S.A. Holden

Cholesterol-loaded-cyclodextrins improve the post-thaw quality of stallion sperm

An unacceptable proportion of stallion sperm do not survive the freeze-thaw process. The hypothesis of this study was that adding cholesterol to a stallion semen extender would stabilise the sperm membrane, resulting in an improved post-thaw semen quality in terms of increased sperm viability, membrane integrity and fluidity, and reduced oxidative stress. Semen was collected from three stallions and diluted in four extenders: TALP; TALP+0.75mg methyl-β-cyclodextrin-cholesterol (MβCD)/mL (MβCD0.75); TALP+1.5mg MβCD-cholesterol/mL (MβCD1.5); and Equipro. Following 15min incubation, samples were centrifuged and diluted to 100×10(6)sperm/mL, frozen in 0.5mL straws and stored in liquid nitrogen. Sperm from each treatment was assessed for progressive linear motility (PLM) and acceptable membrane integrity under hypotonic conditions on a phase contrast microscope at 1000× while viability, membrane fluidity and superoxide generation were assessed by flow cytometry. The MβCD1.5 and MβCD0.75 treatments had a greater proportion of viable sperm than the TALP treatment (P<0.01). There was no effect of treatment on PLM or membrane integrity. The MβCD1.5 treatment had a greater proportion of viable sperm positive for membrane fluidity than the TALP treatment (P<0.05). The MβCD1.5 and MβCD0.75 treatments had a lesser proportion of viable sperm positive for superoxide generation than the TALP treatment (P<0.001). This study has demonstrated that adding cholesterol to stallion sperm prior to cryopreservation increases post-thaw viability, with these viable sperm being of better quality in terms of increased membrane fluidity and reduced superoxide generation.

The impact of storage temperature and sperm number on the fertility of liquid-stored bull semen

In Ireland, liquid bull semen is stored at unregulated ambient temperatures, typically at 5×106 spermatozoa per dose, and inseminated within 2.5 days of collection. In Experiment 1, the effect of storage temperature (5, 15, 22, 32°C and fluctuations (Flux) between these temperatures) on progressive motility, viability, acrosomal status, DNA fragmentation and osmotic resistance was assessed. In Experiment 2, the field fertility of liquid semen at 5, 4 and 3×106 spermatozoa per dose, up to Day 2 after collection, was assessed in comparison to frozen-thawed semen at 20×106 spermatozoa per dose (n=35328 inseminations). In Experiment 1, storage at 15°C resulted in the highest progressive motility (PP6 spermatozoa per dose on Day 2 of storage was reduced in comparison to frozen-thawed semen (P<0.01). In conclusion, liquid semen is versatile between storage temperatures of 5 and 22°C, but demonstrates reduced fertility on Day 2 of storage at lower sperm numbers in comparison to frozen-thawed semen.

In vitro characterisation of fresh and frozen sex-sorted bull spermatozoa.

This study sought to compare the in vitro characteristics of fresh and frozen non-sorted (NS) and sex-sorted (SS) bull spermatozoa. Experiment 1: Holstein-Friesian ejaculates (n=10 bulls) were split across four treatments and processed: (1) NS fresh at 3×106 spermatozoa, (2) X-SS frozen at 2×106 spermatozoa, (3) X-SS fresh at 2×106 spermatozoa and (4) X-SS fresh at 1×106 spermatozoa. NS frozen controls of 20×106 spermatozoa per straw were sourced from previously frozen ejaculates (n=3 bulls). Experiment 2: Aberdeen Angus ejaculates (n=4 bulls) were split across four treatments and processed as: (1) NS fresh 3×106 spermatozoa, (2) Y-SS fresh at 1×106 spermatozoa, (3) Y-SS fresh at 2×106 spermatozoa and (4) X-SS fresh at 2×106 spermatozoa. Controls were sourced as per Experiment 1. In vitro assessments for progressive linear motility, acrosomal status and oxidative stress were carried out on Days 1, 2 and 3 after sorting (Day 0=day of sorting. In both experiments SS fresh treatments had higher levels of agglutination in comparison to the NS fresh (P<0.001), NS frozen treatments had the greatest PLM (P<0.05) and NS spermatozoa exhibited higher levels of superoxide anion production compared with SS spermatozoa (P<0.05). Experiment 1 found both fresh and frozen SS treatments had higher levels of viable acrosome-intact spermatozoa compared with the NS frozen treatments (P<0.01).

Effect of seminal plasma from high- and low-fertility bulls on cauda epididymal sperm function

The aim of this study was to characterise the effect of seminal plasma (SP) from bulls of high or low fertility on sperm function. First, the effect of SP on the motility of fresh cauda epididymal spermatozoa (CES) and frozen-thawed ejaculated spermatozoa was assessed (Experiment 1a). Seminal plasma was then collected from bulls of known high and low fertility. Pooled CES were incubated in the SP from each bull, diluted and assessed for motility and viability on Days 1, 2, 3 and 5 after packaging as fresh semen (Experiment 1b). Also assessed were motility, kinematics, viability and mitochondrial membrane potential after thawing (Experiment 1c) as well as hypotonic resistance (Experiment 2) and fertilisation potential using in vitro fertilisation (Experiment 3). Seminal plasma increased the motility of CES (P<0.05); however, there was no effect of SP on the motility and viability of fresh CES or on CES post-thaw motility, viability and mitochondrial membrane potential (P>0.05). The hypotonic resistance of CES was reduced by SP (P<0.05), irrespective of whether the SP was from high- or low-fertility bulls. Seminal plasma from high- or low-fertility bulls had no effect on cleavage or blastocyst rates (P>0.05). In conclusion, SP affects the physiological function of CES but there is no difference between SP from high- or low-fertility bulls.

In vitro addition of docosahexaenoic acid improves the quality of cooled but not frozen–thawed stallion semen

The aim of the present study was to assess the effect of the addition of docosahexaenoic acid (DHA) on the in vitro quality of cooled and frozen-thawed stallion semen. In Experiment 1, semen from 10 stallions was collected (three ejaculates per stallion). Semen was diluted to 100×106 spermatozoa mL-1 with 0.02mM vitamin E (VE) and 0, 1, 10 or 20ng mL-1 DHA and frozen. Semen was thawed and total motility (TM), rapid progressive motility (PM), acrosome integrity, membrane fluidity and morphology were assessed. In Experiment 2, semen from three stallions was collected (three ejaculates per stallion) and frozen as in Experiment 1, but VE and DHA were added after thawing. TM and PM were assessed at 30, 60 and 120min and viability, acrosome integrity and membrane fluidity were evaluated at 30min. In Experiment 3, semen from five stallions was collected (one to three ejaculates per stallion), diluted to 20×106 spermatozoa mL-1 and stored at 4°C. After 1, 24, 48 and 72h, TM, PM, viability, membrane fluidity and lipid peroxidation were assessed. The addition of DHA had no effect on frozen semen (Experiments 1 and 2) but improved TM, PM and membrane fluidity in cooled stallion semen.