No abstract

The use of transcervical artificial insemination in sheep is limited because of the anatomy of the cervix, which restricts the passage of an inseminating pipette into the uterine lumen. There is a degree of natural cervical relaxation at estrus that enables greater penetration with an inseminating pipette. We hypothesize that this relaxation may be regulated by cervical prostaglandin synthesis and remodeling of the cervical extracellular matrix. The present study investigated the changes in prostaglandin endoperoxide synthase 2 (PTGS2) mRNA expression and the proportion of smooth muscle and collagen in the sheep cervix during the estrous cycle. Sheep cervices were collected at four stages of the estrous cycle: prior to the LH surge, during the LH surge, after the LH surge, and during the luteal phase. The expression of cervical PTGS2 mRNA was determined by in situ hybridization, and the proportion of smooth muscle and collagen in the cervix was investigated by Masson trichrome staining. The expression of PTGS2 mRNA in the sheep cervix was greatest prior to the LH surge, when estradiol concentrations were also greatest. The increase in PTGS2 mRNA expression was associated with an increase in the proportion of collagen in the sheep cervix. We propose that prior to the LH surge, estradiol may stimulate PTGS2 mRNA expression and hence prostaglandin E2 synthesis in the sheep cervix to regulate cervical relaxation, most likely through the rearrangement of collagen bundles within the cervical extracellular matrix.

No abstract

This study compared protocols for cryopreservation of ejaculated, papain-treated alpaca spermatozoa. This included different concentrations of egg yolk (EY; 5, 10 or 15%) and glycerol (2, 5 or 10%), diluent types (SHOTOR, lactose, skim milk or INRA-96™), freeze rates (2, 4 or 8 cm above liquid nitrogen; LN), thaw rates (37 °C for 1 min or 42 °C for 20 sec) and storage vessels (pellets, 0.25 mL straws or 0.5 mL straws). Spermatozoa were assessed pre-freeze and 0, 30, 60 and 90 min post-thaw. Forty-one hembras were inseminated with either fresh, papain-treated or frozen-thawed spermatozoa. Motility was affected by EY concentration (P < 0.001), diluent type (P < 0.001), freeze rate (P = 0.003) and storage vessel (P = 0.001). Viability was affected by EY concentration (P < 0.001), diluent type (P < 0.001), storage vessel (P = 0.002) and thaw rate (P = 0.03). For artificial insemination (AI), semen was diluted 1:3 in a lactose-based diluent, with 5% EY and glycerol. Freezing was in 0.5 mL straws, 2 cm above LN for 4 min then thawing at 37 °C for 1 min. Pregnancy rates of those ovulated (n = 26) were not different (1/5 fresh, 1/4 papain-treated, 0/17 frozen-thawed; P = 0.10). Pregnancy can be achieved after AI with papain-treated spermatozoa. Further work is needed to determine the optimal dose, timing and location for insemination.

In camelids, the development of assisted reproductive technologies is impaired by the viscous nature of the semen. The protease papain has shown promise in reducing viscosity, although its effect on sperm integrity is unknown. The present study determined the optimal papain concentration and exposure time to reduce seminal plasma viscosity and investigated the effect of papain and its inhibitor E-64 on sperm function and cryopreservation in alpacas. Papain (0.1 mg mL–1, 20 min, 37°C) eliminated alpaca semen viscosity while maintaining sperm motility, viability, acrosome integrity and DNA integrity. Furthermore E-64 (10 µM at 37°C for 5 min after 20 min papain) inhibited the papain without impairing sperm function. Cryopreserved, papain-treated alpaca spermatozoa exhibited higher total motility rates after chilling and 0 and 1 h after thawing compared with control (untreated) samples. Papain treatment, followed by inhibition of papain with E-64, is effective in reducing alpaca seminal plasma viscosity without impairing sperm integrity and improves post-thaw motility rates of cryopreserved alpaca spermatozoa. The use of the combination of papain and E-64 to eliminate the viscous component of camelid semen may aid the development of assisted reproductive technologies in camelids.