Effects of cholesterol-loaded cyclodextrins on the quality of frozen-thawed equine epididymal sperm

Equine epididymal sperm are known to be severely sensitive to cryopreservation, in terms of sperm quality and pregnancy rate. The objective of this study was to examine the effects of cholesterol loaded cyclodextrins (CLCs) on the quality of stallion epididymal sperm during cryopreservation.In experiment I, sperm were treated with different concentrations of CLCs: (1) 0 mg (control), (2) 1.5 mg, (3) 3 mg, and (4) 6 mg per 120 × 10⁶ sperm. The sperm viability and amount of cholesterol were determined at 15, 30 and 45 min after CLC treatment using viability markers (Ethidium homodimer-1 and Calcein AM) and gas chromatography, respectively. In experiment II, CLC treated sperm (1.5 mg CLC per 120 × 10⁶ sperm) were fixed and stained with filipin to examine the cholesterol distribution. In experiment III, sperm were treated with CLCs at concentrations of 1.5, 3.0, 6.0 mg per 120 × 10⁶ sperm for 15 min, then equilibrated with freezing extender at 4 °C for 1 h prior to cryopreservation. Epididymal sperm without CLC loading (0 mg) were used as the control group. The sperm quality was examined at post-equilibration and 10 min, 2 h and 4 h after freezing and thawing.The cholesterol was successfully loaded into the plasma membrane of stallion epididymal sperm. The amount of cholesterol was increased in a manner of dose and time dependence, and the filipin–sterol complexes were increasingly labeled over the sperm head. CLCs at 1.5 mg/120 × 10⁶ sperm significantly improved sperm quality during sperm equilibration and cryopreservation compared to other doses of CLCs and non-CLC control. An increasing concentration and incubation time of CLCs was detrimental to sperm quality.It is concluded that cholesterol loading to the sperm plasma membrane via CLCs decreases chilling sensitivity and also improves epididymal sperm cryopreservability.     

Sperm distribution and fertilization after unilateral and bilateral laparoscopic artificial insemination with frozen-thawed goat semen

Generally, laparoscopic artificial insemination (LAI) provides a higher success rate than of cervical insemination in goats. However, the sperm distribution after LAI in goats remains unknown, particularly when frozen-thawed semen is used. This study evaluated the distribution of frozen-thawed goat spermatozoa after LAI and compared the effects of sperm numbers and deposition sites (unilateral and bilateral sites) on pregnancy rate. In experiment 1, the frozen-thawed spermatozoa were stained either with CellTracker Green CMFDA (CT-Green) or CellTracker Red CMPTX (CT-Red), and in vitro evaluations of viability and motility were performed. In experiment 2, the labeled spermatozoa were deposited via LAI into the left (CT-Green) and right (CT-Red) uterine horns (n = 4). After ovariohysterectomy (6 hours after insemination), the distributions of green- and red-colored spermatozoa were assessed via tissue section, flushing, and the oviductal contents were also collected. Experiment 3 was designed to test the pregnancy rates in a group of 120 does after LAI using different numbers of spermatozoa (60 and 120 × 10⁶ sperm per LAI) and different deposition sites. The results demonstrated that the fluorochromes used in this study did not impair sperm motility or viability. Frozen-thawed goat spermatozoa can migrate transuterinally after LAI, as evidenced by the observations of both CT-Green– and CT-Red–labeled spermatozoa in both uterine horns. Lower numbers of spermatozoa (60 × 10⁶) that are inseminated unilaterally (either ipsilateral or contralateral to the site of ovulation) can efficiently be used for LAI in goats (with a 56.67% pregnancy rate).     

Different DNA methylation patterns detected by the Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) technique among various cell types of bulls

Background  

The purpose of this study was to apply an arbitrarily primed methylation sensitive polymerase chain reaction (PCR) assay called Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) to investigate the methylation profiles of somatic and germ cells obtained from Holstein bulls.