In vitro characteristics of X- and Y-bearing ram spermatozoa sorted by bovine serum albumin (BSA) column and TLR7/8 ligand R848.

The quality of the separated fractions in sex-sorted semen is very important for the success of the artificial insemination. This study aimed to evaluate some in vitro characteristics (DNA quantity, kinematic parameters and enzymes activity) of X- and Y-bearing ram spermatozoa sorted by bovine serum albumin (BSA) column and toll-like receptors (TLR)7/8 ligand R848. The ejaculates from six rams were collected by artificial vagina and subjected to a computer-assisted semen analysis (CASA). Total motility and percentage of the sperms with rapid and medium progressivity or non-progressivity in whole ejaculates and in X and Y fractions were analyzed. Activity of the enzymes ALP, GGT, CK, LDH and accumulation of lactate in the seminal plasma of ejaculates and in the environmental fluid of sexed spermatozoa were measured by biochemical analyzer. DNA was isolated from precipitated spermatozoa, and its quantity was measured. For both protocols the DNA mass from X-bearing fractions was higher, than from Y-bearing fractions. The high total motility of X- and Y-bearing spermatozoa as well as greater percent sperms with progressive motility were observed after use of BSA protocol. The application of TLR7/8 ligand R848 protocol led to reducing of Y-sperm motility and enhancement of non-progressivity in both fractions, which corresponded to the determined high amount of the extracellular lactate. For both methods, the significantly reduced activity of enzymes in the X and Y spermatozoa environmental fluids was established. Both protocols produce X- and Y-sperm fractions with satisfactory quality (over 80% total motility and over 50% rapid and medium progressive spermatozoa in each fraction).

Canine sperm vitrification with nonpermeable cryoprotectants and coconut water extender.

This study was aimed to assess the efficiency of coconut water extender with addition of soy lecithin and sucrose as nonpermeable cryoprotectants for canine semen vitrification, using a simple method that yields a high survival rate of spermatozoa for clinical use. Twelve ejaculates from 12 adult normozoospermic dogs were collected separately by digital manipulation and only the second semen fraction was used in this study. After evaluation of volume, concentration, viability, total and progressive motility, velocity parameters and morphology, semen was diluted with a coconut water extender (50% (v/v(volume per volume)) coconut water, 25% (v/v) distilled water and 25% (v/v) 5% anhydrous monosodium citrate solution) with addition of soy lecithin and fructose at 1% and 0.25M sucrose until final concentration of 100x106 spermatozoa/ml. After equilibration at 5ºC for 60 minutes, semen was vitrified by "direct dropping method" into liquid nitrogen in spheres with a volume of 30 µl. After a week of storage the spheres were devitrified as three of them were dropped into 0.5 mL of CaniPlus AI medium (Minitüb, Germany), which was previously warmed in a water bath at 42ºC for 2 minutes and evaluated about the above mentioned parameters. It was found that vitrification resulted in a lower percentage of viable sperms, normal morphology, total and progressive motilities (p<0.05), but most of velocity parameters (VCL, VSL, VAP, LIN, ALH and BCF) did not differ (p>0.05) compared to fresh semen samples. In conclusion, our results demonstrate that vitrification with coconut water extender with addition of 1% soy lecithin and 0.25M sucrose as cryoprotectants, has an excellent potential for routine canine sperm cryopreservation.

Influence of Ovarian Status and Steroid Hormone Concentration on Day of Timed Artificial Insemination (TAI) on the Reproductive Performance of Dairy Cows Inseminated with Sexed Semen.

This study aimed to evaluate the effect of the ovarian status and steroid hormone concentration on the day of TAI on the reproductive performance of dairy cows subjected to estrus synchronization treatment and timed artificial insemination with sexed semen. Seventy-eight cyclic Holstein cows pre-treated with PGF2α-GnRH were divided in two groups-I (Preselect-OvSynch, n = 38) and II (OvSynch+PRID-7-day+eCG, n = 40)-and inseminated with sexed semen. The presence of preovulatory follicle (PF) with or without corpus luteum (CL), the PF diameter, the estradiol (E2) and progesterone (P4) concentrations on the day of TAI, the pregnancy rate (PR) and embryo loss were determined. On the day of TAI, 78.4% of all the pregnant cows presented a PF (mean size 1.80 ± 0.12 cm) without CL, low P4 (0.59 ± 0.28 ng/mL) and high E2 (12.35 ± 2.62 pg/mg) concentrations. The positive correlation between the size of the PF and the level of E2 in the pregnant cows from group II was stronger than that of group I (R = 0.82 vs. R = 0.52, p < 0.05). The pregnancy rate on day 30 (57.5% vs. 36.8%) and day 60 (50% vs. 26.3%; p < 0.05) and the embryo losses (13% vs. 28.5%) showed better effects of treatment in group II. In conclusion, the ovarian status and the steroid hormone concentration on the day of TAI influence the pregnancy rates of dairy cows subjected to estrus synchronization and timed artificial insemination with sexed semen.