Cysteine supplementation pre-freeze and post-thaw improves integrity and reduces oxidative stress in cryopreserved ram spermatozoa.

Cryopreserved ram sperm is highly sensitive to oxidative stress by reactive oxygen species (ROS) which impair sperm function and integrity. Antioxidants such as cysteine can mitigate the effect of ROS, although the optimal concentration or timing of supplementation is unknown. This study aimed to determine the effect of concentration and timing of cysteine supplementation on the integrity and function of cryopreserved ram spermatozoa. Nine ejaculates were collected from three Texel rams then cryopreserved and supplemented with cysteine (0, 0.5, or 1.0 mg/mL) added pre-freeze (PF), post-thaw (PT) or pre-freeze and post-thaw (PF + PT) generating seven treatments: 1) control 0 mg/mL, 2) PF 0.5 mg/mL, 3) PF 1 mg/mL, 4) PT 0.5 mg/mL, 5), PT 1.0 mg/mL, 6) PF + PT 0.5 mg/mL and 7) PF + PT 1.0 mg/mL. Sperm motility, viability, acrosome integrity, ROS production and penetrability through artificial cervical mucus were assessed post-thaw. Cysteine supplementation reduced ROS production which thereby improved spermatozoa motility, viability, acrosome integrity and penetrability (p < 0.001) Sperm integrity for all parameters was greatest in spermatozoa treated PF + PT with 1.0 mg/mL cysteine, although treatment pre-freeze or post-thaw also improved integrity beyond the control. This study has identified that 1.0 mg/mL cysteine is most beneficial and has highlighted the importance of preventing oxidative stress in spermatozoa post-thaw. These finding can help to mitigate the detrimental effect of cryopreservation on spermatozoa and aid the development of cryopreservation protocols in sheep.

An Investigation of Equine Sperm Quality Following Cryopreservation at Low Sperm Concentration and Repeated Freeze-Thawing.

Stallion spermatozoa are typically cryopreserved at 200 to 300 million sperm/ml; however recent advances such as intracytoplasmic sperm injection (ICSI) requires only one spermatozoon, wasting many, after thawing a whole straw. Cryopreserving at concentrations less than the current standard or refreezing thawed spermatozoa could maximize the use of genetically valuable animals and reduce waste. This investigation aimed to identify if lowering the sperm concentration for cryopreservation affected post-thaw quality after one and two freeze-thaw cycles. Nine ejaculates were collected from three fertile, "good freezer" stallions (post-thaw motility ≥35%) for experiment 1. Each ejaculate was split into eight treatments: five, 10, 20, 50, 100, 200, 300, 400 million sperm/ml and cryopreserved. Post-thaw: motility, viability, acrosome integrity and oxidative stress were assessed. Experiment 2, straws from experiment 1 (300 million sperm/ml) were thawed, diluted to 20 million sperm/ml or left undiluted (control) and refrozen. Post-thaw motility and viability were assessed. In experiment 1 sperm concentration did not affect post-thaw total motility (TM), progressive motility (PM) or viability at 50 to 400 million sperm/ml (P > .05). Whilst sperm concentrations of five to 20 million/ml did differ (post-thaw TM and PM). Both refreezing and reducing spermatozoa concentration, decreased TM, PM and viability (P < .05) after two freeze-thaw cycles. These results suggest cryopreserving at sperm concentrations as low as 50 million/ml maintains spermatozoa quality in good freezer stallions. Spermatozoa maintained some motility and viability when initially cryopreserved at 20 million sperm/ml and after two freeze-thaw cycles but research should investigate more optimal conditions.

The expression of prostaglandin endoperoxide synthase 2 messenger RNA and the proportion of smooth muscle and collagen in the sheep cervix during the estrous cycle.

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.

The effects of the prostaglandin E analogue Misoprostol and follicle-stimulating hormone on cervical penetrability in ewes during the peri-ovulatory period.

Two experiments in parous Welsh Mountain ewes determined the pattern of natural cervical relaxation over the peri-ovulatory period and investigated FSH and Misoprostol as cervical relaxants to facilitate transcervical passage of an insemination pipette into the uterine cavity. Following synchronisation of oestrus using progestagen sponges and PMSG (500 IU) the depth of cervical penetration was determined using a modified cattle insemination pipette as a measuring device. Penetration of the cervix was least at the time of sponge removal and increased to a maximum at 72 h after sponge removal and then declined. Intra-cervical administrations of either ovine FSH (Ovagen; 2mg) or Misoprostol (1mg; a Prostaglandin E(1) analogue) facilitated cervical penetration. Ovagen given 24h after sponge removal allowed transcervical intrauterine penetration in 100% of ewes at 54 and 60 h after sponge removal while Misoprostol given 48 h after sponge removal allowed trans-cervical penetration in 100% of ewes at 54 h. A combination of Ovagen and Misoprostol was as effective but not more so than Ovagen or Misoprostol alone. These results show that there is natural relaxation of the cervix at oestrus and that maximum relaxation occurs 72 h after sponge removal, which is too late for the correct timing of insemination. The intra-cervical administration of FSH or Misoprostol enhanced relaxation of the cervix and both were able to relax the cervix to allow intrauterine penetration 54 h after sponge removal, the optimum time for insemination. The results also show that FSH is biologically active after intracervical, topical application.

The anatomy of the sheep cervix and its influence on the transcervical passage of an inseminating pipette into the uterine lumen.

The anatomy of the sheep cervix is highly variable between animals and may explain the differing success of transcervical AI between individuals. This study aims to quantify the variation in cervical morphology between ewes and investigate the relationship between cervical anatomy and cervical penetration. Two series of reproductive tracts were collected. Series A: 132 adult anoestrous ewes, and series B: 165 cycling adult ewes and ewe lambs which were identified as luteal or non-luteal based on the presence of a corpus luteum. The morphology of the cervical external os was classified as slit, papilla, duckbill, flap or rose. An inseminating pipette was inserted into the lumen and the depth of penetration recorded. The cervix was opened longitudinally, its length recorded, the number of cervical rings counted and the arrangement of those rings graded. The maximum depth of cervical penetration was affected by cervical grade (series A: P=0.021; series B: P=0.037) and the stage of the oestrous cycle (P=0.008). Grade 1 cervices were more penetrable than grade 2, with grade 3 the least penetrable and non-luteal cervices could be penetrated further than luteal cervices. The distribution of os types differed with age, with rose types more common in adult ewes, and papilla os types more common in ewe lambs. These results indicate that the depth of cervical penetration is affected by the anatomy of the cervical lumen. Cervices with a less convoluted lumen (grade 1) were more penetrable. Non-luteal cervices are likely to have higher oestradiol concentrations than luteal, stimulating cervical relaxation and enabling deeper penetration. The difference in os types with age may be contributable to a morphological alteration at parturition.