3 Animal Protein-Free Semen Extender for Fixed-Time Insemination of Beef Cows

Abstract

Current semen cryopreservation protocols include the use of animal products, such as egg yolk, in semen extenders, which raises important biosecurity concerns related to the transmission of infectious agents. Therefore, there is a need for alternatives to animal proteins in semen extenders. Cholesterol-cyclodextrin (CC) has been used as an adjunct in semen extenders to facilitate the delivery of exogenous cholesterol into sperm plasma membranes. The purpose of this study was to determine the fertility potential of semen extended with cholesterol of plant origin (PhytoChol, Wilshire Technologies, NJ, USA) in a fixed-time insemination program for beef cows. Semen was collected by electroejaculation from mature Simmentals bulls (n = 4 bulls, 4 replicates/bull) and analysed using computer-assisted semen analysis (CASA). Ejaculates from different bulls with ≥200 × 10⁶ sperm mL⁻¹ and ≥60% total motility were pooled. The pooled ejaculates were distributed into 3 treatments and diluted to 50 × 10⁶ mL⁻¹ in Tris-citric-acid base extenders containing either egg yolk (20% vol/vol egg yolk, control), 0.5 mg of CC per mL of semen, or 1.0 mg of CC per mL of semen. Glycerol (7% vol/vol) was added to each extender. Extended semen was loaded into 0.5-mL straws and frozen to –196°C. Post-thaw sperm motility was analysed using CASA. Ovulation was synchronized among multiparous beef cows using 1 of 3 protocols: (1) 5-day intravaginal progesterone-releasing device (PRID) and prostaglandin F_2α PGF_2α) treatment on the day of PRID removal (n = 37 on random days of cycle); (2) oestradiol + progesterone treatment and 7-day PRID followed by PGF_2α on the day of PRID removal (n = 37 on random days of cycle); or (3) PGF_2α alone (n = 19 on Day 7 to 9 from ovulation). Cows were treated with gonadotropin-releasing hormone (GnRH) and assigned randomly to be inseminated with 1 of the 3 semen treatments 72 h after PGF_2α treatment. Ovulations were confirmed and pregnancies were diagnosed 28 to 35 days after insemination by ultrasonography. Post-thaw sperm motility was compared among extenders by analysis of variance. Pregnancy rates were compared among groups by generalized linear mixed model. Total motility (52 ± 3%, 59 ± 3%, and 62 ± 7%) and progressive motility (47 ± 3%, 54 ± 3%, and 58 ± 6%) did not differ among the egg yolk, 0.5 mg CC, and 1.0 mg CC extenders, respectively. There were no differences in pregnancy rates among synchronization groups; therefore, data were combined to compare the effects of semen extender. Four cows were excluded due to improper synchronization treatment. Pregnancy rates of cows inseminated with egg yolk (n = 31), 0.5 mg CC (n = 31), and 1.0 mg CC (n = 27) semen were 32, 74, and 52%, respectively (P < 0.05). Fertility in cattle inseminated using semen extended with cholesterol of plant origin has not been previously reported. We conclude that plant cholesterol may be used to replace animal proteins (egg yolk or milk origin) in bovine semen cryopreservation.

Research supported by grants from the Saskatchewan Agricultural Development Fund, Agriculture and Agri-Food Canada, and Alberta Livestock and Meat Agency.