation, acrosome reaction, and fertilization, thus its role in male fertility cannot be ignored. Recent studies have further elucidated sperm metabolism, placing greater emphasis on the importance of mitochondrial energy production for some species. Additionally, the dogma of mitochondrial reactive oxygen species production is changing and is being described by some as an indicator of increased mitochondrial function, potentially representing the most fertile sperm. Further, the mitochondrial genome, specifically mitochondrial DNA copy number, has been indicated as a potential biomarker for sperm quality and fertility in several species. Methods to study the sperm mitochondria are also evolving, allowing for researchers to learn more about the bioenergetics and status of this important organelle. Because of the importance of mitochondrial function for sperm function in most species, it is crucial to better understand the mechanisms involved in order to improve our knowledge of sperm physiology as well as improve handling and storage techniques for the industry.